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CN101500591A - Compositions and methods using same for the detection of viruses - Google Patents

Compositions and methods using same for the detection of viruses Download PDF

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Publication number
CN101500591A
CN101500591A CNA2006800389723A CN200680038972A CN101500591A CN 101500591 A CN101500591 A CN 101500591A CN A2006800389723 A CNA2006800389723 A CN A2006800389723A CN 200680038972 A CN200680038972 A CN 200680038972A CN 101500591 A CN101500591 A CN 101500591A
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virus
protease
substrate
test
cutting
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阿萨夫·以斯拉
多里特·阿拉德
吉拉德·韦里德
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MND Diagnostics Ltd
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MND Diagnostics Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

An isolated peptide is provided. The isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 and 47, said amino acid sequence being no more than 14 amino acids in length. Also provided are compositions which comprise the peptides and use of same in the detection of viruses.

Description

Be used to detect the compositions of virus and use said composition to detect the method for virus
Technical field
The present invention relates to be used for detect quickly and easily the new compositions of the cleavage activity of catalytic molecule.The present invention also provides diagnostic test and the test kit that adopts these compositionss.
Background technology
No matter to healthy correlation factor (is the labelling of disease or health risk, the labelling or the factor of normal and pathogenic course or disease, or the indication of exotic disease substance and its by-product) fast, detection specific and that cost is to one's profit has growing demand.The outburst of propagation repeatedly along with the infectious disease that have swepts the globe such as HIV, SARS, bird flu, west Nile fever, drug resistance pathogenic bacterium disease etc. makes the demand more remarkable.In the face of the popularity of increase day by day and the threat of being very popular property illness outbreak, the earlier detection of virulence factor is for sufficient concern and prevent most important.Yet, the many pathogen detection of employing and cost, complexity and inefficiencies of classifying method of can supplying at present force the policy of carrying out waste time and energy extremely expensive and poor efficiency, inoculate such as isolate, become extinct vector and preventative vaccine, and its success rate remains query.In addition, a large amount of labellings of disease and pathological process have been identified, some of them comprise catalytic activity such as the telomerase in various cancers, the terminal deoxynucleotidyl transferase in leukemia (TdT), and the β in Alzheimer-and gamma-secretase (secretase).
1918, the world experienced spanish influenza, caused surpassing 2,000 5 million people's death altogether in Europe.Hereafter the fear to medical emergency occurs once more.Only before the several years, fatal SARS virus makes All Around The World economy that the danger of paralysis be arranged in the outburst of east, the world.The outburst of described SARS is retarded and finally be controlled, but this only be take the measure of very disruptive (comprise suspected patient only because heating and with the long-term isolation of healthy population) afterwards.At present, the importance of improving one's methods of people of the world's fast detecting dangerous pathogens is reminded in the outburst of bird flu threat once more.
In 21 century, the flowability owing to travel by air middle very big probability that infects and individual, crowd increase day by day makes that novel global communicate illness transmission danger is very high.The method that being used to of adopting at present detected the pathogenicity factor comprises: immune detection, such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA) (EIA) and immunofluorescence assay (IFA), it depends on the antipathogen detection of antibodies of carrying out in 10～20 days after primary infection; RT-PCR, it is very expensive time-consuming and mistake usually occurs; And the tissue culture infectiousness, equally also be time-consuming expensive.Therefore, needing badly can be at the fast simple diagnostic method of airport and the infected or potential infectiousness individuality of other public territorys detection.Described method must have the highly variable feature in the face of current virokine, can accurately detect under the situation that forms highly pathogenic species that can make a variation.Need the multiple viral pathogen of one-time detection to get rid of probability and the more formation of strongly-typed virus that mixes cross infection.The typing quick and precisely of described pathogenic factor also is need be top-priority.Need be at hospital institution or a large amount of pathogenic factors of fast detecting at home, this will be provided for selecting the accurate data of Therapeutic Method, and prevent the mixing and the infection of the Different Kinds of Pathogens factor, and be easy to the trickle adjustment of on-site supervision and therapeutic scheme.
Many organisms have the characteristic catalytic activity relevant with the moment of growth or differentiation, metabolism etc.Equally, pathological process usually has the characteristic enzymatic activity, can be used in the diagnosis of described pathological process, such as cancer markers and cardiac enzymes.Based on catalytic activity be used to diagnose and classify and the method that detects is open, for example be used for the diagnosis of malignant bacteria and classification (referring to, Maiden etc. for example, J.Clin.Micro, 1996; 34:376-84 and authorize No. 5,888,760, the United States Patent (USP) of Godsey etc.), (referring to for example Kundu etc., ChemBioChem 2002 for the DNA-photolyase (phytolyases) during cancer forms; 3:1053-60), beta-secretase activity in Alzheimer (referring to No. the 200302555th, the U.S. Patent application of Hazuda etc.), metabolic activity mapping in living cells (referring to Boonacker etc., J of Histochem and Cytochem 2001; 49:1473-86), caspase and other enzymes relevant with apoptosis (referring to No. the 20020150885th, the U.S. Patent application of Weber etc.) and virus detect (referring to No. the 4th, 952,493, the United States Patent (USP) of for example authorizing Kettner etc.).
The clear and definite specific substrate that effective measurement of these catalytic activitys and clinical practice requirement can easily be detected.The specific proteins enzymatic activity that a kind of potential this diagnostic catalytic activity is a viral infection.
Between the replicative phase of many viruses such as SARS virus, human immunodeficiency virus, human papillomavirus, herpesvirus, rhinovirus, picornavirus, coronavirus, hepatitis C virus etc., viral genetic is transcribed the formation polyprotein, and it finally is cut into two or more biological activity protein.It is key component in the viral life cycle that described viral polyprotein is cut into body protein.Many viruses, the encoding viral protease that comprises adenovirus, baculovirus, cowpea mosaic virus, picornavirus, retrovirus retrovirus and togavirus family etc., this protease cuts described viral polyprotein to form the required activated protein of virus replication at some specific cleavage sites.For example, the processing of the polyprotein between the replicative phase of orchardgrass mottle virus (adzuki bean mosaic virus genus) is described in Makinen, K etc., J.Gen.Virol.2000; Among the 81:2783-89, its content is introduced by reference at this.As a reference, the non-exhaustive list of the known embodiment of some virus proteases and other enzymes provides hereinafter.
The protease of some encoding virals only cuts the polyprotein of specific virus.Other cuttings surpass the polyprotein of one type virus.The specificity of protease effect stems from the interactive property of described protease in the cutting zone of described polyprotein.In addition, change at the cutting speed of these positions peptide sequence according to the polyprotein around the described cleavage site.
The cleavage site of being discerned by these virus proteases along described virus protein has shown the aminoacid sequence that contains high conservative, this pointed out with its be included in the diagnosis and Therapeutic Method in probability.
For example, the United States Patent (USP) of authorizing Kettner etc. discloses the peptide substrates that is used to detect the virus-specific proteinase activity for the 4th, 952, No. 493, and it designs according to the conservative cleavage site of being discerned by virus protease.Aminoacid sequence according to the virus-specific cleavage site of determining by the order-checking of virus polypeptide, perhaps determined the cleavage site of these peptide substrates, and can compare by comparison with other virus polypeptide sequences according to described virus genomic coded sequence.The conservative replacement that the similar residue to other biological of some amino acid residue carries out is also thought sustainable.
The U.S. Patent application of Tan etc. discloses the application that the substrate that is used for test sample parasite, protozoacide, virus and other proteinase activities merges substrate in conjunction with reorganization fluorescence for No. 20050214890, and wherein said detection is based on the protease recognition mode of many substrates.The design of described cutting and/or identification substrate is derived from known total cutting and/or binding sequence.It then is owing to measure many substrates the time that the enhancing of target protein enzyme detects.
Yet, thereby describe, advise or mention without any a kind of said method and select viral substrate to obtain optimized affinity and finally realize analyzing simultaneously fast and the specific recognition of neotype strain of various product, many viruses.
The protease detection efficiency that optimized design by described substrate cutting sequence improves will provide excellent agility, sensitivity and the economy of detection and the characterization of viral infection.In addition, the method for designing of substrate cutting sequence and screening and the exploitation that product will be used for antiviral drugs thereof.
Therefore there is the extensively demand of approval, and it will be very favorable obtaining optimization substrate and method, the cleavage activity that described optimization substrate and method are used to detect the catalytic molecule is characterized in that the disease and the course of infection of cleavage activity to detect specifically fast, and does not have above-mentioned defective.
Summary of the invention
According to an aspect of the present invention, the invention provides isolated peptides, described isolated peptides contains the aminoacid sequence that is selected from by SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46 and 47 groups of forming, and the length of described aminoacid sequence is no more than 14 aminoacid.
According to a further aspect in the invention, the invention provides the compositions of the substrate that contains virus protease, but described substrate links to each other with at least one test section and contains described aminoacid sequence.
According to another feature in the preferred implementation of the following description of the present invention, but described at least one test section is that FRET is right, and the cutting of described substrate generation comes from the right signal of described FRET.
According to another feature in the preferred implementation of the following description of the present invention, described compositions further contains separating part.
According to a further aspect of the invention, the invention provides the compositions that general formula is X-Y-Z,
Wherein:
Y contains the substrate of virus protease, and described substrate contains aminoacid sequence, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
But X contains the test section; With
Z contain can with the separating part that combines mutually separating of two phase-separated system;
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease.
According to another feature in the preferred implementation of the following description of the present invention, but described test section X contains the marking agent that is selected from the group of being made up of enzyme, fluorogen, chromophore, albumen, proenzyme, chemiluminescent substance and radiosiotope.
According to another feature in the preferred implementation of the following description of the present invention, described separating part Z is selected from by immunoconjugator, magnetic knot and closes the group that part, peptide bound fraction, affinity bound fraction, nucleic acid moiety are formed.
According to a further aspect of the invention, the invention provides the compositions that general formula is X-Y-Z,
Wherein:
Y contains the substrate of virus protease, and described substrate contains aminoacid sequence, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
X or Z contain underlined, but perhaps test section and/or can separate through separating part cutting or uncut compositions in suitable mode.
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease.
According to another feature in the preferred implementation of the following description of the present invention, wherein said labelling, X or Z partly contain and are selected from the marking agent that, pearl, peptide, proenzyme and radiosiotope, immunoconjugator, magnetic knot is closed the group that part, peptide bound fraction, affinity bound fraction, nucleic acid moiety form by enzyme, fluorogen, chromophore, albumen, chemiluminescent substance, quencher, FRET.
According to another aspect of the present invention, the invention provides the method for at least a virus in the test sample, described method comprises
(a) in sample and the described compositions at least one contacted under the condition of the cutting that can carry out described substrate; With
(b) cutting of the described substrate of monitoring, the cutting of wherein said substrate are the indications that at least a virus exists in the described sample.
According to another feature in the preferred implementation of the following description of the present invention, step (a) comprises described sample contacted with at least two kinds of substrates of different virus protease, and any described cutting do not occur and then indicates and do not have virus in the described sample in wherein said at least two kinds of substrates.
According to another feature in the preferred implementation of the following description of the present invention, described sample is selected from by mucus, saliva, throat cleanout fluid, nose cleanout fluid, spinal fluid, expectorant, urine, seminal fluid, perspiration, feces, blood plasma, blood, bronchovesicular liquid (broncheoalveolar fluid), vaginal secretion, tear and biopsy thing.
According to another feature in the preferred implementation of the following description of the present invention, the detection of cleavage activity described in the described sample is the diagnosis to medical condition.
According to another feature in the preferred implementation of the following description of the present invention, described monitoring is to adopt homogeneous determination (homogeneous assay) to realize.
According to another feature in the preferred implementation of the following description of the present invention, described monitoring is to adopt out-phase to measure (heterogeneous assay) to realize.
According to a further aspect of the invention, the invention provides the diagnostic kit that is used at least a virus of test sample, described test kit contains at least a compositions and is used to detect the reagent of the cutting of described substrate.
According to a further aspect of the invention, the invention provides the diagnostic kit that contains packaging material and be used to detect the multiple compositions of multiple virus existence, wherein the general formula of every kind of described compositions is,
X-Y-Z
Wherein:
Y contains the substrate of virus protease, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
X or Z contain underlined, but perhaps test section and/or can separate through separating part cutting or uncut compositions in suitable mode,
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease,
But each among wherein said X or the Z comprises at least one special test section, and described packaging material comprise that label or package insert are used for the multiple virus of test sample to indicate described test kit.
According to a further aspect of the invention, the invention provides the diagnostic kit that contains packaging material and be used to detect the multiple compositions of multiple virus existence, wherein the general formula of every kind of described compositions is,
X-Y-Z
Wherein:
Y contains the substrate of virus protease, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
But X contains the test section; With
Z contain can with the separating part that combines mutually separating of two phase-separated system;
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease,
Among the wherein said X each is special detectable, and described packaging material comprise that label or package insert are used for the multiple virus of test sample to indicate described test kit.
According to another feature in the preferred implementation of the following description of the present invention, described multiple compositions links to each other with single solid support.
According to another feature in the preferred implementation of the following description of the present invention, described special detection is to realize by the location of the addressable on described single solid support.
According to another feature in the preferred implementation of the following description of the present invention, but described special detection is to realize by different test sections.
According to another feature in the preferred implementation of the following description of the present invention, each in the described multiple compositions links to each other with solid support.
According to another feature in the preferred implementation of the following description of the present invention, described solid support is configured as pearl.
According to another feature in the preferred implementation of the following description of the present invention, described pearl is selected from by molecular group of coloured pearl, magnetic pearl, label pearl and fluorescent bead.
According to another feature in the preferred implementation of the following description of the present invention, be to breathe test kit, contain at least two kinds of viruses that are selected from the group of forming by coronavirus, SARS, HMPV (human stroma lung virus), influenza virus A+B, bird flu virus, adenovirus, RSV (respiratory syncytial virus), rhinovirus, parainfluenza virus.
According to another feature in the preferred implementation of the following description of the present invention, be the breathing test kit that contains Hantaan and LaCrosse's encephalitis (La Crosse Encephalitis).
According to another feature in the preferred implementation of the following description of the present invention, be the gastrointestinal test kit, contain at least two kinds of viruses that are selected from the group of forming by rotavirus, adenovirus 40/41, hepatitis A virus, hepatitis C virus, hepatitis E virus, Calicivirus and CMV (cytomegalovirus).
According to another feature in the preferred implementation of the following description of the present invention, described diagnostic kit is the meningitis test kit, contains at least two kinds of viruses that are selected from by enterovirus (1-～80), west nile virus, herpes simplex virus 1,2 and 6 groups of forming.
According to another feature in the preferred implementation of the following description of the present invention, described diagnostic kit is the meningitis test kit, contains at least two kinds of viruses that are selected from the group of being made up of togavirus, banzi virus and rabies virus.
According to another feature in the preferred implementation of the following description of the present invention, described diagnostic kit is the sexually transmitted disease (STD) test kit, contains at least two kinds of viruses that are selected from the group of being made up of HIV Strain, herpes simplex virus 1, herpes simplex virus 2, HSV-1, HSV-2, HPV (human papillomavirus) and HTLV-1.
According to another feature in the preferred implementation of the following description of the present invention, described diagnostic kit is traveller's test kit, contains at least two kinds of viruses that are selected from the group of being made up of hepatitis A virus, hepatitis B virus, hepatitis C virus, HIV, herpesvirus 1 and 2.
According to another feature in the preferred implementation of the following description of the present invention, described diagnostic kit is veterinary's test kit, contains at least two kinds of viruses that are selected from the group of being made up of rabies virus and canine distemper virus.
According to another feature in the preferred implementation of the following description of the present invention, described at least a sample comprises several samples.
According to another feature in the preferred implementation of the following description of the present invention, described at least a virus comprises multiple virus.
According to another feature in the preferred implementation of the following description of the present invention, described virus is adenovirus and described substrate contains SEQ ID NO:1 or 2.
According to another feature in the preferred implementation of the following description of the present invention, described virus is α virus and described substrate contains SEQ ID NO:3.
According to another feature in the preferred implementation of the following description of the present invention, described virus is rubella virus and described substrate contains SEQ ID NO:4.
According to another feature in the preferred implementation of the following description of the present invention, described virus is HIV and described substrate contains SEQ ID NO:5.
According to another feature in the preferred implementation of the following description of the present invention, described virus is HTLV and described substrate contains SEQ ID NO:6,7 or 8.
According to another feature in the preferred implementation of the following description of the present invention, described virus is arteritis virus and described substrate contains SEQ ID NO:9.
According to another feature in the preferred implementation of the following description of the present invention, described virus is coronavirus and described substrate contains SEQ ID NO:10.
According to another feature in the preferred implementation of the following description of the present invention, described virus is sars coronavirus and described substrate contains SEQ ID NO:11 or 12.
According to another feature in the preferred implementation of the following description of the present invention, described virus is Orbivirus and described substrate contains SEQ ID NO:13.
According to another feature in the preferred implementation of the following description of the present invention, described virus is CMV virus and described substrate contains SEQ ID NO:14 or 15.
According to another feature in the preferred implementation of the following description of the present invention, described virus is herpesvirus and described substrate contains SEQ ID NO:16.
According to another feature in the preferred implementation of the following description of the present invention, described virus is banzi virus and described substrate contains SEQ ID NO:17.
According to another feature in the preferred implementation of the following description of the present invention, described virus is dengue virus and described substrate contains SEQ ID NO:18,19 or 20.
According to another feature in the preferred implementation of the following description of the present invention, described virus is west nile virus and described substrate contains SEQ ID NO:21,22 or 23.
According to another feature in the preferred implementation of the following description of the present invention, described virus is yellow fever virus and described substrate contains SEQ ID NO:24,25 or 26.
According to another feature in the preferred implementation of the following description of the present invention, described virus is Japanese encephalitis virus and described substrate contains SEQ ID NO:27,28 or 29.
According to another feature in the preferred implementation of the following description of the present invention, wherein said virus is that Ticks passes virus (Tick bone virus) and described substrate contains SEQ ID NO:30,31 or 32.
According to another feature in the preferred implementation of the following description of the present invention, described virus is hepatitis C virus and described substrate contains SEQ ID NO:33,34 or 35.
According to another feature in the preferred implementation of the following description of the present invention, described virus is Pestivirus and described substrate contains SEQ ID NO:36.
According to another feature in the preferred implementation of the following description of the present invention, described virus is hepatitis A virus and described substrate contains SEQ ID NO:37 or 38.
According to another feature in the preferred implementation of the following description of the present invention, described virus is HRV and described substrate contains SEQ ID NO:39 or 40.
According to another feature in the preferred implementation of the following description of the present invention, described virus is enterovirus and described substrate contains SEQ ID NO:41,42,43,44,45,46 or 47.
According to another feature in the preferred implementation of the following description of the present invention, described virus is HRV virus and described substrate contains SEQ ID NO:143～151.
According to a further aspect of the invention, the invention provides the method that design motivation is learned optimized virus protein zymolyte, described method comprises:
(a) in a plurality of cutting sequences of the polyprotein of at least a described Strain, differentiate demonstrate cutting sequence that the fastest cutting power of described protease learns and
(b) differentiate the total cutting of the family's scope sequence that demonstrates the fastest cutting power, the total cutting of described family scope sequence is used for design motivation and learns optimized described protease substrate.
According to another feature in the preferred implementation of the following description of the present invention, described virus protease is the protease of encoding viral.
According to another feature in the preferred implementation of the following description of the present invention, described virus is selected from the group of being made up of DNA viruses and RNA viruses.
According to another feature in the preferred implementation of the following description of the present invention, described virus is selected from by tectivirus section (Tectiviridae), papovaviridae (Papovaviridae), porcine circovirus section (Circoviridae), Parvoviridae (Parvoviridae) and Hepadnaviridae (Hepadnaviridae), Cystovirus section (Cystoviridae), birnavirus section (Birnaviridae), Reoviridae (Reoviridae), coronaviridae (Coronaviridae), flaviviridae (Flaviviridae), Togaviridae (Togaviridae), arteritis virus (Arterivirus), Astroviridae (Astroviridae), Caliciviridae (Caliciviridae), Picornaviridae (Picornaviridae), marmor upsilon section (Potyviridae), Retroviridae (Retroviridae), orthomyxovirus section (Orthomyxoviridae), filamentous virus section (Filoviridae), Paramyxoviridae (Paramyxoviridae), Rhabdoviridae (Rhabdoviridae) and Bunyan Viraceae (Bunyaviridae), Adenoviridae (Adenoviridea), herpetoviridae (Herpesviridae), the group that Picornaviridae (Picornaviridae) is formed.
According to another feature in the preferred implementation of the following description of the present invention, described virus protease is selected from by serine protease, metalloproteases, aspartic protease, cysteine proteinase, 3C protease, the PA transcriptase, adenine protease, 2A protease, chymase (chimotrypsin) or trypsin, NS3 for example, NS2, the NS-pro cysteine proteinase, the nsP2 cysteine proteinase, nsP23pro, C protein enzyme, SFV NS, the HIV aspartic protease, nsp4 arteritis virus protease, HCMV protease, NS2-3, NS3-4Ap protease, the group that HTLV-1PR forms.
According to another feature in the preferred implementation of the following description of the present invention, described method further may further comprise the steps:
(c) design contains a plurality of cutting sequences of the total cutting of described family scope sequence; With
(d) in described a plurality of cutting sequences, differentiate to have the cutting sequence that the fastest cutting power of described protease is learned.
According to another feature in the preferred implementation of the following description of the present invention, described design comprises that design has the described cutting sequence of optimal dissolution degree, temperature sensitivity and/or pH sensitivity.
According to another feature in the preferred implementation of the following description of the present invention, the described discriminating of step (a) comprises empirical experiment.
According to another feature in the preferred implementation of the following description of the present invention, the described discriminating of step (a) comprises data mining.
According to a further aspect of the invention, the invention provides a kind of isolated peptides, described isolated peptides contains and is selected from the NO:1 by SEQ ID, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, the aminoacid sequence of 46 and 47 groups of forming, the length of described aminoacid sequence is no more than 14 aminoacid, and contains the active analogies of the corresponding virus protease of inhibition.
According to a further aspect of the invention, the invention provides the purposes of described peptide in the manufacturing that is used for the treatment of medicine for treating viral infections through discriminating.
According to a further aspect of the invention, the invention provides and comprise the pharmaceutical composition of described isolated peptides as active component.
The present invention is by being provided for detecting the compositions of virus and the shortcoming that method has successfully overcome existing scheme.
Except as otherwise noted, as used herein technology and scientific and technical terminology have with those skilled in the art the identical implication generally understood.Though can be used for practice of the present invention and test to similar or suitable method described herein and material, below describe suitable method and material.Under the situation of conflict, with description of the present invention, comprise definition, be main.In addition, described material, method and embodiment only are descriptive, are not to be intended to be used to limit.
Description of drawings
With reference to the accompanying drawings, only present invention is described herein by by way of example.At length concrete now with reference to described accompanying drawing, it is emphasized that shown details is by by way of example and only is purpose for the descriptive discussion of preferred implementation of the present invention, and be to be the description of the most useful of principle of the present invention and concept nature aspect and the easiest understanding and stating in order to it is believed that.In this, do not carry out anyly attempting to demonstrate than representing the more detailed CONSTRUCTED SPECIFICATION of the present invention of the necessary CONSTRUCTED SPECIFICATION of basic comprehension of the present invention, the incidental description of described accompanying drawing makes understanding various ways of the present invention is how to embody in practice with those skilled in the art know that.
In described accompanying drawing:
Fig. 1 a～b contains HRV 163C and the plasmid pMND1 (1a) of SARS 3C1 protease and illustrating of pMND2 (1b) respectively.
Fig. 2 is the time course from the expression of the reorganization 3C protease of SARS among the escherichia coli E.coli and HRV.Induce the clone of conversion with 0.4mM IPTG, on Bioanalyzer 2100lab-on-chip, adopt Protein 200 plus chip kit (protein 200 adds chip agent box) (Agilent Technologies, Inc, Palo Alto CA) by the electrophoretic analysis WCL.Swimming lane 1-MW gradient; Swimming lane 2～4-be respectively and inducing 0,1 and 6 hour the pMND-2 in back; Swimming lane 4～6-be respectively and inducing 0,1 and 6 hour the pMND-1 in back.Notice the stable increase that the expression of SARS3CL and HRV 16 3C protease increases in time.
Fig. 3 compares with natural cleavage site (Ori 2), the pattern description of the excellent substrate characteristic of the PEP1 substrate through designing.Reaction contains 5 μ M fluorogenic substrates and 500nM reorganization 3C protease, is monitored at 340/490 ± 15nm by luminoscope.Enzyme concentration is 500pM～1 μ M Pep-1-Filled Ellipse; Ori 2-solid diamond; Pep1 contrast-Filled Rectangle; Ori 2 contrast-solid triangles.Excellent substrate cutting when noticing Pep 1.
Fig. 4 is the pattern description of HRV 3C protease (250nM) and the excellent kinetics of synthetic Pep 1 substrate under the concentration of substrate of 3nM～4 μ M.
Fig. 5 is the specific pattern description of described synthetic Pep 1 substrate by the cutting of HRV 3C protease.Incubation is carried out in fluorescence Pep 1 substrate and E.coli lysate (closed square), reorganization SARS 3CL lysate (filled circles), reorganization HRV 3C lysate (solid triangle) and contrast (no lysate) (solid diamond).Notice the absolute specificity of 1 couple of HRV 3C of Pep protease.
Fig. 6 is the pattern description of Pep 1 substrate by the effective cutting of HRV 3C protease under nose cleanout fluid condition.Fluorescence Pep 1 substrate and reorganization HRV 3C are not having in the presence of the 50 volume % nose cleanout fluid (closed square) and are having in the presence of the 50 volume % nose cleanout fluid (sample E0052) (filled circles) to react.Contrast is for only there being nose cleanout fluid (E0052) (solid diamond).Notice the effect of described nose cleanout fluid that do not exist to the substrate cutting.
Fig. 7 is the sketch map that can be used to cut the compositions of the present invention of event detection.
Fig. 8 is the sketch map that shows described system elementary event, and described system adopts the compositions described in above-mentioned Fig. 7.
Fig. 9 is the schematic representation that shows the separating mechanism of multiple virus.
Figure 10 is the schematic representation that detects three kinds of molecules of the cleavage reaction that carries out three types simultaneously.
Figure 11 describes the sketch map of NS3 protease to the continuous hydrolysis of hepatitis C virus polyprotein.
The specific embodiment
The present invention relates to be used to detect the compositions of virus and use said composition to detect the method for virus.
Can understand principle of the present invention and operation better with reference to described accompanying drawing and appended explanation.
Before explaining at least one embodiment of the present invention in detail, be to be understood that not to be that the present invention is used be confined to set forth in the following description or illustrational in an embodiment details.The present invention can have other embodiments or can implement in many ways or carry out.Equally, it should also be understood that phrase and the term that adopt in this place only should be as not restrictive for descriptive purpose.
The diagnostic assay field that is used to detect actute infection changes fast, fade to pathogen detection from antibody test, by fading to based on point-of care based on clinical laboratory, detect by single analyte and to fade to multiple analyte and detect, and more be conceived to the strategy that adopts less invasive collection of biological sample to detect.Novel mensuration is measured sensitive more usually than tradition, and can provide more information and described pathogen or described host are characterized replying of pathogen.From the publilc health angle, the appearance of the molecular epidemiology that can follow the tracks of pathogen according to the gene order of uniqueness or antigen attribute has been revolutionary have been changed the epidemiologist and how to have investigated and assessed epidemic diseases and evaluation region disease.In addition, the use of point of care (POC) equipment can exert an influence to infecting detection and supervision, and will improve our and accurately judge nosogenetic ability.
By differentiating that viral enzyme activity detects virus, obtained proposing, such as No. the 20050048473rd, No. the 4th, 952,493, the United States Patent (USP) of authorizing DoritArad and U.S. Patent application before.These diagnostic assays based on enzymatic activity are excellent more than analysis of molecules such as polymerase chain reaction (PCR) chemical examination, and this is because the latter bothers very much, need carry out several hours usually, and the cost costliness.In addition, the PCR method that provides at present provides 40% false positive results and negative findings (because they also differentiate the virus of inactivation) and makes described method inefficiency and also dangerous.Usually, the testing result of virus protease indicates the existence of living in the health and have active viral infection in the live body sample.This with can detect and the irrelevant DNA of live virus or the pcr analysis of RNA sequence, the antibody test that detects the immunoreactive existence of antiviral health is measured and is formed sharp contrast.On the contrary, the TPPA that can detect virus antigen is not to represent the existence of live virus all the time, and this is because of virus antigen even can changes and make a variation for specific antibodies in a disease life cycle.
When design is of the present invention, the best substrate that inventor's hypothesis is used for protease activity determination will be the total cleavage site of polyprotein (being the natural substrate of described protease), and it is the fastest that its cutting power is learned.The substrate that meets this sequence will be used in the enzymatic diagnosis test to differentiate many virus of sharing same protease fast widely sporadicly.
Such as hereinafter description (and in the embodiment 1 of hepatitis C NS3 protease illustrated in greater detail), the inventor has identified these Dynamics Optimization substrates of a large amount of virus families (being DNA and RNA viruses, referring to embodiment 2).So the substrate of HRV that identifies and enterovirus can be used for successfully detecting in the corresponding virus of nose liquid and cerebrospinal fluid (CSF) sample and with result that RT-PCR analyzes (referring to embodiment 4 and 5) and compares.Yet just as mentioned, the accuracy that RT-PCR detects is shady, and protease of the present invention detects and compares RT-PCR and detect and have clear superiority, and is as indicated above.
Therefore, according to an aspect of the present invention, the invention provides the method for designing of the optimum substrate of kinetics of virus protease.
Phrase " the optimum substrate of kinetics " refers to corresponding to being cut (by K (1M) by described protease the most apace as used herein, k (cat) and k (cat)/K (m) defines, and supposes that described enzyme observes Michaelis-Menten kinetics) the conserved amino acid sequence of natural cleavage site.
Described substrate can be amino acid substrate (that is, containing aminoacid sequence) or its analogies.
The protease example of protease (below provide) of encoding viral is provided phrase as used herein " virus protease ".Described virus can be any virus of expressing protein hydrolytic enzyme (be preferably do not show the host protein enzyme action cut specificity).
The following realization of the method for this aspect of the present invention: (a) in a plurality of cutting sequences (referring to Figure 11) of the polyprotein of at least a Strain, identify and show the cutting sequence that the fastest cutting power of described protease is learned, (b) identify the total cutting of the family's scope sequence that demonstrates the fastest cutting power, the total cutting of described family scope sequence can be used to design the kinetics optimum substrate of described virus protease.
Adopt any known method in this area can realize empirical kinetics sign, generally include by recombinating or synthetic method prepares soluble fluorescent substrate (for example based on HPLC mensuration).Alternatively, also can use the cell bank [for example, referring to Boulware and Daugherty2006 PNAS 103:20-7583:7588] of peptide substrates.Also can be referring to " Hydrolysis of a seriesof synthetic peptide substrates by the human rhinovirus 14 3C proteinase; cloned and expressed in E.coli " such as Orr D.C., J.Gen.Vir. roll up 70, page or leaf 2931-42 (1989), its content is introduced by reference at this.
Can adopt data in literature substituting or additivity to excavate description list to reveal the cutting sequence that the fastest cutting power is learned, as the detailed description in the embodiment 1 of following examples part.
In case obtain to show the cutting sequence that the fastest cutting power is learned, then can identify and show the total cutting of the family's scope sequence that the fastest cutting power is learned.
" family's scope total " as used herein refers at the modal aminoacid on each position of the sequence series of comparison corresponding to the fastest cutting sequence of the polyprotein that belongs to same virus family.
This is by using common bioinformatics instrument to adopt sequence alignment algorithm such as BLAST (Basic Local Alignment Search Tool (base area comparison search tools) obtains by www.ncbi.nlm.nih.gov/BLAST) or Smith-Waterman algorithm (referring to the embodiment 1 of following examples part) to realize.
In case identify described family scope consensus sequence, can design the peptide that contains this consensus sequence, if desired, can make its sequence be adapted to show interesting biochemical property.Example comprises optimal dissolution degree, temperature stability and pH stability.These peptides are considered to substrate of the present invention.
The term of Shi Yonging " peptide " comprises native peptides (or catabolite, the synthetic peptide of synthesis mode or recombinant peptide) and intends peptide (being generally the synthetic peptide of synthesis mode) herein, and as the class peptide and the half class peptide of peptide analogues, it has for example makes described peptide more stable or more can penetrate into modifications such as cell in health.These modifications include but are not limited to: that N is terminal modified, C is terminal modified, peptide bond is modified (including, but not limited to CH 2-NH, CH 2-S, CH 2-S=O, O=C-NH, CH 2-O, CH 2-CH 2, S=C-NH, CH=CH or CF=CH), backbone modification and residue modify.Preparing the method for intending peptide compounds knows in this area, and at for example Quantitative Drug Design, CA.Ramsden Gd., Chapter 17.2, describe in detail in the F.Choplin Pergamon publishing house (1992), it is introduced as describing in detail fully at this by reference at this.More details in this respect provide following.
Peptide bond in the described peptide is (CO-NH-) can be for example by the N-key (N (CH that methylates 3)-CO-), ester bond (C (R) H-C-O-O-C (R)-N-), ketone methene key (CO-CH 2-), (NH-N (R)-CO-) replace, wherein R is for example methyl, carba key (CH of any alkyl to α-azepine key 2-NH-), hydroxy ethylene key (CH (OH)-CH 2-), thioamides key (CS-NH-), alkene double bond (CH=CH-), trans amide key (NH-CO-), peptide derivant (N (R)-CH 2-CO-), wherein R is " normally " side chain, natural being present on the described carbon atom.
These modifications can occur in along on any key of described peptide chain and even simultaneously (2～3) take place in many places.
Natural aromatic aminoacid, Trp, Tyr and Phe can be replaced by synthetic alpha-non-natural amino acid such as phenylglycine, Tic, naphthyl alanine (Nal), phenylisoserine, Soviet Union's ammonia alcohol, the ring-derivant that methylates of Phe, halo derivatives or the adjacent methyl-Tyr of Phe.
Except that above-mentioned, peptide of the present invention also can comprise one or more modified aminoacid or one or more non-amino acid monomer (for example fatty acid, complex carbohydrate etc.).
The term " aminoacid " that uses in this description and following claim part or " several amino acids " are interpreted as and comprise described 20 kinds of natural amino acids; Usually after translation, carry out the aminoacid modified in the body, comprise for example hydroxyproline, phosphoserine and phosphothreonine; And other non-common amino acids including, but not limited to 2-aminoadipic acid, oxylysine, different chain lysine, norvaline, nor-leucine and ornithine, in addition, term " aminoacid " comprises D-and L-aminoacid.
Following table 1 and table 2 have been listed and can be used for natural amino acid of the present invention (table 1) and unconventional or modified aminoacid (for example synthetic, table 2).
Table 1
Aminoacid Trigram is called for short One digital code
Alanine Ala A
Arginine Arg R
Agedoite Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Iie I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
Above arbitrary amino acid Xaa X
Table 2
Unconventional aminoacid Code name Unconventional aminoacid Code name
Butyrine Abu The L-N-methylalanine Nmala
Alpha-amido-alpha-methyl butyric acid ester Mgabu The L-N-methylarginine Nmarg
Amino-cyclopropane- Cpro The L-N-methylasparagine Nmasn
Carboxylate The L-N-methylaspartic acid Nmasp
Aminoisobutyric acid Aib L-N-methyl cysteine Nmcys
Amino normal borneol base- Norb L-N-methyl glutamine Nmgin
Carboxylate L-N-methyl glutamic acid Nmglu
Cyclohexylalanine Chexa The L-N-methylhistidin Nmhis
The cyclopenta alanine Cpen L-N-methyl isoleucine Nmile
The D-alanine Dal The L-N-methylleucine Nmleu
The D-arginine Darg The L-N-methyllysine Nmlys
The D-aspartic acid Dasp The L-N-methylmethionine Nmmet
The D-cysteine Dcys L-N-methyl nor-leucine Nmnle
The D-glutamine Dgln L-N-methyl norvaline Nmnva
D-glutamic acid Dglu L-N-methyl ornithine Nmorn
The D-histidine Dhis L-N-methylbenzene alanine Nmphe
The D-isoleucine Dile The L-N-methylproline Nmpro
The D-leucine Dleu L-N-methyl serine Nmser
D-lysine Dlys The L-N-methylthreonine Nmthr
The D-methionine Dmet The L-N-methyl tryptophan Nmtrp
The D-ornithine Dorn The L-N-methyl-tyrosine Nmtyr
The D-phenylalanine Dphe The L-N-methylvaline Nmval
The D-proline Dpro L-N-Methylethyl glycine Nmetg
The D-serine Dser L-N-methyl-tert-butyl group glycine Nmtbug
The D-threonine Dthr The L-nor-leucine Nle
The D-tryptophan Dtrp The L-norvaline Nva
D-tyrosine Dtyr Alpha-Methyl-aminoisobutyric acid esters Maib
The D-valine Dval Alpha-Methyl-γ-An Jidingsuan ester Mgabu
D-Alpha-Methyl alanine Dmala The Alpha-Methyl Cyclohexylalanine Mchexa
D-Alpha-Methyl arginine Dmarg Alpha-Methyl cyclopenta alanine Mcpen
D-Alpha-Methyl agedoite Dmasn Alpha-Methyl-Alpha-Naphthyl alanine Manap
D-Alpha-Methyl aspartate Dmasp The Alpha-Methyl penicillamine Mpen
D-Alpha-Methyl cysteine Dmcys N-(the amino butyl of 4-) glycine Nglu
D-Alpha-Methyl glutamine Dmgln N-(2-amino-ethyl) glycine Naeg
D-Alpha-Methyl histidine Dmhis N-(3-aminopropyl) glycine Norn
D-Alpha-Methyl isoleucine Dmile N-amino-alpha-methyl butyric acid ester Nmaabu
D-Alpha-Methyl leucine Dmleu The Alpha-Naphthyl alanine Anap
D-Alpha-Methyl lysine Dmlys N-benzyl glycine Nphe
D-Alpha-Methyl methionine Dmmet N-(2-carbamyl ethyl) glycine Ngln
D-Alpha-Methyl ornithine Dmorn N-(carbamyl ethyl) glycine Nasn
D-Alpha-Methyl phenylalanine Dmphe N-(2-carboxy ethyl) glycine Nglu
D-Alpha-Methyl proline Dmpro N-(carboxyl methyl) glycine Nasp
D-Alpha-Methyl serine Dmser N-cyclobutyl glycine Ncbut
D-Alpha-Methyl threonine Dmthr N-suberyl glycine Nchep
D-Alpha-Methyl tryptophan Dmtrp The N-Cyclohexylglycine Nchex
The D-alpha-methyltyrosine Dmty N-ring decyl glycine Ncdec
D-Alpha-Methyl valine Dmval N-cyclo-dodecyl glycine Ncdod
D-α-methylalnine Dnmala N-ring octyl group glycine Ncoct
D-Alpha-Methyl arginine Dnmarg N-cyclopropyl glycine Ncpro
D-Alpha-Methyl agedoite Dnmasn N-ring undecyl glycine Ncund
D-Alpha-Methyl aspartate Dnmasp N-(2, the 2-diphenyl-ethyl) glycine Nbhm
D-Alpha-Methyl cysteine Dnmcys N-(3, the 3-diphenyl propyl) glycine Nbhe
The D-N-methylleucine Dnmleu N-(3-indyl ethyl) glycine Nhtrp
The D-N-methyllysine Dnmlys N-methyl-γ-An Jidingsuan ester Nmgabu
N-methylcyclohexyl alanine Nmchexa The D-N-methylmethionine Dnmmet
D-N-methyl ornithine Dnmorn N-methylcyclopentyl alanine Nmcpen
Sarcosine Nala D-N-methylbenzene alanine Dnmphe
N-methylamino isobutyrate Nmaib The D-N-methylproline Dnmpro
N-(1-methyl-propyl) glycine Nile D-N-methyl serine Dmnser
N-(2-methyl-propyl) glycine Nile D-N-methyl serine Dnmser
N-(2-methyl-propyl) glycine Nleu The D-N-methylthreonine Dnmthr
The D-N-methyl tryptophan Dnmtrp N-(1-Methylethyl) glycine Nva
The D-N-methyl-tyrosine Dnmtyr N-methyl Alpha-Naphthyl alanine Nmanap
The D-N-methylvaline Dnmval N-methyl penicillanate amine Nmpen
γ-An Jidingsuan Gabu N-(right-hydroxy phenyl) glycine Nhtyr
L-tert-butyl group glycine Tbug N-(sulfidomethyl) glycine Ncys
The L-ethyl glycine Etg Penicillamine Pen
The high phenylalanine of L- Hphe L-Alpha-Methyl alanine Mala
L-Alpha-Methyl arginine Marg L-Alpha-Methyl agedoite Masn
L-Alpha-Methyl aspartate Masp L-Alpha-Methyl tert-butyl group glycine Mtbug
L-Alpha-Methyl cysteine Mcys L-Methylethyl glycine Metg
L-Alpha-Methyl glutamate Mgln L-Alpha-Methyl glutamate Mglu
L-Alpha-Methyl histidine Mhis The high phenylalanine of L-Alpha-Methyl Mhphe
L-Alpha-Methyl isoleucine Mile N-(2-methyl sulfur ethyl) glycine Nmet
D-N-methyl glutamine Dnmgln N-(3-guanidine radicals propyl group) glycine Narg
D-N-methyl glutamate Dnmglu N-(1-hydroxyethyl) glycine Nthr
The D-N-methylhistidin Dnmhis N-(hydroxyethyl) glycine Nser
D-N-methyl isoleucine Dnmile N-(imidazole radicals ethyl) glycine Nhis
The D-N-methylleucine Dnmleu N-(3-indyl ethyl) glycine Nhtrp
The D-N-methyllysine Dnmlys N-methyl-γ-An Jidingsuan ester Nmgabu
N-methylcyclohexyl alanine Nmchexa The D-N-methylmethionine Dnmmet
D-N-methyl ornithine Dnmorn N-methylcyclopentyl alanine Nmcpen
Sarcosine Nala D-N-methylbenzene alanine Dnmphe
N-methylamino isobutyrate Nmaib The D-N-methylproline Dnmpro
N-(1-methyl-propyl) glycine Nile D-N-methyl serine Dnmser
N-(2-methyl-propyl) glycine Nleu The D-N-methylthreonine Dnmthr
The D-N-methyl tryptophan Dnmtrp N-(1-Methylethyl) glycine Nval
The D-N-methyl-tyrosine Dnmtyr N-methyl Alpha-Naphthyl alanine Nmanap
The D-N-methylvaline Dnmval N-methyl penicillanate amine Nmpen
γ-An Jidingsuan Gabu N-(right-hydroxy phenyl) glycine Nhtyr
L-tert-butyl group glycine Tbug N-(sulfidomethyl) glycine Ncys
The L-ethyl glycine Etg Penicillamine Pen
The high phenylalanine of L- Hphe L-Alpha-Methyl alanine Mala
L-Alpha-Methyl arginine Marg L-Alpha-Methyl agedoite Masn
L-Alpha-Methyl aspartate Masp L-Alpha-Methyl tert-butyl group glycine Mtbug
L-Alpha-Methyl cysteine Mcys L-Methylethyl glycine Metg
L-Alpha-Methyl glutamine Mgln L-Alpha-Methyl glutamate Mglu
L-Alpha-Methyl histidine Mhis L-Alpha-Methyl homophenylalanin Mhphe
L-Alpha-Methyl isoleucine Mile N-(2-methyl sulfur ethyl) glycine Nmet
L-Alpha-Methyl leucine Mleu L-Alpha-Methyl lysine Mlys
L-Alpha-Methyl methionine Mmet L-Alpha-Methyl nor-leucine Mnle
L-Alpha-Methyl norvaline Mnva L-Alpha-Methyl ornithine Morn
L-Alpha-Methyl phenylalanine Mphe L-Alpha-Methyl proline Mpro
L-Alpha-Methyl serine mser L-Alpha-Methyl threonine Mthr
L-Alpha-Methyl valine Mtrp The L-alpha-methyltyrosine Mtyr
L-Alpha-Methyl leucine Mval?Nnbhm L-N-methyl homophenylalanin Nmhphe
N-(N-(2, the 2-diphenyl-ethyl) N-(N-(3, the 3-diphenyl propyl)
Carbamoyl methyl-glycine Nnbhm Carbamoyl methyl (1) glycine Nnbhe
1-carboxyl-1-(2,2-diphenyl-ethyl amino) cyclopropane Nmbc
Because peptide of the present invention is preferred for clinical, it requires described peptide is soluble form, therefore peptide of the present invention preferably includes one or more non-naturals or natural polar amino acid, includes, but are not limited to can improve because it contains the side chain of hydroxyl the serine and the threonine of peptide dissolubility.
Peptide of the present invention can synthesize by any technology known to the skilled in the synthetic field of peptide.Synthetic for solid-phase peptide, the summary of many technology can be at Stewart, J.M. and Young, J.D. (1963), " Solid Phase Peptide Synthesis, " W.H.Freeman Co. (SanFrancisco); And Meienhofer, J (1973). " Hormonal Proteins and Peptides, " volume 2, p.46, find in the Academic publishing house (New York).For the synthetic summary of classical solution, can be referring to Schroder, G. and Lupke, K. (1965) .The Peptides, volume 1, Academic publishing house (New York).
Summarize it, the peptide synthetic method comprises with one or more aminoacid or through the aminoacid of appropriate protection and being added to successively on the peptide chain in the growth.Usually, first amino acid whose amino or carboxyl are by appropriate blocking group protection.By under being suitable for forming the condition of amido link, adding the next aminoacid in the sequence; this amino acid whose relative group (complimentary group) (amino or carboxyl) is subjected to appropriate protection, and described protected or modified aminoacid can or link to each other with the inert solid holder or uses in solution.Slough blocking group from initiate amino acid residue then, and add next aminoacid (appropriate protection), or the like; Traditionally, this process also is accompanied by washing step.Amino acid needed at all according to after correctly being linked in sequence, take off remaining any blocking group (and any solid support) successively or simultaneously, to obtain final peptide compounds.By this universal process is carried out simple modification; can once add aminoacid to the chain in the growth more than one; for example, after deprotection, form pentapeptide by with shielded tripeptides and the dipeptides coupling (not making under the racemic condition of chiral centre) that is subjected to appropriate protection, or the like.
Peptide is synthetic further at United States Patent (USP) the 6th, 472, and is open in No. 505.The method for optimizing for preparing peptide compounds of the present invention comprises that the solid-phase peptide that adopts solid support is synthetic.Extensive peptide is synthetic by Andersson Biopolymers 2000,55 (3), and 227-50 is described.
Adopt above-mentioned instruction to identify and finally to be used to detect any virus, and thereby be used in substrate among many researchs and the clinical practice.
The example of the best substrate that instruction according to the present invention identifies is provided among following examples embodiment 2 partly (for example SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47).
The peptide that instruction according to the present invention identifies preferably is no more than 20 through being designed to length, is preferably 19, is preferably 18, is preferably 17, is preferably 16, is preferably 15, is preferably 14, is preferably 13, is preferably 12, is preferably 11, is preferably 10, is preferably 9, is preferably 8, is preferably 7, is preferably 6, is preferably 5, is preferably 4, is preferably 3 aminoacid.
The present invention is intended to comprise open and claimed any peptide in the U.S. Patent application 20050048473, and as any known peptide according to principle disclosed herein, it is got rid of from the present invention especially.
Peptide of the present invention can be included in the compositions that wherein further contains the unit that is useful on cutting and detects (these unit are described further below, but for example test section (being also referred to as signal section and quencher part at this)) and separating part.
Therefore, according to a further aspect in the invention, the invention provides the method for at least a virus in the test sample.
According to this aspect of the invention, the virus that can detect be those contain cleavage activity can be as the virus of protease that detect the basis (can't help host cell expressed).According to this aspect of the invention, the non-limiting tabulation of the protease that its activity can be detected including, but not limited to, serine protease, metalloproteases, aspartic protease, cysteine proteinase, 3C protease, PA transcriptase, adenine protease, 2A protease, chymase or trypsin.For example NS3, NS2, NS-pro cysteine proteinase, nsP2 cysteine proteinase, nsP23pro, C protein enzyme, SFV NS, HIV aspartic protease, nsp4 arteritis virus protease, HCMV protease, NS2-3, NS3-4Ap protease, HTLV-1PR.
According to this aspect of the invention, the non-limiting tabulation of the virus that can detect provides in the embodiment 2 of following examples part.
Term used herein " detection " refers to differentiate the existence of described virus, with described virus taxis and the diagnosis medical condition relevant with described virus.
" diagnosis " used herein refers to this severity of disease be determined in the medical condition classification, the monitoring course of disease, prediction disease consequence and/or rehabilitation prospect.
Therefore, for detecting adenovirus, described substrate can contain SEQ ID NO:1 or 2.
For detecting α virus, described substrate contains SEQ ID NO:3.
For detecting rubella virus, described substrate contains SEQ ID NO:4.
For detecting HIV, described substrate contains SEQ ID NO:5.
For detecting HTLV, described substrate contains SEQ ID NO:6,7 or 8.
For detecting arteritis virus, described substrate contains SEQ ID NO:9.
For detecting coronavirus, described substrate contains SEQ ID NO:10.
For detecting sars coronavirus, described substrate contains SEQ ID NO:11 or 12.
For detecting Orbivirus, described substrate contains SEQ ID NO:13.
For detecting CMV virus, described substrate contains SEQ ID NO:14 or 15.
For detecting herpesvirus, described substrate contains SEQ ID NO:16.
For detecting banzi virus, described substrate contains SEQ ID NO:17.
For detecting dengue virus, described substrate contains SEQ ID NO:18,19 or 20.
For detecting west nile virus, described substrate contains SEQ ID NO:21,22 or 23.
For detecting yellow fever virus, described substrate contains SEQ ID NO:24,25 or 26.
For detecting Japanese encephalitis virus, described substrate contains SEQ ID NO:27,28 or 29.
Pass virus for detecting Ticks, described substrate contains SEQ ID NO:30,31 or 32.
For detecting hepatitis C virus, described substrate contains SEQ ID NO:33,34 or 35.
For detecting Pestivirus, described substrate contains SEQ ID NO:36.
For detecting hepatitis A virus, described substrate contains SEQ ID NO:37 or 38.
For detecting HRV, described substrate contains SEQ ID NO:39 or 40.
For detecting enterovirus, described substrate contains SEQ ID NO:41,42,43,44,45,46 or 47.
Be to be understood that if different virus has similar aminoacid sequence, single protease can cut these viral polyproteins.Therefore, the present invention utilizes this specificity to provide to single Virus Type or to multiple Virus Type specific detection method.It should also be understood that method of the present invention also can be designed for the discriminating of non-family correlated virus, such as the virus that causes same symptoms (multiple virus detects).Below the embodiment that further is described is arranged in embodiment 3 and 6.
The term of Shi Yonging " sample " refers to contain or can be by any biological sample of viral infection (permissive) (for example tissue culture's sample or body fluids/tissue sample) herein.Be preferably various exocrine secretions (for example nose cleanout fluid sample), intestinal, genitourinary tract, tear, saliva, seminal fluid, perspiration, feces and milk that described biological sample refers to body fluid such as whole blood, serum, blood plasma, cerebrospinal fluid, urine, lymph fluid, respiratory tract, and leukocyte, malignant tissue, amniotic fluid and chorionic villi.
The method of this aspect of the present invention is by contacting described sample under the condition that allows the described substrate of cutting with any substrate described herein; And monitor the cutting of described substrate and realize that the cutting of wherein said substrate is the indication that has described at least a virus in the sample.
The mensuration that this area is any to become known for monitoring the cutting of Proteolytic enzyme substrate can be used this aspect according to the present invention.
Be preferably described substrate cutting sequence and be included in the compositions, described compositions further is provided for the unit that detects, but at least one test section for example.Example is described in detail in the U.S. Patent application 20050048473 of Dorit Arad, and it is introduced by reference at this.
As used herein, but the test section refer to can by directly (for example fluorescence, radiosiotope) or indirectly (for example proenzyme) detect any molecule of (visual, counting etc.).
Therefore, the cutting of monitoring Proteolytic enzyme can be by being used to detect the homogeneous determination realization of virus.Selected substrate warp synthesizes and links to each other with the end of signal section at described cutting zone, and links to each other at the other end of described cutting zone with the quencher part.Be to be understood that and (depend on and detect structure) that all described parts also can be positioned at an end of described cutting sequence as required.
The phrase of Shi Yonging " homogeneous determination " do not need to refer to signal section and other are analyzed the isolating mensuration of composition herein.
Be purpose of the present invention, " quencher part " is to reduce or to eliminate any material of the signal that described signal section sends.For example, described quencher part can be by absorbing the signal that sent by described signal section or working by energy delivery mechanism.Distance between described signal section and described quencher part can reduce the existence of described quencher part basically or eliminates the signal that is sent by described signal section, unless described substrate is cut in certain position and causes described signal section partly to separate with quencher.
In one embodiment, described signal section and quencher part is separated by being no more than 3 or 5 aminoacid.In another embodiment, described signal section and quencher part separates by being no more than 10 amino acid residues.In another embodiment, described signal section and quencher part separate by being no more than 15 amino acid residues, and in going back an embodiment, described signal section and quencher part separate by being no more than 20 amino acid residues.Instruct according to these, can coupling can be as other parts (for example, also in out-phase mensuration) of detecting unit, as described further below all.Equally, be to be understood that any detecting unit (for example part etc.) can be directly or indirectly with substrate or order coupling or by amino acid modified any one aminoacid that is coupled to described peptide cutting sequence self.
Described compositions contacts with the sample that is used for the Test Virus existence.If there is virus in sample, described virus protease also exists.This protease cutting substrate, and can be observed the signal variation that comes from signal section.So homology fluorescence (homogenous fluorescent) and colorimetric determination are known for those skilled in the art.Referring to for example: Biochemistry, Allinger, Wang Q.M. etc., " A continuous calorimetric assay for rhinovirus-14 3C protease usingpeptide p-nitroanilides as substrates " Anal.Biochem. volume 252, page or leaf 238-45 (1997), with " In vitro elucidation of substrate specificity andbioassay of proprotein convertase 4 using intramolecularly quenchedfluorogenic peptides " Biochem.J. such as Basak S. volume 380, page or leaf 505-14 (2004).
In another embodiment of the present invention, described signal section is the chemiluminescence signal part.Described chemiluminescence signal part links to each other with a side of the cutting zone of described substrate, and fluorescence is accepted quencher and partly linked to each other with the opposite side of described cutting zone.United States Patent (USP) the 6th, 243, No. 980, its content is introduced by reference at this, has described such detection system, relates to using chemiluminescently 1, and 2-dioxetanes hydride compounds is as described signal section.If there is not described virus protease in sample, will the cutting of described substrate can not take place.Come from 1, the energy delivery that the 2-dioxetane decomposes to described fluorescence is accepted part, and with described 1, the different wavelength in emission spectra zone of 2-dioxetane discharges down.If described substrate is through cutting, described fluorescence is accepted part with described 1, and the 2-dioxetane separates, thereby observes the chemiluminescence that comes from described dioxetanes hydride compounds.
In another embodiment, described signal section is that fluorescent chemicals and described quencher partly are the fluorescent chemicalses with excitation spectrum that the emission spectra with described signal section overlaps.Herein, described two parts are separated with the certain distance that conforms to the fluorescence resonance energy transmission, thereby make described fluorescence part can be used as the resonance energy donor.
In another embodiment, quencher group such as non-fluorescent absorption dyestuff is accepted the quencher part in order to replace fluorescence.Suitable quencher group is at United States Patent (USP) the 6th, 243, described in No. 980, and its content is introduced by reference.
Adopt so detection method, can allow to take place described substrate by under the condition of described protease when having virus in sample (if) cutting, described specimen contacts with described substrate.In one embodiment, temperature is controlled.For example, described temperature can be controlled at 37 ℃ so that the optimum condition of enzyme reaction to be provided.Utilize the checkout equipment that adapts with the labelling that is adopted to detect the segmental signal of substrate of the cutting of hanging oneself then.
Another aspect of the present invention provides the out-phase mensuration that is used to detect virus." out-phase mensuration " is that wherein said solid phase is parsed into the isolating mensuration of phase-splitting with other in the mensuration process.Detailed description that out-phase is measured and example provide in the embodiment 6 of following examples part.
In the case, described substrate is included in the compositions with following general formula.
X-Y-Z
Wherein:
Y contains the substrate of virus protease, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
But X contains the test section; With
Z contain can with the separating part that combines mutually separating of two phase-separated system;
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease.
But described test section X can directly or indirectly be detected, and can contain marking agent such as enzyme, fluorogen, chromophore, albumen (such as the epi-position label), chemiluminescent substance and radiosiotope.
Separating part Z can be incorporated into the phase of separating of two phase-separated system (for example solid phase and liquid phase) directly or indirectly.The example of described separating part Z comprises that immunoconjugator, magnetic knot close part, peptide bound fraction, affinity bound fraction, nucleic acid moiety.More examples provide in following examples 6.
Compositions of the present invention can with described sample incubation before, simultaneously or carry out incubation with described separation system afterwards.
Utilize out-phase of the present invention to measure and monitor any embodiment realization that cutting can be passed through embodiment 6, in Fig. 7～10, schematically describe.
Make described test section not link to each other but should measure with described separating part.
In one embodiment, but test section of the present invention is a proenzyme.Therefore described enzyme can be activated and detect (by the catalytic activity test of this enzyme) when substrate cuts.So the example of proenzyme is other enzymes in thrombinogen (factor II) or this cascade.
In described herein any embodiment, any part can directly link to each other with described peptide or link to each other indirectly by the spacer molecule that all has coupling functional group at every end by covalent bond.So the example of connexon comprises alkyl, ethylene glycol, ether, polyethers, polynucleotide and peptide molecule.
The solid phase that is applicable to described out-phase mensuration is including, but not limited to test tube, titer plate, microtitre hole, pearl, test strips (dipstick), polymer particles, magnetic particle, NC Nitroncellulose, chip array and other solid phases well known to those skilled in the art.The signal section that uses in described out-phase is measured can be any labelling well known by persons skilled in the art.Described labelling comprises radioactivity, calorimetric, fluorescence and luminescent marking.
The heterogeneous chemical luminescent detecting that is used to detect protease is at United States Patent (USP) the 56th, 243, describes in No. 980, and its content is introduced by reference at this.In one embodiment, homogeneity of the present invention or out-phase assay method automatization carry out, thereby can obtain the result and need not the healthcare givers and contact the experimenter who has infected described viral disease that it is believed that who accepts test.For example, described experimenter tests (such as, but be not limited to P3 type chamber) in the toilet.Described experimenter can obtain or obtain diagnostic kit before entering the room, described test kit can comprise the solid phase of the labelling peptide that is coated with described type discussed above.For example, described solid phase is used the submerged tissue of peptide before can being, perhaps is derived from the test bar that is used to test conceived that type.Described experimenter can sampling (such as saliva sample) on the preparation point of described solid phase.
The solid phase that will contain described sample is then carried out incubation to carry out described enzyme reaction.In one embodiment, described reaction temperature is controlled at 37 ℃ so that the optimum condition of described enzyme reaction to be provided.When described incubation is finished, can use remote control or can outside described room, manually operated mechanical system spectrophotometer, measure described sample to be tested.The quantized color or the UV that can measure described sample detect.After test, described sample can be abandoned by automatic system that can abandon described sample or operated from a distance handle.
Be exist (such as in order to detect the virus relevant, referring to embodiment 3) of the multiple virus of one-time detection, can use any method known in the art or the above-mentioned method that detects multiple virus that is applicable to specific symptoms or specific host.
The non-limit example of these means below is provided.
Microplate-in the X orifice plate.The different particular peptide sequences corresponding to different virus is contained in each hole.Along with adding described clinical sample, with standard microplates reader monitoring reaction and note which hole and show enzymatic activity under suitable wavelength.Because a kind of particular peptide is contained in each hole, therefore the data that provide according to microplate can illustrate there is which kind of viral enzyme in described clinical sample.The existence of described enzyme has confirmed the existence of described virus.
Medisel chip technology (2002 Anticancer Res.22:2663-9 such as Schiffenbauer)-employing Medisel technology can be fixed described particular peptide (corresponding to interested virus) on chip.Along with adding described clinical sample, use the laser beam monitoring reaction.Because each point contains a kind of particular peptide on the described chip, the data that provide according to Medisel equipment can illustrate there is which kind of viral enzyme in described clinical sample.The existence of described enzyme has confirmed the existence of described virus.
Separation-particular peptide on the post (corresponding to interested virus) can utilize the unique dna introns to be connected on the pearl of commercial source.React by adding described clinical sample.In case the cutting (by the specific virus enzyme in described clinical sample) of particular peptide takes place, quencher is released and described pearl sends fluorescence.Then by on post, separating described pearl, and under suitable wavelengths, measure the fluorescence (corresponding to every kind of different virus) of every type pearl with the standard fluorescence meter with the hybridization of described unique dna introns.Only there is the pearl that is cut by described viral enzyme to send fluorescence.Therefore can illustrate and in described clinical sample, have which kind of viral enzyme.The existence of described enzyme has confirmed the existence of described virus.
Pearl FACS separation-only separating analogous with post, when every kind of specific peptide linked to each other with the pearl with different colours, described separating step was finished by FACS.In the method, described introns can be DNA or peptide or intend peptide or carbohydrate or any organic principle introns [Gonzalez (2005) Clin.Biochem.38:966-72].
Can be used for additive method of the present invention at Tozzoli etc., (2006) Clin.Chem.Lab Med.44:837-42; Abreu (2005) Ann.N.Y.Acad.Sci.1050:357-63; Buliard (2005) Ann.Biol.Clin. (Paris) 63:51-8; Yinnaki (2004) J.ImmunoassayImmunochem.25:345-57; Rouquette (2003) 120:676-81; Toellner (2006) Clinical Chemistry 52:1575-1583; Among Horejsh (2005) the Nucl.Acids Res.33 description is arranged, wherein the content of each piece is by reference this whole introducing.
The peptide cutting sequence of differentiating according to instruction of the present invention can be used for detecting the new virus strain.Such as the epiphytotics detection of for example SARS, it shows the homology with protovirus family (such as coronavirus).This depends on the fast adaptability of described detection method to the new virus that causes pandemic disease.
Therefore, in case the genome of emergent virus differentiated and know its dubbing system, the sequence homology between the sequence that can be by checking described emergent virus and the sequence of known viruse is determined virus protease and by the zone of the viral polyprotein that described protease cut.According to this comparison, can realize selection to the optimum Cutting sequence.
The test kit that contains peptide of the present invention also is provided.Different test kit compositions can be packaged in the container separately and before use and mix immediately.So independently component packagedly make it long preservation and do not lose the function of described active component.Also imagined the embodiment that two or more composition wherein is housed in same container.An exemplary kit can comprise one or more following reagent: be used for the washing buffer reagent that out-phase is measured; The negative control reagent that does not contain the protease that can cut substrate; Contain the positive control that can cut the protease of described substrate; (d) signal that is used to manifest the detectable signal that comes from described signal section produces reagent; (d) sample collection unit is such as syringe, throat swab or other sample collection apparatus.
For multiple virus detection kit, wherein detect one or more viruses as mentioned above, each many plates detection kit will be carried out decision design according to common theme, such as the different virus that causes same or similar disease; Infect the virus of homologue or organ; Has virus that close kind system is related such as the virus that classifies as same family, subfamily etc.; Can be in identical body fluid such as saliva, nasal discharge, blood, urine, feces etc. detected virus; The virus of common and wide-scale distribution; Virus by identical body fluid communication or the like.
The reagent that comprises in described test kit can provide in the container of any kind, makes that kept and the not adsorbed or change by the material of described container the service life of heterogeneity.For example, the sealed glass ampoule bottle can contain freeze-dried reagent or the buffer agent of packing under neutrality, non-reactive gas such as nitrogen.Ampoule bottle can be made of such as glass, organic polymer such as Merlon, polystyrene etc. any suitable material; Pottery, metal or any other are generally used for holding the material of similar reagent.Other examples of suitable vessel comprise and can and can contain the envelope of paper tinsel shape lining such as aluminum or alloy by the simple bottle of the material manufacture similar to ampoule bottle.Other containers comprise test tube, bottle, flask, bottle or syringe etc.Container can have aseptic contact opening, such as the bottle with the stopper that can be pierced through by hypodermic needle.Other containers can have separated two chambers of film that removed easily, described film are removed described composition is mixed.Removable film can be glass, plastics, rubber etc.
Test kit also can be furnished with illustrative material.Description can be printed on paper or other base materials, and/or provides in electronically readable medium mode, such as floppy disk, CD-ROM, DVD-ROM, Zip dish, video-tape, audiotape etc.Detailed description can not link to each other with described test kit physics; The substitute is, the user can visit the specified internet site by the manufacturer of described test kit or retail trader, perhaps provides with E-mail mode.
The present invention further imagination peptide of the present invention is used to design and disturbs described virus protein hydrolysing activity and thereby the viral interference therapeutic agent that duplicates and infect.Be preferably, described peptide sequence is through modifying thereby can combining and suppress its activity (for example irreversible inhibitor) with described protease.As long as the identification that still can keep protease by introducing at least one non-peptide bond (as mentioned above), is so intended peptide and be can be used for utilizing the sequence replacing that can not cut can cut sequence.
Therapeutic peptide of the present invention can join through discriminating and can be used for treating in the pharmaceutical composition of interested virus disease.
The present invention further imagination utilizes compositions screening antiviral agent of the present invention.
Therefore, the invention provides the compositions and the test kit that can be used in the peptide in therapeutic and the diagnostic application and contain described peptide.
After research following examples, other purposes of the present invention, advantage and novel feature are conspicuous for those of ordinary skills, and described embodiment is intended to limit.In addition, described above and the claimed of the present invention various embodiments of claim part and aspect in following examples, find experiment support.
Embodiment
Begin now with reference to following examples, described embodiment and above description have been described the present invention with non-limiting way jointly.
Usually, the experimental arrangement of term that herein uses and employing in the present invention comprises molecule, biochemistry, microorganism and recombinant DNA technology.These technology are described in detail in the literature.Referring to for example " Molecular Cloning:A laboratory Manual " Sambrook etc., (1989); " Current Protocols in Molecular Biology ", volume I-III, Ausubel, R.M. edits. (1994); Ausubel etc., " Current Protocols in Molecular Biology ", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, " A Practical Guide toMolecular Cloning ", John Wiley ﹠amp; Sons, New York (1988); Watson etc., " Recombinant DNA ", Scientific American Books, New York; Birren etc. (editor) " Genome Analysis:A Laboratory Manual Series ", volume 1-4, Cold SpringHarbor Laboratory publishing house, New York (1998); In United States Patent (USP) 4,666,828; 4,683,202; 4,801,531; The method of describing in 5,192,659 and 5,272,057; " Cell Biology:A Laboratory Handbook ", volume I-III, Cellis, J.E., editor (1994); " CurrentProtocols in Immunology ", volume I-III, Coligan J.E., editor (1994); Stites etc. (editor), " Basic and Clinical Immunology " (the 8th edition), Appleton ﹠amp; Lange, Norwalk, CT (1994); Mishell and Shiigi (editor), " Selected Methods in CellularImmunology ", W.H.Freeman and Co., New York (1980); Operational immunoassay are described in detail in patent and scientific literature, referring to for example United States Patent (USP) 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; " Oligonucleotide Synthesis " Gait, M.J., editor (1984); " Nucleic Acid Hybridization " Hames, B.D., and HigginsS.J., editor (1985); " Transcription and Translation " Hames, B.D. and Higgins S.J., editor (1984); " Animal Cell Culture ＂ Freshney, R.I., editor (1986); " Immobilized Cells and Enzymes " IRL publishing house, (1986); " APractical Guide to Molecular Cloning " Perbal, B., (1984) and " Methods inEnzymology " volume 1-317, Academic publishing house; " PCR Protocols:A Guide ToMethods And Applications ", Academic publishing house, San Diego, CA (1990); Marshak etc., " Strategies for Protein Purification and Characterization-ALaboratory Course Manual " CSHL publishing house (1996); All these are introduced by reference in full at this.Other common lists of references provide in this description.Program wherein it is believed that to knowing in this area, and provides for reader's convenience.All information that wherein contain are introduced herein by reference.
Embodiment 1
The total cutting of hepatitis C NS3 protease sequence is differentiated
The discriminating of the cutting sequence through optimizing of the NS3 protease of hepatitis C has below been described.
Stage 1: obtain the mechanism that described polyprotein is cut by its virus protease, main points are orders (1,4 of cutting incident in the viral life cycle, referring to the Figure 11 that takes from hepatitis C virus (HCV): Models structure and genome organization (model structure and genomic organization structure), Vol 5; On November 19th, 2003, Cambridge University publishing house).
Stage 2: utilize data base such as Swiss prot., Pubmed, Gene bank and OWL that all cutting sequences of the Strain of identical virus as much as possible are compared, and utilize FASTA software to its compare (referring to following table 1).
Table 1
Figure A200680038972D00411
Figure A200680038972D00421
E1<＝>NS1 Cell HCV 633202 FSGVDA|ETYIT 99
E1<＝>NS1 Cell HCV 221615 ISQAEA|ALENL 100
E2<＝>NS1 Cell HCV 22129793 IAQAEA|ALENL 101
E2<＝>NS1 Cell HCV 2764398 ISNVEA|AVERL 102
E2<＝>NS1 Cell HCV(6a33) 57791994 LGQAEA|ALEKL 103
E2<＝>NS1 Cell HCV(JPUT971017) 9757542 IAQAEA|TLENL 104
E2<＝>NS1 Cell HCV?2 496367 PQRAYA|LDTEM 105
E2<＝>NS2 Cell HCV 9843677 DARVCA|CLWMM 106
NS1<＝>NS2 Cell HCV 1381032 PQRAYA|LDTEV 107
NS1<＝>NS2 Cell HCV 22129793 PPRAYA|LDREM 108
NS1<＝>NS2 Cell HCV 2764398 PHRAYA|MDNEQ 109
NS1<＝>NS2 Cell HCV(6a33) 57791994 PQRAYA|LDQEL 110
NS1<＝>NS2 Cell HCV(HC-G9) 464178 PQQAYA|LDAAE 111
NS1<＝>NS2 Cell HCV(JPUT971017) 9757542 LLLADA|RVCVA 112
NS1<＝>NS2 Cell HCV(NZL1) 1183029 PPRAYA|MDREM 113
NS1<＝>NS2 Cell HCV?1b 5420377 PPQAYA|MDREM 114
NS3<＝>NS4A NS3-4Ap HCV 221615 DLEIVT|STWVL 115
NS3<＝>NS4A NS3-4Ap HCV 2764398 DLEVIT|STWVL 116
NS3<＝>NS4A NS3-4Ap HCV 9843677 DLEVMT|STWVL 117
NS3<＝>NS4A NS3-4Ap HCV(6a33) 57791994 DLEVTT|STWVL 118
NS3<＝>NS4A NS3-4Ap HCV(JPUT971017) 9757542 DLEVTT|SAWVL 119
NS3<＝>NS4A NS3-4Ap HCV(NZL1) 1183029 DLEIMT|STWVL 120
NS3<＝>NS4A NS3-4Ap HCV(Tr?Kj) 1435035 DEMEEC|SQHLP 121
NS3<＝>NS4A NS3-4Ap HCV(VAT96) 6521009 AGVAGA|LVAFK 122
NS4A<＝>NS4B NS3-4Ap HCV 22129793 DEMEEC|ASHLP 123
NS4A<＝>NS4B NS3-4Ap HCV 221615 DEMEEC|SRHIP 124
NS4A<＝>NS4B NS3-4Ap HCV 2764398 DEMEEC|ASKAA 125
NS4A<＝>NS4B NS3-4Ap HCV(6a33) 5779199 DEMEEC|SQAAP 126
NS4A<＝>NS4B NS3-4Ap HCV 9757542 DEMEEC|ASRAL 127
NS4A<＝>NS4B NS3-4AP HCV(NZL1) 1183029 ECTTPC|SGSWL 128
NS4B<＝>NS5A NS3-4Ap HCV 221607 DCSTPC|SGSWL 129
NS4B<＝>NS5A NS3-4AP HCV 2764398 ESTTPC|SGSWL 130
NS4B<＝>NS5A NS3-4AP HCV 59479 DTATPC|ATSWL 131
NS4B<＝>NS5A NS3-4AP HCV 9843677 DCTAPC|AGSWL 132
NS4B<＝>NS5A NS3-4AP HCV(6a33) 57791994 DCPVPC|SGSWL 133
NS4B<＝>NS5A NS3-4Ap HCV(HC-G9) 464178 DYPSPC|SDDWL 134
NS4B<＝>NS5A NS3-4Ap HCV (JPUT971017) 9757542 DYPSPC|NGDWL 135
NS4B<＝>NS5A NS3-4Ap HCV(Tr?Kj) 1435035 EDVVCC|SMSYT 136
NS4B<＝>NS5A NS3-4Ap HCV(VAT96) 6521009 DDVVCC|SMSYS 137
NS5A<＝>NS5B NS3-4Ap HCV 2764398 QSVVCC|SMSYS 138
NS5A<＝>NS5B NS3-4Ap HCV(6a33) 57791994 GSEVCC|SMSYS 139
NS5A<＝>NS5B NS3-4Ap HCV(NZL1) 1183029 DDIVCC|SMSYT 140
NS5A<＝>NS5B NS3-4Ap HCV(VAT96) 6521009 EDVVCC|SMSYS 141
NS5A<＝>NS5B NS3-4Ap HCV?2 496367 DSVICC|SMSYS 142
Step 3: according to replacing or the site of Dynamics Optimization obtains to cut the most fast sequence by carrying out data mining according to list of references listed in the following table 2.Described site is NS5A/B (3,4,5) under the situation of HCV NS3 protease.
Table 2
1.Grakoui, A., D.W.McCourt, C.Wychowski, S.M.Feinstone and C.M.Rice.1993.A second hepatitis C virus-encoded proteinase.Proc.Natl.Acad.Sci.USA 90:10583-10587.
2.Reed?KE，Grakoui?A，Rice?CM.Hepatitis?C?virus-encoded?NS2-3protease：cleavage-site?mutagenesis?and?requirements?for?bimolecularcleavage.
3.J?Virol.1995?Jul；69(7)：4127-36.
4.Bartenschlager, R., L Ahlborn-Laake, J.Mous and H.Jacobsen.1994.Kinetic and structural analysis of hepatitis C virus polyproteinprocessing.J.Virol.68:5045-5055.
5.Failla, C., L.Tomei and R.De Francesco.1994.Both NS3 and NS4Aare required for proteolytic processing of hepatitis C virus nonstructural protein.J.Virol.68:3753-3760.
6.Grakoui, A., C.Wychowski, C.Lin, S.M.Feinstone and C.M.Rice.1993.Expression and identification of hepatitis C virus polyprotein products.J.Virol.67:1385-1395.
7.Pacini?L，Vitelli?A，Filocamo?G，Bartholomew?L，Brunetti?M，Tramontano?A，Steinkuhler?C，Migliaccio?G.In?vivo?selection?of?proteasecleavage?sites?by?using?chimeric?Sindbis?virus?libraries.J?Virol.2000?Nov；74(22)：10563-70.
8.AA Kolykhalov, EV Agapov and CM Rice Specificity of the hepatitisC virus NS3 serine protease:effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.J.Virol.1994 Nov 68 (11), 7525-7533.
9.Urbani?A，Bianchi?E，Narjes?F，Tramontano?A，De?Francesco?R，Steinkuhler?C，Pessi?A.Substrate?specificity?of?the?hepatitis?C?virus?serineprotease?NS3.J?Biol?Chem.1997?Apr?4；272(14)：9204-9.
Table 3-is based on the active and selected cleavage site of the peptide substrate of NS4A/B, NS5A/B sequence a
Figure A200680038972D00441
aWhen existing (+) or do not have (-), 150mM NaCl measures kinetic parameter
, nor-leucine (noreleucine)
4-is trans for table
Figure A200680038972D00451
Table 5-cis
Figure A200680038972D00452
Figure A200680038972D00461
Table 6
The cutting power mathematic(al) parameter of the decapeptide that P6 and P1 ' modify-corresponding to described NS4A/NS4B cleavage site
The residue of runic printing has been indicated the change with respect to wild-type sequence.Data are the meansigma methods ± S.D. of at least three different measurings.
Figure A200680038972D00462
Table 7
The residue of runic printing has been indicated the change with respect to wild-type sequence.Data are the meansigma methods ± S.D. of at least three different measurings.
Figure A200680038972D00463
The protease of cis-effect only cuts adjacent cleavage site.The protease of trans-effect works to cleavage site far away.
Cis-
1.P1-have threonine.
2.P4-accept the residue that great majority have nonpolar fatty family side chain.
3.P2-demonstrate preference to charged residue.
4. the specificity that described protease cis-cleavage site NS4A/C connects is greatly degenerated, and to indicate described cutting be to determine by polyprotein is folding (6).
Trans
1.P1-cysteine is extremely important and can not be replaced.
2.P3-only accept valine, glutamic acid, threonine and isoleucine.Substitute Glu with Asn or Lys and do not have effect.
3.P4-can tolerate most of residues.
4.P2-demonstrate to leucic preference.Substitute Glu with Asn or Lys and do not have effect.
5.P5-the charged residue of preference (negative charge residue → aspartate).
6.P6-acidic residues is important.(tyrosine)
7.P3-residue helps effective substrate identification (gln).
8.P4 '-residue helps effective substrate identification (hydrophobic residue → Leu).
There is not effect 9.P7-replace Phe with His or Arg.
There is not effect 10.P5-substitute Glu with Lys.
11.P2 '-replace Gln with Lys, do not have effect.
12.P1 '-replace Ser with Ile, Thr, Arg, Ala, Asp or His, make and can effectively cut.Yet, replace inhibition cutting (7) significantly with Pro.
13. effective external split requirement is from the peptide substrates of between P6 to P4 ' at least 10 residues.
Step 4: according to drawing the consensus sequence site, and sum up the consensus sequence scheme by step 4 according to the variant that step 3 allowed from two of step 3 site and replacements the most fast.For HCVNS3 protease, put all data in order and obtain result such as following sequence:
(T/S/ acidic amino acid) X (E/T/I/N/K/D/V) (C/F) C-/-(S/D) (Y/ hydrophobic amino acid) (SEQ ID NO:33) of X (M/norL)
Embodiment 2
The Dynamics Optimization substrate that instruction according to the present invention identifies
Following code name is used to represent the expression formula of consensus sequence.
Hydrophobic amino acid: G, A, V, L, I, M, W, F, Y, H
Basic amino acid: Q, N, K, H, R
HB donor: K, R, S, C, T, Q, N, Y
Acidic amino acid: E, D, Y
Aromatic amino acid: Y, F, H, W
-/-: cleavage site
Adenoviridae (1-9):
Ape: adenovirus 25, adenovirus 24, adenovirus 23, adenovirus 22, adenovirus 21, adenovirus 19.
Pig: adenovirus C, adenovirus B, adenovirus A, adenovirus 5, adenovirus 4, adenovirus 3, adenovirus 2, adenovirus 1.
Sheep: adenovirus B, adenovirus A, adenovirus 5, adenovirus 4, adenovirus 3, adenovirus 2, adenovirus 1.
Mus: adenovirus A, adenovirus 1.
People: adenovirus F, adenovirus E, adenovirus D, adenovirus C, adenovirus B, adenovirus A, adenovirus 9, adenovirus 8, adenovirus 7, adenovirus 6, adenovirus 51, adenovirus 50, adenovirus 5, adenovirus 49, adenovirus 48, adenovirus 47, adenovirus 46, adenovirus 45, adenovirus 44, adenovirus 43, adenovirus 42, adenovirus 41, adenovirus 40, adenovirus 4, adenovirus 39, adenovirus 38, adenovirus 37, adenovirus 36, adenovirus 35, adenovirus 34, adenovirus 33, adenovirus 32, adenovirus 31, adenovirus 30, adenovirus 3, adenovirus 29, adenovirus 28, adenovirus 27, adenovirus 26, adenovirus 25, adenovirus 24, adenovirus 23, adenovirus 22, adenovirus 21, adenovirus 20, adenovirus 2, adenovirus 19, adenovirus 18, adenovirus 17, adenovirus 16, adenovirus 15, adenovirus 14, adenovirus 13, adenovirus 12, adenovirus 11, adenovirus 10, adenovirus 1.
Horse: adenovirus B, adenovirus A, adenovirus 2, adenovirus 1.
Dog: adenovirus 2, adenovirus 1, adenovirus.
Cattle: adenovirus C, adenovirus B, adenovirus A, adenovirus 9, adenovirus 3, adenovirus 2, adenovirus 10, adenovirus 1.
Figure A200680038972D00491
Togaviridae (10-33)
α virus: Aura virus, crust horse forest virus (Barmah Forest virus), Eastern equine encephalitis virus, MID (Middelburg virus), ndumu virus (Ndumu virus), BEB (Bebaru virus), hole tribute subvirus (Chikungunya virus), GET (Getah virus), Ma Yaluo virus (Mayaro virus), A Niang-Ni Ang virus (O ' nyong-nyong virus), ross river virus (Ross River virus), Semliki Forest virus (Semliki forest virus), UNA (Una virus), Trocara virus, Cabassou virus, Mucambo virus, Pixuna virus, Venezuelan equine encephalitis virus, Fort Morgan virus (Fort Morgan virus), Highlands J virus, sindbis alphavirus (Sindbis virus), WEEVirus, Whataroa virus, α virus HBb 17, Norway Salmonoidei α virus (Norwegian salmonid alphavirus), Rio Negro virus, sea dog parasite virus (Seallouse virus).
Figure A200680038972D00492
Rubella virus: rubella virus, rubella virus (BRDI strain), rubella virus (BRDII strain), rubella virus (Cendehill strain), rubella virus (M33 strain), rubella virus (RN-UK86 strain), rubella virus (THERIEN strain), rubella virus (TO-336 vaccine strain), rubella virus (TO-336 strain), rubella virus (RA27/3 vaccine strain).
Figure A200680038972D00493
Retroviridae (34-60)
Positive and negative viral subfamily, slow virus, the primates slow virus group of transcribing:
Human immunodeficiency virus I: HIV-1 M:C_92BR025, HIV-1 M:C_ETH2220, HIV-1 M:F1_93BR020, HIV-1 M:F1_VI850, HIV-1 M:F2_MP255C, HIV-1M:F2_MP257C, HIV-1 M:G_92NG083, HIV-1 M:G_SE6165, HIV-1M:H_90CF056, HIV-1 M:H_VI991, HIV-1 M:J_SE9173, HIV-1M:J_SE9280, HIV-1 M:K_96CM-MP535, HIV-1 M:K_97ZR-EQTB11, HIV-1 N_YBF106, HIV-1 N_YBF30, HIV-1 O_ANT70, HIV-1O_MVP5180, HIV (human immunodeficiency virus) 3, human immunodeficiency virus type 1 (ARV2/SF2ISOLATE), human immunodeficiency virus type 1 (BH10 ISOLATE), human immunodeficiency virus type 1 (BH5 ISOLATE), human immunodeficiency virus type 1 (BH7 separated strain), human immunodeficiency virus type 1 (BH8 ISOLATE), human immunodeficiency virus type 1 (BRAINISOLATE), human immunodeficiency virus type 1 (BRU ISOLATE), human immunodeficiency virus type 1 (CDC-451 ISOLATE), human immunodeficiency virus type 1 (CLONE 12), human immunodeficiency virus type 1 (ELI ISOLATE), human immunodeficiency virus type 1 (HXB2ISOLATE), human immunodeficiency virus type 1 (HXB3 ISOLATE), human immunodeficiency virus type 1 (separated strain YU2), human immunodeficiency virus type 1 (JH3 ISOLATE), human immunodeficiency virus type 1 (JRCSF ISOLATE), human immunodeficiency virus type 1 (KB-1 separated strain), human immunodeficiency virus type 1 (Lai separated strain), human immunodeficiency virus type 1 (MALISOLATE), human immunodeficiency virus type 1 (MFA ISOLATE), human immunodeficiency virus type 1 (MN ISOLATE), human immunodeficiency virus type 1 (NDK ISOLATE), human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE), human immunodeficiency virus type 1 (NIT-A separated strain), human immunodeficiency virus type 1 (OYI ISOLATE), human immunodeficiency virus type 1 (PV22 ISOLATE), human immunodeficiency virus type 1 (RF/HAT ISOLATE), human immunodeficiency virus type 1 (SC ISOLATE), human immunodeficiency virus type 1 (SF162ISOLATE), human immunodeficiency virus type 1 (SF33 ISOLATE), human immunodeficiency virus type 1 (STRAIN UGANDAN/ISOLATE U455), human immunodeficiency virus type 1 (WMJ1 separated strain), human immunodeficiency virus type 1 (WMJ2 ISOLATE), human immunodeficiency virus type 1 (Z-84 ISOLATE), human immunodeficiency virus type 1 (Z2/CDC-Z34ISOLATE), human immunodeficiency virus type 1 (ZAIRE3 ISOLATE), human immunodeficiency virus type 1 (ZAIRE 6 ISOLATE), human immunodeficiency virus type 1 (ZAIRE HZ321ISOLATE), human immunodeficiency virus type 1 1w12.3 separated strain.
HIV (human immunodeficiency virus) 2 types: HIV (human immunodeficiency virus) 2 types (ISOLATE BEN), HIV (human immunodeficiency virus) 2 types (ISOLATE ROD), HIV (human immunodeficiency virus) 2 types (ISOLATE ST), HIV (human immunodeficiency virus) 2 types (separated strain ST/24.1C#2), HIV-2B_EHO, HIV-2 B_UC1, HIV-2.D205, HIV (human immunodeficiency virus) 2 types (ISOLATED205,7), HIV (human immunodeficiency virus) 2 types (separated strain 7312A), HIV (human immunodeficiency virus) 2 types (ISOLATE CAM2), HIV (human immunodeficiency virus) 2 types (ISOLATE D 194), HIV (human immunodeficiency virus) 2 types (ISOLATE GHANA-1), HIV (human immunodeficiency virus) 2 types (separated strain KR), HIV (human immunodeficiency virus) 2 types (ISOLATE NIH-Z), HIV (human immunodeficiency virus) 2 types (ISOLATE SBLISY).
Figure A200680038972D00511
Retroviridae; Positive and negatively transcribe viral subfamily; The δ retrovirus retrovirus; Primates is had a liking for the T-lymphocyte virus
Human T cell is had a liking for lymphocyte virus 1 type (Caribbean separated strain), human T cell and is had a liking for lymphocyte virus 1 type (separated strain MT-2), human T cell and have a liking for that lymphocyte virus 1 type (ATK strain), human T cell are had a liking for lymphocyte virus 1 type (African separated strain), human T cell is had a liking for lymphocyte virus 1 type (North America separated strain).
Figure A200680038972D00512
Cover the class of virus: Arteriviridae (78-82)
Arteritis virus: equine arteritis virus Bucyrus, lactic acid dehydrogenase increase virus, viral C, lactic acid dehydrogenase and increase viral Plagemann, Lelystad virus, PRRSV 16244B, PRRSVHB-1 (sh)/2002, PRRSV HB-2 (sh)/2002, PRRSV HN1, PRRSV VR2332, ape hemorrhagic fever virus.
Figure A200680038972D00513
The cover the class of virus: Coronaviridae (61-77)
Coronavirus: canine coronavirus, feline coronavirus, human crown 229E, Porcine epidemic diarrhea virus, Transmissible gastroenteritis virus, the non-categorical bovine coronavirus, human coronary virus OC43, murine hepatitis virus, pigs haemagglutinating encephalomyelitis virus, the puffinosis coronavirus, the rat coronavirus, sars coronavirus, infectious bronchitis virus, the turkey coronavirus, Vespertilio coronavirus China 2005, Vespertilio coronavirus strain 61, Vespertilio coronavirus ZS-2005, birds droppings is coronavirus just, the Intestinum Gallus domesticus coronavirus, the duck coronavirus, the goose coronavirus, human coronary virus NO, Psittacula alexandri fasciata coronavirus AV71/99, the pigeon coronavirus
Figure A200680038972D00521
Orbivirus: cattle Orbivirus, breda virus (Breda virus), horse Orbivirus, berne virus (Berne virus), people's Orbivirus, porcine circovirus, porcine circovirus (Strain P10).
Figure A200680038972D00522
Herpetoviridae (83-105);
Betaherperesvirinae: cytomegalovirus
Rhesus cytomegalovirus strain 68-1, nerpes vinrus hominis 5 (Strain 1042), nerpes vinrus hominis 5 (Strain 119), nerpes vinrus hominis 5 (Strain 2387), nerpes vinrus hominis 5 (Strain 4654), nerpes vinrus hominis 5 (Strain 5035), nerpes vinrus hominis 5 (Strain 5040), nerpes vinrus hominis 5 (Strain 5160), nerpes vinrus hominis 5 (Strain 5508), nerpes vinrus hominis 5 Strain AD 169, nerpes vinrus hominis 5 Strain Eisenhardt, nerpes vinrus hominis 5 Strain Merlin, nerpes vinrus hominis 5 Strain PT, nerpes vinrus hominis 5 Strain toledo, nerpes vinrus hominis 5 Strain Towne, the chimpanzee cytomegalovirus, Aotine herpesvirus 1, baboon cytomegalovirus OCOM4-37, ercocebus agilis cytomegalovirus 1, moustache monkey (Cercopithecus cephus) cytomegalovirus 1, dark red wart monkey (Colobus badius) cytomegalovirus 1, black and white wart monkey (Colobus guereza) cytomegalovirus 1, middle Moschus moschiferous Shrew (Crocidura russula) cytomegalovirus 1, machin (Macaca fascicularis) cytomegalovirus 1, mandrill belongs to (Mandrillus) cytomegalovirus, Africa warthog (Phacochoerusafricanus) cytomegalovirus 1, orangutan (Pongo pygmaeus) cytomegalovirus 1, inclusion-body rhinitis virus of pigs, the ape cytomegalovirus.
Figure A200680038972D00531
Alphaherpesvirinae: herpes simplex virus:
Bovid herpesvirus 2 type (Strain BHM-1), bovid herpesvirus 2 type (Strain BMV), cercopithecid herpesvirus 1 (Strain E2490), cercopithecid herpesvirus 16, SA 6 virus (SA8), herpes simplex virus (1 type/Strain 17), herpes simplex virus (1 type/Strain A44), herpes simplex virus (1 type/Strain Angelotti), herpes simplex virus (1 type/Strain CL101), herpes simplex virus (1 type/Strain CVG-2), herpes simplex virus (1 type/Strain F), herpes simplex virus (1 type/Strain HFEM), herpes simplex virus (1 type/Strain HZT), herpes simplex virus (1 type/Strain MGH-10), herpes simplex virus (1 type/Strain MP), herpes simplex virus (1 type/Strain Patton), herpes simplex virus (1 type/Strain R19), herpes simplex virus (1 type/Strain SC16), herpes simplex virus 1 Strain R-15, human herpes virus 7 Strain JI, nerpes vinrus hominis 1 Strain KOS, nerpes vinrus hominis 2 (herpes simplex virus type 2), herpes simplex virus (2 types/Strain B4327UR), nerpes vinrus hominis's 2 Strain 186, nerpes vinrus hominis's 2 Strain 333, nerpes vinrus hominis 2 Strain G, nerpes vinrus hominis 2 Strain HG52, nerpes vinrus hominis 2 Strain SA8, nerpes vinrus hominis 2 Strain SN03, nerpes vinrus hominis 2 Strain SS01, nerpes vinrus hominis 2 Strain ST04.
Flaviviridae; Banzi virus (106-133)
Alfuy virus (Alfuy virus), the Alkhurma hemorrhagic fever virus, apoi virus (Apoivirus), Aroa virus, Ba Jiazha virus (Bagaza virus), the strange virus of class (Banzi virus), crust figure cave virus (Batu Cave virus), Bouboui virus, bukalasa bat virus (Bukalasa bat virus), bussuquara virus (Bussuquara virus), CY1014 virus, Cacipacore virus, triumphant jasmine island virus (Carey Island virus), cell fusion agent virus, cow-bone ridge virus (Cowbone Ridge virus), dakar bat virus (Dakar batvirus), deer ixodism poison (Deer tick virus), dengue virus, edgehill virus (Edge Hill virus), entebbe bat virus (Entebbe bat virus), banzi virus CbaAr4001, banzi virus FSME, flavivirus, Gadgets Gully virus, Greece's goat encephalitis, her melon virus (Iguape virus) of wearing, ILH (Ilheus virus), Israel's turkey meningoencephalomyelitis virus (Israel turkey meningoencephalomyelitis virus), Japanese encephalitis virus, jugra virus (Jugra virus), Jutiapa (Jutiapa virus), kadam virus (Kadam virus), Kamiti river virus (Kamiti River virus), Keshen virus (Karshi virus), Kai Dugu virus (Kedougou virus), KOK (Kokoberavirus), koutango virus (Koutango virus), khoum spirit strategic point virus (Kumlinge virus), Kunjin virus (Kunjin virus), Kyasanur Forest disease virus (Kyasanur forest virus), langat virus (Langat virus), langat virus (Strain TP21), langat virus (Yelantsev Strain), louping-ill virus, Meaban virus, Murdoch's virus (Modocvirus), Montana Mus ear bat leukoencephalitis virus (Montana myotis leukoencephalitisvirus), the U.S. sharp scorching virus of valley head (Murray Valley encephalitis virus), Naranjal virus, Negishi virus, ntaya virus (Ntaya virus), msk haemorrhagia fever virus (Omsk hemorrhagic fever virus), phnom bat virus (Phnom Penh bat virus), Bo Disi khoum virus (Potiskum virus), Bo Wasang virus (Powassan virus), Re Baiwei virus (Rio Bravo virus), ROC (Rocio virus), roval farm virus, russian spring-summer encephalitis virus, Sha's ripple subvirus (Saboya virus), Sal Vieja virus, San Perlita virus, Saumarez Reef virus, match pik virus (Sepik virus), real million viruses far away (Sitiawan virus), sokoluk virus, the scorching virus of Spain's Medulla caprae seuovis, spondweni virus (Spondweni virus), Saint Louis' encephalitis virus, STR (Stratford virus), tamana bat virus (Tamana bat virus), tembusu virus (Tembusu virus), tick-brone encephalitis virus, tick-transmitted flavivirus, Ticks is propagated Bo Wasang (Tick-bornepowassan) virus (Strain 1b), Turkey's sheep encephalitis, Tyuleniy virus, makonde virus, outstanding Soviet Union its virus (Usutu virus), Wang Thong virus, Wei Seersi Blang virus (Wessselsbron virus), west nile virus, Yaounde virus (Yaoundevirus), yellow fever virus, Yokose virus, neat card virus (Zika virus), the mosquito transmitted virus.
Figure A200680038972D00551
Flaviviridae; Hepatitis virus belongs to (134-142); Hepatitis C virus
Hepatitis C virus hypotype 1a, hepatitis C virus hypotype 1b, hepatitis C virus hypotype 1c, hepatitis C virus hypotype 1d, hepatitis C virus hypotype 1e, hepatitis C virus hypotype 1f, hepatitis C virus hypotype 2a, hepatitis C virus hypotype 2b, hepatitis C virus hypotype 2c, hepatitis C virus hypotype 2d, hepatitis C virus hypotype 2f, hepatitis C virus hypotype 2i, hepatitis C virus hypotype 2k, hepatitis C virus hypotype 3a, hepatitis C virus hypotype 3b, hepatitis C virus hypotype 3g, hepatitis C virus hypotype 3k, hepatitis C virus hypotype 4a, hepatitis C virus hypotype 4c, hepatitis C virus hypotype 4d, hepatitis C virus hypotype 4f, hepatitis C virus hypotype 4h, hepatitis C virus hypotype 4k, hepatitis C virus hypotype 5a, hepatitis C virus hypotype 6a, hepatitis C virus hypotype 6b, hepatitis C virus hypotype 6d, hepatitis C virus hypotype 6g, hepatitis C virus hypotype 6h, hepatitis C virus hypotype 6k, hepatitis C virus (separated strain EC1), hepatitis C virus (separated strain EC10), hepatitis C virus (separated strain Glasgow), hepatitis C virus (separated strain HC-J2), hepatitis C virus (separated strain HC-J5), hepatitis C virus (separated strain HC-J7), hepatitis C virus (separated strain HCT18), hepatitis C virus (separated strain HCT27), hepatitis C virus (separated strain HCV-476), hepatitis C virus (separated strain HCV-KF), hepatitis C virus (separated strain Hunan), hepatitis C virus (separated strain TH), hepatitis C virus (separated strain VT204), hepatitis C virus (separated strain VT316), hepatitis C virus (separated strain VT681), hepatitis C virus (separated strain VT886), hepatitis C virus (separated strain VT887), hepatitis C virus (separated strain VT897), hepatitis C virus (separated strain VT898).
Figure A200680038972D00561
Flaviviridae; Pestivirus (143-144);
Border disease virus (Border disease virus), bovine viral diarrhea virus, chamois Pestivirus, classical swine fever virus (Classical Swine Fever virus), classical swine fever virus, hog cholera virus, border disease virus, Pestivirus, swine fever virus (Porcine pestivirus), pronghorn Antilocapra americana Pestivirus.
Figure A200680038972D00562
Picornaviridae; Hepatovirus; Hepatitis A virus (145-151):
Hepatitis A virus (Strain 18F), hepatitis A virus (Strain 24A), hepatitis A virus (Strain 43C), hepatitis A virus (Strain CR326), hepatitis A virus (Strain GA76), hepatitis A virus (Strain HM-175), hepatitis A virus (Strain LA), hepatitis A virus (Strain LCDC-1), hepatitis A virus (Strain MBB), hepatitis A virus (Strain MSM1), ape hepatitis A virus (Strain AGM-27), ape hepatitis A virus (Strain CY-145).
Figure A200680038972D00563
Picornaviridae; Rhinovirus (152-163);
The human rhinovirus 10, the human rhinovirus 100, the human rhinovirus 11, the human rhinovirus 12, the human rhinovirus 13, the human rhinovirus 15, the human rhinovirus 16, the human rhinovirus 18, the human rhinovirus 19, human rhinovirus 1A, human rhinovirus 1B, the human rhinovirus 2, the human rhinovirus 20, the human rhinovirus 21, the human rhinovirus 22, the human rhinovirus 23, the human rhinovirus 24, the human rhinovirus 25, the human rhinovirus 28, the human rhinovirus 29, the human rhinovirus 30, the human rhinovirus 31, the human rhinovirus 32, the human rhinovirus 33, the human rhinovirus 34, the human rhinovirus 36, the human rhinovirus 38, the human rhinovirus 39, the human rhinovirus 40, the human rhinovirus 41, the human rhinovirus 43, the human rhinovirus 44, the human rhinovirus 45, the human rhinovirus 46, the human rhinovirus 47, the human rhinovirus 49, the human rhinovirus 50, the human rhinovirus 51, the human rhinovirus 53, the human rhinovirus 54, the human rhinovirus 55, the human rhinovirus 56, the human rhinovirus 57, the human rhinovirus 58, the human rhinovirus 59, the human rhinovirus 60, the human rhinovirus 61, the human rhinovirus 62, the human rhinovirus 63, the human rhinovirus 64, the human rhinovirus 65, the human rhinovirus 66, the human rhinovirus 67, the human rhinovirus 68, the human rhinovirus 7, the human rhinovirus 71, the human rhinovirus 73, the human rhinovirus 74, the human rhinovirus 75, the human rhinovirus 76, the human rhinovirus 77, the human rhinovirus 78, the human rhinovirus 8, the human rhinovirus 80, the human rhinovirus 81, the human rhinovirus 82, the human rhinovirus 85, the human rhinovirus 88, the human rhinovirus 89, the human rhinovirus 9, the human rhinovirus 90, the human rhinovirus 94, the human rhinovirus 95, the human rhinovirus 96, the human rhinovirus 98, human rhinovirus B, the human rhinovirus 14, the human rhinovirus 17, the human rhinovirus 26, the human rhinovirus 27, the human rhinovirus 3,8001 Finland November nineteen ninety-five of human rhinovirus, the human rhinovirus 35, the human rhinovirus 37, human rhinovirus 6253 Finland in JIUYUE, 1994, human rhinovirus's 9166 Finland's nineteen ninety-five JIUYUE, the human rhinovirus 4, the human rhinovirus 42, human rhinovirus 9864 Finland in JIUYUE, 1996, the human rhinovirus 5, the human rhinovirus 52, human rhinovirus's 7425 Finland's nineteen ninety-five Decembers, 5928 Finland Mays nineteen ninety-five of human rhinovirus, the human rhinovirus 70, the human rhinovirus 72, the human rhinovirus 79, the human rhinovirus 83, human rhinovirus 8317 Finland in Augusts, 1996, the human rhinovirus 86, human rhinovirus 7851 Finland in JIUYUE, 1996, the human rhinovirus 92, the human rhinovirus 93, the human rhinovirus 97, the human rhinovirus 99, Antwerp rhinovirus 98/99 (Antwerp rhinovirus 98/99), human rhinovirus 263 Berlin 2004, human rhinovirus's hanks Strain (Human rhinovirusstrain Hanks), typing human rhinovirus OK88-8162 not, amblyomma americanum virus (Amblyomma americanum), human rhinovirus UC.
Figure A200680038972D00581
Picornaviridae; Enterovirus (164-179)
Bovine enteroviruses, bovine enteroviruses Strain K2577, bovine enteroviruses Strain SL305, bovine enteroviruses 2 types, Coxsackie virus (Coxsackievirus) A16, Coxsackie B virus 3, enterovirus A01-2A-1, enterovirus H02-2A-3, enterovirus H02-2B-1, enterovirus H04-2B-2, enterovirus Hu, enterovirus I01-1-2, enterovirus S01-2A-1, enterovirus S02-1-6, enterovirus S03-1-3, enterovirus S06-1-1, human Coxsackie virus A16, human Coxsackie virus A9, human Coxsackie virus A9B, Coxsackie virus, Enterovirus 69, enterovirus 74, enterovirus 79, enterovirus 81, enterovirus 82, enterovirus 83, enterovirus 86, enterovirus prolongs limit (Enterovirus Yanbian) 96-83csf, enterovirus prolongs limit 96-85csf, human Coxsackie virus A1, human Coxsackie virus A10, human Coxsackie virus A11, human Coxsackie virus A12, human Coxsackie virus A13, human Coxsackie virus A14, human Coxsackie virus A15, human Coxsackie virus A17, human Coxsackie virus A18, human Coxsackie virus A19, human Coxsackie virus A2, human Coxsackie virus A20, human Coxsackie virus A21, human Coxsackie virus A22, human Coxsackie virus A24, human Coxsackie virus A3, human Coxsackie virus A4, human Coxsackie virus A5, human Coxsackie virus A6, human Coxsackie virus A7, human Coxsackie virus A8, human Coxsackie virus B1, human Coxsackie virus B2, human Coxsackie virus B4, human Coxsackie virus B5, human Coxsackie virus B6, human ECHO virus 1, human ECHO virus 11, human ECHO virus 12, human ECHO virus 13, human ECHO virus 14, human ECHO virus 15, human ECHO virus 16, human ECHO virus 17, human ECHO virus 18, human ECHO virus 19, human ECHO virus 2, human ECHO virus 20, human ECHO virus 21, human ECHO virus 24, human ECHO virus 25, human ECHO virus 26, human ECHO virus 27, human ECHO virus 29, human ECHO virus 3, human ECHO virus 30, human ECHO virus 31, human ECHO virus 32, human ECHO virus 33, human ECHO virus 4, human ECHO virus 5, human ECHO virus 6, human ECHO virus 7, human ECHO virus 8, human ECHO virus 9, human intestine's virus 68, human intestine's virus 69, human intestine's virus 70, human intestine's virus 71, human intestine's virus 73, human intestine's virus 74, human intestine's virus 75, human intestine's virus 77, human intestine's virus 78, human intestine's virus 89, human intestine's virus 90, human intestine's virus 91, human intestine's virus B, human poliovirus virus 1, human poliovirus virus 2, human poliovirus virus 3, pig enterovirus 10, pig enterovirus 9, pig enterovirus J10, pig enterovirus J4, pig enterovirus J6, ape picornavirus 7, ape picornavirus 7 ', swine vesicular disease virus, wild type poliovirus 3 types.
The data mining of sequence provided herein is based on the list of references at this description ending place row.
Embodiment 3
Exemplary multi-plate detection kit
Table 8-breathes test kit
Strain
Other coronavirus of coronavirus SARS
HMPV (human stroma lung virus)
Influenza virus A+B bird flu virus
Adenovirus
RSV (respiratory syncytial virus)
Rhinovirus
Parainfluenza virus: 1,2,3
Special breathing test kit
Hantaan
LaCrosse's encephalitis
Table 9-gastrointestinal test kit:
Strain
Rotavirus
Adenovirus 40/41
Hepatitis A virus
Hepatitis C virus
Hepatitis E virus
Calicivirus
CMV (cytomegalovirus)
Table 10-meningitis test kit:
Strain
Enterovirus (1～80) (comprising poliovirus 1,2,3)
West nile virus
Dan Chunpaozhenbingdu1 ﹠2﹠6
Special meningitis test kit
Togavirus (east/west horse)
Banzi virus (St. Louis encephalitis, Japanese encephalitis ...)
Rabies virus
Table 11-sexually transmitted disease (STD) test kit:
Strain
The HIV Strain
Herpes simplex
1 type virus
Herpes simplex
2 type viruses
HSV-1
HSV-2
HPV (human papillomavirus)
HTLV-1
Table 12-traveller test kit:
Strain
Hepatitis A virus
Hepatitis B virus
Hepatitis C virus
HIV
Herpesvirus
1﹠2 type
Table 13-veterinary test kit:
Strain
Rabies virus
Canine distemper virus
The example that can adopt the disease that the mentioned reagent box detects below is provided.
Breathe test kit:
Pertussis does not indicate organism
Pneumococcal infection is under the situation of elsewhere classification (in condition classif.elsewhere); Specifically do not indicate the place
Adenovirus infection is under the situation of elsewhere classification; Specifically do not indicate the place
Acute alcoholism in the excessive drinking (ac.alcoholic intoxic.in alcoholism), of short duration drinking behavior
Acute nonsuppurative otitis media does not specifically indicate the place
Acute serous otitis media
Acute mucoid otitis media
Acute sanguinous otitis media (acute sanguinous otitis media)
Acute anaphylaxis otitis media serosa
Acute anaphylaxis MOM
The courageous and upright otitis media of acute anaphylaxis
Acute suppurative otitis media does not have spontaneous myringorupture
Acute suppurative otitis media is followed spontaneous myringorupture
Chronic tubotympanic suppurative otitis media
Not concrete indicated chronic suppurative otitis media
Not concrete indicated suppurative otitis media
Not concrete indicated otitis media
Acute mastoiditis without complications
Subperiosteal abscess of mastoid
Acute mastoiditis is accompanied other complication
The perforation of ear drum does not specifically indicate
Central perforation of tympanic membrane
Diaphragm-operated attic perforation
Diaphragm-operated other edge-punchings
Diaphragm-operated a plurality of perforation
Total perforation of tympanic membrane
Atrophic flaccid tympanic membrane
Atrophic nonflaccid tympanic membrane
Not concrete indicated tympanum disease
Acute nasopharyngitis (flu)
Acute antritis
Acute frontal sinusitis
Acute ethmoidal sinusitis
Acute sphenoiditis
Other acute sinusitis
Acute sinusitis does not specifically indicate
Acute pharyngitis
Acute tonsillitis
Acute laryngitis
Acute tracheitis, not mentioned blocking
Acute tracheitis, the companion is blocked
Acute laryngotracheitis, not mentioned blocking
Acute laryngotracheitis, the companion is blocked
Acute epiglottitis, not mentioned blocking
Acute epiglottitis, the companion is blocked
Croup
Acute pharyngolaryngitis
The acute upper respiratory tract infection at other a plurality of positions
Specifically do not indicate the upper respiratory tract infection at position
Acute bronchitis
Acute bronchiolitis
Pneumonia due to the adenovirus
Pneumonia due to the respiratory syncytial virus
Pneumonia due to the parainfluenza virus
Non-classified other the virogenetic pneumonia in other places
Viral pneumonia does not specifically indicate
Pneumococcal pneumonia
Pneumonia due to the kerekou pneumonia diphtheria
Pneumonia due to the pseudomonas
Pneumonia due to the hemophilus influenza (h.influenzae)
Bacterial pneumonia does not specifically indicate
Pneumonia in the cytomegalovirus infection
Pneumonia in the pertussis
Pneumonia in the anthrax
Pneumonia in the aspergillosis
Pneumonia in the other system mycosis
Pneumonia in other infectious diseases of other places classification
Bronchopneumonia does not specifically indicate organism
Pneumonia does not specifically indicate organism
The influenza of companion's pneumonia
Accompany the influenza of other respiratory tract performances
Accompany the influenza of other performances
Bronchitis does not specifically indicate acute or chronic
Other chronic bronchitiss
Extrinsic asthma, not mentioned status asthmaticus
Extrinsic asthma, companion's status asthmaticus
Intrinsic asthma, not mentioned status asthmaticus
Intrinsic asthma, companion's status asthmaticus
Asthma does not specifically indicate type, not mentioned status asthmaticus
Asthma does not specifically indicate type, companion's status asthmaticus
Pneumonia due to the suction (food, vomitus or n.o.s.)
The empyema of companion's fistula
Empyema, not mentioned fistula
Pleuritis, not mentioned effusion or current tuberculosis
The pleuritis of companion's effusion is due to other antibacterials outside the tbc
The leural effusion of other bright not forms is outside the expel stagnation nuclearity
Not concrete indicated leural effusion
Pulmonary abscess
Mediastinal abscess
The pulmonary collapse
Respiratory failure
The respiratory system other diseases, other places are unfiled
Not concrete indicated respiratory system disease
Toxic gastroenteritis and colitis
Other and not concrete indicated noninfectious gastroenteritis and colitis
Cyanosis
Adnormal respiration does not specifically indicate
Overventilation
Orthopnea
Other dyspnea and adnormal respiration
Cough
Suffocate
The gastrointestinal tract test kit:
Cholera due to the vibrio cholera (vibrio cholerae)
Cholera due to the Vibrio cholera El Tor (vibrio cholerae eltor)
Cholera does not specifically indicate
Typhoid fever
Paratyphoid fever
Paratyphoid fever
Paratyphoid fever
Paratyphoid fever does not specifically indicate
The Salmonella gastroenteritis
The Salmonella septicemia
Local Salmonella infection does not specifically indicate
The Salmonella meningitis
The Salmonella pneumonia
Salmonella arthritis
Salmonella osteomyelitis
Other local Salmonella infections
The Salmonella infection that other are expressed
Salmonella infection does not specifically indicate
Shigella dysenteriae (Shigella dysenteriae)
Shigella flexneri (Shigella flexneri)
Shigella bogdii (Shigella boydii)
Shigella sonnei (Shigella sonnei)
The shigella infection that other are expressed
Shigellosis does not specifically indicate
Staphylococcal food poisoning
Botulism
Alimentary toxicosis due to the bacillus perfringens (clostridium perfringens) (Clostridium welchii (c.welchii))
Poison due to other clostridiums
Alimentary toxicosis due to the vibrio parahaemolyticus (vibrio parahaemolyticus)
Alimentary toxicosis does not specifically indicate
Acute amebic dysentery, not mentioned abscess
Chronic intestinal amebiasis, not mentioned abscess
Amebic nondysenteric colitis
Amebic liver abscess
Amebic abscess of lung
Cerebral amebic abscess
The ameba skin ulcer
The ameba at other positions infects
Amebiasis does not specifically indicate
Balantidiasis
Giardiasis
Coccidiosis
Intestinal trichomoniasis
The protozoon property intestinal diseases that other are expressed
Not concrete indicated protozoon property intestinal diseases
Intestinal infects due to the Arizona class paracolon (paracolon bacilli)
Intestinal infects due to the Aerobacter aerogenes (aerobacter aerogenes)
Intestinal infects due to the mycetozoan (proteus) (proteus mirabilis (mirabilis)) (proteus morganii (morganii))
Intestinal infects due to the staphylococcus
Intestinal infects due to the pseudomonas
Infect by intestinal due to other antibacterials of expressing
Bacterial enteritis does not specifically indicate
Intestinal infects due to the non-classified other biological body in elsewhere
Infectious colitis, enteritis and gastroenteritis
Be assumed to colitis, enteritis and the gastroenteritis in infectiousness source
Infectious diarrhea
Be assumed to the diarrhoea in infectiousness source
The meningitis test kit
Specifically do not indicate bacteremia
Bacteremia
Septicemia
Pneumococcal septicemia
Bacterial meningitis
The Diplococcus pneumoniae meningitis
Meningitis in Fen Lei other bacterial diseasees elsewhere
Express meningitis due to the antibacterial by other
Meningitis does not specifically indicate
Suppurative arthritis
Cellulitis around the eye socket
Mastoiditis and associated conditions
Acute mastoiditis
Acute mastoiditis, no complication
Pneumococcal peritonitis
Herpetic meningoencephalitis
Herpetic septicemia
Sexually transmitted disease (STD) (std) test kit
Herpes simplex
The herpes simplex dermatitis of eyelid
The herpes simplex disciform keratitis
Herpes simplex iridocyclitis
Herpes simplex meningitis
The herpes simplex EOM
Herpes simplex, companion's ophthalmic complications
Herpes simplex is accompanied other ophthalmic complications
Herpes simplex, the complication of accompanying other to express
Herpes simplex, the complication of accompanying other to express
Herpes simplex is accompanied not concrete indicated complication
Herpes simplex is accompanied not concrete indicated ophthalmic complications
Herpes simplex, not mentioned complication
Herpes zoster
The herpes zoster dermatitis of eyelid
Herpes zoster iridocyclitis
The herpes zoster keratoconjunctivitis
Herpes zoster, companion's meningitis
Herpes zoster, companion's ophthalmic complications
Herpes zoster is accompanied other neurological complications
Herpes zoster is accompanied other neurological complications
Herpes zoster is accompanied other ophthalmic complications
Herpes zoster, the complication of accompanying other to express
Herpes zoster, the complication of accompanying other to express
Herpes zoster is accompanied not concrete indicated complication
Herpes zoster is accompanied not concrete indicated neurological complication
Herpes zoster, not mentioned complication
Herpetic gingivostomatitis
Herpetic infection of penis
Herpetic meningoencephalitis
Herpetic septicemia
Herpetic vulval ulcer
Herpetic vulvovaginitis
Herpetic whitlow
Embodiment 4
Protease clone and determination of activity
Material and experimental technique
Protease assay:
HRV 3C: under the room temperature, (carry out the active conventional external test of HRV 3C in PH 7.5 and the cumulative volume=1ml) at 25mM HEPES, 150mM NaCl, 1mM EDTA, 5% glycerol.Monitor in the sample, in the cell lysates, in the transformant extract (reorganization) and the HRV 3C proteinase activity in the reorganization HRV16 3C sample of purification.Fluorogenic substrate ( pep 1,5,6,8, Ori 2; EDANS/DANCYL) use in 4 μ M concentration usually.Detectable substrate is including, but not limited to Ori 2, PEP1.Utilize the exometer monitoring reaction at excitation/emission 340/490 ± 15nm corresponding to the excitation/emission feature of EDANS/DABCYL group.Enzyme concentration changes between 1 μ M～500pM.
Alternatively, at 30 ℃, the volume of employing in 96 orifice plates is that the substrate with ayapanin-4-acetic acid (MOC) fluorogen and dinitrophenol,DNP (DNP) quencher labelling of 100 μ l carries out the 3C protease assay, wherein contains 25mM Tris HCl (pH 8.0), 150mMNaCl, 1mM EDTA (pH 8.0), 6mM DTT, 2 μ M～6 μ M substrates, 2% DMSO, 416nM 3C protease and the inhibitor as needing then to add.By exciting and monitor fluorescence (cutoff is 10nm) at 328nm in the emission of 393nm.Adopt following equation that data are analyzed with nonlinear regression analysis program EnzFitter (BioSoft):
K i＝(I/((V max×S)/V 0)/K s)-I-S
Employed concentration of substrate is lower than the K of described substrate m(16.8 μ M) therefore do not need to revise S/K m.
What add in described reaction comprises DTT, glycerol, Na 2SO 4, BSA and nose cleanout fluid.
Enterovirus (non-polio enterovirus NPEV) determination of activity: at 4 ℃ with fresh CSF sample (preserving 1～2 day) supersound process 3 * 20 seconds at 4 ℃.The described cell lysates of 100 μ l is joined in 2 * reaction buffer of 100 μ l, and the Pep 1 that adopts 4 μ M is as substrate.Described reaction is at room temperature carried out, and monitors under excitation/emission 340/490 ± 15nm respectively with exometer.
Tissue culture is measured: H1 HeLa cell is grown in the DMEM culture medium that is supplemented with 5% FBS, 1% pen-strep and 1% non essential amino acid.The H1 Hela cell MOI that converges culture bottle is the HRV serotype 14﹠amp of 1～10PFU/ cell; 1A infects.In infection back 48 hours, perhaps with trypsin treatment or scrape harvesting.Cell also is suspended in the reaction buffer for three times again with the PBS washing.Made cell rupture in ultrasonic 3 * 15 seconds on ice, subsequently at 4 ℃, the centrifugal 10min of 13000rpm.Cell lysates (containing solubility 3C pro) through cleaning is used for described mensuration.
The clone of SARS and HRV 16 3C protease: with SARS and HRV protease clone as the source that is used to test with the protease of test kit.
HRV 16: primer is positioned at genomic 4320～4869bp position through design.Forward primer is added with 5 ' primer extension, and CACC goes in the topoisomerase cloning vehicle to assist directed cloning.Reverse primer is in order to be directed into 3 ' primer end with termination codon (TGA).Utilize the desired PCR product of these primers to be 556bp.Employed template is derived from the carrier that contains HRV 16 cDNA (GenBank accession number gi:3915817).
HRV 16 forward primers: 5 ' CACCGGTCCAGAAGAAGAAT, 3 ' SEQID NO:48
HRV 16 reverse primers: 5 ' TCATTGTTGTTCAGTGAAGTAT, 3 ' SEQID NO:49
SARS: primer is positioned at 9985～10902bp position of genome (SARS 3CL, GenBank registration number gi:37999886) through design.Forward primer is added with 5 ' primer extension, and CACC is to assist directed cloning.Reverse primer is in order to be directed into 3 ' primer end with termination codon (TAA).Utilize the desired PCR product of these primers to be 925bp.Employed template stems from (the Av Gay from Dr.Av-Gay; Vancouver; Canada) the cDNA library that obtains.
SARS forward primer: 5 ' CACCAGTGGTTTTAGGAAAATGGC, 3 ' SEQ ID NO:50
SARS reverse primer: 5 ' TTATTGGAAGGTAACACCAGAGC, 3 ' SEQID NO:51
In the standard reaction mixture, utilize and proofread and correct pcr amplification (1 * reaction buffer, 0.5mM dNTP, 100pmol primer and 1 pfu of unit that polymerase (Pfu) carries out gene.Adopt following recycle scheme: 94 ℃, 3min; 35 circulations subsequently (94 ℃, 1min; 59 ℃, 1min; 72 ℃, 1min) and at 72 ℃, 10min finishes).PCR product from SARS 3CL and HRV 16 3C protease directly is cloned into pET 151/D-TOPO (Invitrogen, Carlsbad, CA) (Fig. 1 a and 1b).For HRV 16 3C protease, find one among 35 clones of screening and be positive.For SARS 3CL protease, in 15 of screening, there are 3 to be positive.
Experimental result
The clone of SARS and HRV 16 3C protease: as mentioned above SARS 3CL and HRV16 protease clone are gone into pET 151/D-TOPO (Invitrogen, Carlsbad CA) as the protease source that is used for described experiment and test kit.Fig. 1 a and 1b illustrate SARS3CL (pMND2) and HRV 16 3C (pMND1) plasmid respectively.For HRV 16 3C protease, find one among 35 clones of screening and be positive.For SARS 3CL protease, in 15 of screening, there are 3 to be positive.In coming from the WCL of transform bacteria, detect reorganization HRV 16 3C and SARS 3CL proteinase activity (Fig. 2).
Adopt the excellent substrate activity of optimum Cutting sequence peptide substrate in the external test
The peptide design: for being provided for detecting and characterizing the optionally the most effective substrate of institute's protein of interest enzyme, the cutting with peptide substrates natural and through designing in external test compares.Peptide substrates is used in the mensuration of HRV 3C protease through design.Peptide substrates sequence or design according to natural cleavage site sequence, perhaps the method according to this invention is selected.Usually, by carrying out the multisequencing comparison of a plurality of known HRV cleavage sites, and determine best aminoacid, determine the substrate sequential design at ad-hoc location according to its bioinformatics character.Following table 14 has been described the 3C protease substrate and the source thereof of adopting in external test.
Table 14
SEQ ID NO Title Sequence Target design
143 Ori2 (DABCYL)-S-A-I-F-Q-G-P-I-S-M-D (EDANS)-K Original cleavage site based on HRV 16
144 PEP1 (DABCYL)-L-E-A-L-F-Q-G-P-D (EDANS)-S-Q Design based on a plurality of HRV cleavage sites and bioinformatics
145 PEP3 E-A-L-F-Q-pNA Based on HRV cleavage site and PEP1
146 PEP4 D-S-L-E-V-L-F-Q-pNA Based on the HRV cleavage site
147 PEP5 (DABCYL)-L-E-V-L-F-Q-G-P-D (EDANS)-S-Q Design based on a plurality of HRV cleavage sites and bioinformatics
148 PEP6 (DABCYL)-T-S-A-V-L-Q-S-G-F-R-D (EDANS)-K Original cleavage site based on SARS
149 PEP7 : T-S-A-V-L-Q-pNA Original cleavage site based on SARS
150 PEP8 (DABCYL)-L-E-A-L-F-Q-A-A-D (EDANS)-S-Q-NH 2 Do not cut through being designed to by HRV protease.Also having N-terminal simultaneously modifies being designed to and blocks described end and reduce background
151 PEP9 (DABCYL)-L-E-A-L-F-Q-G-P-D (EDANS)-S-Q-NH 2 Design according to a plurality of HRV cleavage sites and bioinformatics, also have N-terminal simultaneously and modify being designed to and block described end and reduce background
Determine the best peptide substrates of 3C protease: be to determine the optimum Cutting substrate of 3C protease, the recombinated cutting of 3C protease (referring to above embodiment 1) and the cutting of described natural substrate sequence Ori 2 of described synthetic peptide substrates Pep1～Pep9 (referring to above table 14) compares.Showing the most dynamic (dynamical) substrate in Pep1～Pep9 is Pep1, and it designs on the basis of a plurality of HRV 3C cleavage sites and bioinformatics.When Fig. 3 has described and has compared with Ori 2, the excellent kinetics of described 3C protease assay.Fig. 4 has described in the concentration of substrate of 0.003 μ M～4 μ M, when adopting 250nM 3C protease, and the kinetics that reorganization 3C protease cuts Pep1.For all concentration of substrate of being tested, all observe the linear kinetics of at least 3 minutes courses of reaction.
Determine the optimum condition of the 3C protease assay of the best peptide substrates of employing:, assessed the sensitivity that reaction condition is changed for definite optimum condition that uses the 3C protease assay of described substrate through designing.Main component such as DTT, glycerol, the Na of described standard buffer solution have been changed 2SO 4With the concentration of BSA, and measured influence to reaction rate (RFU/min).Described result points out that the optimum response buffer that uses with the Pep1 substrate comprises 6mM DTT, 5% glycerol, 0.8MNa 2SO 4With 0.1～1mg BSA/ml.
Determine the sensitivity of the 3C protease assay of the best peptide substrates of employing: estimate that the enzyme concentration in clinical sample is lower usually.The lower limit that adopts for measuring the 3C proteinase activity of described peptide substrates through design to detect, with the recombinase concentration determination of certain limit the Pep1 of 4 μ M.The lower limit that protease detects in this mensuration is through being defined as 0.5nM～1.0nM 3C protease.
Determine to adopt the specificity of the 3C protease assay of best peptide substrates: it is essential that for cleavage activity in the real clinical sample of assessment desirable cleavage activity is to the specificity through the substrate of design.Be the specificity of test HRV 3C protease, tested the enzyme cross reactivity of Pep1 substrate and SARS protease and escherichia coli (E.coli) lysate Pep1.
Fig. 5 has shown in the HRV 16 lysates HRV 3C protease specificity to the Pep1 cutting, and with SARS 3CL lysate or E.coli lysate do not have any can detected cleavage reaction.
Adopt the H1HeLa cell culture to infect and carry out more specificity test, and test designed substrate and so detecting the active effectiveness of 3C under the condition to simulate real original position HRV.The lysate through cleaning and supersound process of the HeLa cell that HRV infects is tested with substrate Pep1, Ori2 and Pep6.Described result points out that the HeLa cell has very high background for the nonspecific protease activity.
Non-specific influence when utilizing the HeLa cell to carry out work for attempting to eliminate has been assessed multiple known protein enzyme inhibitor (EDTA, EGTA, PMSF and aprotinin (Aprotinin)) to the influence of HeLa cell lysates to the cutting of designed substrate.Following table 15 has been described and has been adopted Pep1 as the albumen cleavage reaction of the substrate relative insensitivity to inhibitor PMSF and aprotinin.EDTA/EGTA partly suppresses the cutting of described 3C protease to Pep1.
Table 15
Contrast (the H1 cell lysates is only arranged, do not have inhibitor) The tablet (Sigma) that does not contain EDTA completely PMSF 2mM Aprotinin 8 μ g/ml EDTA/EGTA 10mM
Reaction rate RFU/min 119 110 119 115 65
Suppress % (with respect to contrast) - 7.5％ 0％ 3.3％ 45％
Determine the effectiveness of the 3C protease assay of the best peptide substrates of employing under clinical condition: the clinical settings of HRV protease assay is characterised in that and has mucosa secretions.In order under real clinical condition, to determine the effectiveness of 3C protease, compared the cutting of 3C protease of when existing the nose that adds to clean sample and not exist the nose of adding to clean sample, recombinating to Pep1 to the cutting of designed substrate.Fig. 6 shows that nose cleans sample to not influence of kinetics, shows the described suitability that is reflected in the clinical practice.
Accurately detect rhinovirus infection-be to test the suitability that designed peptide substrates is used in clinical setting by the cutting of cleaning optimum Cutting sequence peptide substrate in the sample at clinical nose, clean sample (utilize mucus withdrawal device test kit-have the Mucosafe withdrawal device-Maersk Medical A/S-Denmark of filter) and under the situation that virus exists, assess obtaining nose from the experimenter, carry out the protease catalytic activity with method of the present invention then and test.The part of each sample is measured (IFA) employing by direct immunofluorescence and is purchased monoclonal antibody (Chemicon International, Inc., Temecula CA), utilizes tissue culture to detect the existence of RSV, influenza A and B virus, parainfluenza virus and adenovirus.Remainder is kept at-80 ℃, until being used for the analysis that HRV exists.
At present, " the gold medal standard " that does not exist HRV to detect.Because viral susceptibility makes that tissue culture is not is a kind of routine test, and do not have and to test for the commercial immunoassay of adopting.Therefore select RT-PCR to analyze, (the The Central Laboraotry of Virology of virusology central laboratory of department of infectious diseases of internal medicine portion of hospital of University of Geneva at Geneva, Switzerland, Division of InfectiousDiseases, Department of Internal Medicine, University Hospitals of Geneva) carries out sample analysis.
Clinical nose cleanout fluid test: utilize 24 parts of clinical samples (nose cleanout fluid) to measure.The synthetic peptide substrates Pep1 of use has calculated the 3C protease ratio (passing through Bradford) alive (than work=RFU/min/mg protein) with respect to total protein concentration.
Following table 16 has shown by RT-PCR and the comparative result by HRV in proteinase activity (MND mensuration) test sample.17 parts in 24 duplicate samples are defined as the protease positive relevant with described RT-PCR result or protease feminine gender (being greater than or less than 0.5RFU/min/mg protein respectively than living).In 7 duplicate samples of remainder, 5 parts negative in RT-PCR, but positive according to protease assay, and 2 parts positive in RT-PCR, but negative according to protease assay.Suppose the inerrancy of described RT-PCR, the sensitivity compatibility of statistical analysis (table 17) demonstration 75% of these PRELIMINARY RESULTS and 70% the specificity compatibility.
Table 16
Sample RT-PCR Other viruses Than RFU/min/mg alive MND result
2 μ M substrates Threshold value (Trashhold)=0.5
* E0260 N 0.49084 N
* E0265 P 0.532999377 P
* E0269 N 0 N
* E0281 N RSVPIAD 0.296603037 N
** E0282 N 0.588661728 P
* E0283 N 0 N
** E0300 N CMV 2.94504 P
* E0306 N 0 N
* E0312 N 0 N
** E0313 N HPMV 1.10124359 P
* E0314 N 0.332420741 N
* E0317 P 6.932318182 P
H1?HeLa 0.191111111
4 μ M substrates
Sample Threshold value (Trash hold)=0.53
* E0320 P CMV 19.30126825 P
** E0335 N 21.87407626 P
* E0336 P 6.405326675 P
** E0347 N PI 16.08482439 P
* E0370 N 0.658017493 N
* E0424 P 9.198241407 P
*** E0474 P 0.120745921 N
*** E0497 P 0.334046092 N
* E0499 N 0 N
* W0006 N 5.290901444 N
* W0007 N AD 3.075 N
* W0011 P 9.290322581 P
H1?HeLa 0.375
The P-HRV positive, N-HRV feminine gender, PI-parainfluenza virus, AD-adenovirus
*-relevant with RT-PCR, *The negative MND positive of-RT-PCR, * *The positive MND feminine gender of-RT-PCR.
Table 17
Figure A200680038972D00731
Yet the accuracy that RT-PCR detects is shady, adopts the protease of method of the present invention to detect to have than RT-PCR and detects more obvious advantage.RT-PCR only detects the RNA of virus and therefore also can detect the virus of inactivation.Therefore, possible is that positive sample negative in proteinase activity is due to the fact that in RT-PCR, and promptly described protease assay only detects the virus that is in activity form.In addition, be to be understood that the sample tested preserved 3～4 years at-80 ℃ before use.Therefore, the positive and sample that protease assay is negative of test may contain because long preservation and the 3C protease of inactivation in RT-PCR.Once more, protease detects may be sensitiveer more than RT-PCR, and it is negative and in the positive sample of protease that this can be reflected in test among the RT-PCR.
Therefore, the 3C protease assay of employing optimum Cutting sequence peptide substrate successfully detects the HRV16 in the clinical sample.
Embodiment 5
Cutting by optimum Cutting sequence peptide substrate in the clinical CSF sample accurately detects enterovirus infection
Non-polio enterovirus (NPEV) infects at the beginning of annual autumn late summer very common, and is to be only second to the most common rhinoviral human viral infection's factor.Surpass 90% aseptic meningitis case and caused by NPEV, classical symptom is for heating, serious headache, Wry Neck, cervical pain, nausea and vomiting, to the light sensitivity and erythra may occur.
The detection of NPEV is undertaken by PCR and tissue culture at present among the CSF.NPEV PCR compares Virus culture can provide the result more delicately shorter time in 5～24 hours after receiving sample.Yet tissue culture and RT-PCR all spend big and require complicated instrument, and are not suitable for field diagnostic fast.Adopt the enterovirus of the protease assay of optimum Cutting sequence peptide substrate to detect for test and render a service, human cerebrospinal fluid (CSF) sample utilizes method of the present invention and measure NPEV in definitiveness tissue culture measures.
Clinical CSF test: table 18 has been described the excellent susceptiveness of method of the present invention.In order in CSF, to detect accurately, delicately, select for use Pep9 as described optimal sequence peptide substrate.Select Pep9 for use based on following reason: at first, the protease of NPEV and HRV belongs to identical family, though adopt non-optimum determining condition, but still can obtain the result.Secondly, because HRV is not present among the CSF, therefore there is not the risk of cross reactivity.Obtain fresh CSF by lumbar puncture.
Table 18
The CSF sample Cell/mm 3 MND result The result of tissue culture
N1 450 Positive Positive
N2
4 Negative Negative
N3
2 Negative Negative
N4
1 Negative Negative
N5 148 Negative Negative
N6 194 Negative Negative
N7
4 Negative Negative
In 7 parts of fresh CSF samples, tissue culture's measurement result and based on obtaining complete dependency between the result of the detection of proteinase activity.Notice that the positive correlation between described result of tissue culture and the protease testing result is not subjected to cell density (450 cells/mm 3～1 cell/mm 3) influence.Therefore, thus under clinical condition, can use the protease that undertaken by the cutting of optimum Cutting sequence peptide substrate in the CSF sample to detect to carry out enterovirus accurately and fast detecting.
In a word, these results show that the optimum peptide substrates according to relatively enzyme kinetics design and selection can be used for detecting the target enzyme with diagnostic significance, and it has excellent sensitivity and accuracy.These detections with target enzyme of diagnostic significance can greatly improve common disease such as enterovirus and rhinovirus, and the suspicious pathogenic former quick diagnosis such as SARS and bird flu of popular or being very popular property.
Embodiment 6
Detect based on isolating protease
The present invention also provides according to the specific substrates of carrying out in reaction and has cut the novel method that detects one or more reacting substances (enzyme or particular chemicals) simultaneously, has described the separation based on affinity.The existence of the necessary specific reaction of described detection has been indicated in the detection of described cutting.In one embodiment, an application as the method is the certain enzyme that is used to detect the specific reaction of catalysis.In another embodiment, described enzyme is the part of the living things system that need detect.In another embodiment, described living things system is virus or antibacterial or other pathogen.Described detection method is constituted by two steps:
Separating of the molecule of the first step-process processing in reaction and the molecule of not process processing.Second goes on foot-only the molecule through processing is detected.
The described separation mechanism or the special affinity that can utilize two parts be in conjunction with for example antibody-substrate, and nucleic acid hybridization perhaps can utilize and fixed surface being connected of film, chip, pearl etc. for example.Described detection step or can be based on affinity or any other mode for example fluorescence method, colorimetry, enzyme process, perhaps the two.
The substrate that carries out specific reaction to be measured comprises three parts (X, Y, Z) and C (Fig. 7).Core element (fragment Y, for example the best substrate of kinetics is determined such as an above description) end with specificity cleavage site is connected in tag molecule (fragment X gets final product the test section), its objective is and detects the substrate that is cut.The other end with will link to each other through the substrate and the structure fragment (fragment Z, separating part) separated of unprocessed substrate of processing.When the cutting of molecule Y, form the substrate cleaved products: (1) is contained the label fragment (TS is also referred to as X-Y ' herein) of X part and part Y and the isolated fragment that (2) contain Z and part Y (SS is also referred to as Y ＂-Z) herein.Causing described molecule cutting and its detection is that desirable process can be enzyme reaction, chemical reaction, degeneration or any other process.
The substrate of in Fig. 8, describing and its cutting part reaction through design.In case cut, described Z part (isolated fragment) is used to separate substrate and the crude substrate through processing.Therefore, the TS (but containing the test section) of described substrate through processing is only arranged but combine with the part that has the high affinity of detection molecules.Therefore only detect described affinity cohesive process for the substrate that is cut.Detection can be based on existing, known or novel method.Can only detect molecule (Fig. 8) in this way through processing.
In another embodiment, also may adopt said method to detect the cutting of many substrates simultaneously.If this probability takes place when following situation, i.e. similar but its specificity cutting molecule (Y) difference of the isolating construction of described substrate (Z).In this case, have can be with its core element (Y) but the different test sections (X) of continuous uniqueness for every kind of substrate.Cutting is taking place and after the separation between processing and the crude substrate, the TS of substrate that described difference (with through processing) is only arranged is by affinity combine (according to said method).Any molecule that contains Z (the not SS of the substrate of the substrate of process processing or process processing) keeps by described isolating construction.Can realize the separation between the different TS of different substrates by design film, chip etc., the part that has affinity with variant TS is contained in each predetermined area on described film, chip, and for example each place only can be in conjunction with a kind of TS.Separated subsequently solution contacts with this chip or film and the affinity combination takes place.Be connected with different TS by affinity by knowing which predetermined area, can identify which substrate through processing.Because every kind of substrate is specific for the described enzyme that causes described substrate cutting, therefore might identify which kind of enzyme has cut its corresponding substrate, therefore reason out which kind of protease and be present in the described solution, and which kind of pathogen or factor (Fig. 9) corresponding to corresponding enzyme reasons out be.
Reverse PH system (RPHS)-in this embodiment, described C fragment be in the buffer solution for all substrates total specific molecular.A fragment corresponding to various substrates is the dye molecule entity, and its different colours is to different PH sensitivities.After cutting, described buffer solution passes through the filtration that has the pillar of affinity with fragment C.Any molecule (the fragment SS of the substrate of crude substrate or process processing) that contains fragment C will be retained in (by the affine part corresponding to fragment C) on the described post.Only have the described TS fragment (not containing fragment C) of substrate through processing can be along with entering the cell with several junior units, wherein each junior unit has different PH.Once described TS fragment (containing A) contacts with described junior unit (different PH), described junior unit changes color according to the character of Segment A.Which kind of substrate this indicates through processing.
Separation mechanism
The purpose of described separation mechanism is to separate described TS and SS makes that but only described TS can be allowed to combine with the part that has affinity corresponding to described test section.Detect at the same time under the situation of many substrates, described separation mechanism also has other functions, promptly removes complete substrate failing for these substrates to cause under the situation of cutting.
For said system, can adopt any separation method.The simple declaration of these separation methods below is provided.
1. fixedly separated system (ISS)-in this system, Z is by pearl, NC Nitroncellulose film, biotin-avidin or other affinitys pair introns that are connected with fixed surface.In buffer solution after the cutting, anyly do not remove by from described buffer solution, separating described fixed surface (by extraction, centrifugal, filtration etc.) through the substrate of processing or through the SS of the substrate of processing, only stay TS through the substrate of processing.In this mode, also may monitor the kinetics of every kind of substrate.
2. dynamic split system (DSS)-in this system, Z be in the described buffer solution for all substrates total or unique specific molecular.After cutting, described buffer solution contacts with the film or the chip of process particular design.Said film is for vertically and in the bottom it has the part that has affinity corresponding to Z.The adjacent part of described film comprises the different location corresponding to the X of different substrates.By capillary force or electric power the length of described buffer solution along described film or chip is advanced then.Any molecule (the not SS of the substrate of the substrate of process processing or process processing) that contains Z will be retained in the bottom (by the affinity part corresponding to Z) of described film.Only there is TS (not containing Z) to move up and combine (Figure 10) by affinity along film with its predetermined area through the substrate of processing.
3. affinity filtration system (AFS), in this system, the pillar of described buffer solution through having an affinity with Z filters, and any molecule (not through the substrate of processing or through the SS of the substrate of processing) that therefore contains Z will be retained in the pillar.Effluent will only contain the TS through the substrate of processing.
The example that affinity is right
The affinity that is used for this system is right to being any affinity as known in the art.The right example of affinity is including, but not limited to biotin-avidin, antibody-substrate; Receptor-substrate; Justice-antisense DNA/RNA chain based on nucleic acid hybridization; PH dependency color molecule; But fluorescence-test section can be based on FRET or other fluorescence detection methods.
The detection method example
Antibodies/receptors-substrate-based on any existing or novel immuno-chemical method of fluorescence or color is suitable.
Labelling-test section but (X) can be following molecule or be connected in following molecule: it is measurable, visible or can easy detected molecule to produce color, fluorescence, FRET or any other.
DNA hybridization-such as any existing or novel hybridizing method based on fluorescence or color probe is suitable.
Enzyme reaction-test section but (X) can link to each other with the enzyme of energy catalysis color or fluorescence or the reaction that any other is measurable, visible or any other is easy to detect.
Protease based on the antibody affinity detects
Detected the existence of a large amount of viruses in the clinical sample according to its specific proteins enzymatic activity.Many virus families adopt the pith of specific proteinase activity as its life cycle.For example rhinovirus (3C protease), enterovirus (3C protease) and SARS (3CL protease) all adopt the cutting sequence unique for its virus family.
In this embodiment, described substrate is the peptide cutting sequence (corresponding to Y) that at one end links to each other with biotin (corresponding to Z).The other end links to each other with the molecule (corresponding to X) that specific antibodies is had affinity.The cutting of this substrate will cause separate (as mentioned above) of SS and TS.
Described reactant mixture contains dissimilar above-mentioned substrates.All described substrates at one end contain biotin moiety.The difference of described peptide is the cutting sequence corresponding to specific proteases, and the antibody substrate of described uniqueness can be associated in specificity cutting sequence (for example A1, A2, A3......).In this way, every kind of substrate can be associated with different virus.
Described reactant mixture contacts with clinical sample and can cut.After cutting, on film or chip, analyze described reactant mixture, said film or chip shape such as strip also aim at this purpose and special design (Figure 10).It has the zone of containing avidin in the bottom.Has the different location of the antibody [for example anti-A1 (corresponding to anti-X1), anti-A2 (corresponding to anti-X2), anti-A3 (corresponding to anti-X3) ...] that contains corresponding to the different antibodies substrate of different substrates more than the zone for it at this.By capillary force or electric power the length of described buffer solution along described film or chip is advanced then.Any molecule that contains biotin (unprocessed substrate or through the SS of the substrate of processing) will be retained in described film the bottom (by with the affinity of avidin).Only have through the TS (not containing biotin) of the substrate of processing can along described film move up and with its predetermined area by affinity combine (for example, the anti-A1 of A1-, the anti-A2 of A2-etc. are corresponding to above-mentioned name).
Because the different protease of each place representative, therefore may determine which kind of protease is present in the described clinical sample by the existence of described substrate through processing.The existence of respective specific virus has been confirmed in the existence of described protease.
Protease based on nucleic acid hybridization detects
The positive-sense strand of DNA or RNA and antisense strand have very high affinity each other.Described system has utilized this character.This system designs according to the criterion described in the above-mentioned antibody embodiment.
Difference is that described affinity combination is based on nucleic acid hybridization.In this embodiment, described substrate is the end peptide cutting sequence (corresponding to Y) continuous with separating ssDNA chain (corresponding to Z).The other end links to each other with the ssDNA chain (corresponding to X) of uniqueness.The cutting of this substrate causes separate (as mentioned above) of SS and TS.
Described reactant mixture contains dissimilar above-mentioned substrates.All substrates have identical separation ssDNA chain.Their difference is the cutting sequence corresponding to specific proteases, and can cut unique ssDNA (for example A1ssDNA, A2ssDNA, A3ssDNA......) that sequence is associated with specificity.In this way, every kind of substrate can be associated with different virus.
Described reactant mixture contacts with clinical sample and can cut.After cutting, on film or chip, analyze described reactant mixture, said film or chip shape be specially designed strip (Figure 10) as aiming at this purpose.It has the zone of containing antisense DNA isolation chain in the bottom.It has the different location of the antisense DNA chain (for example antisense A1ssDNA, antisense A2ssDNA, antisense A3ssDNA......) that contains corresponding to unique ssDNA of different substrates more than the zone at this.By capillary force or electric power the length of described buffer solution along described film or chip is advanced then.Any molecule that contains described separation ssDNA chain (unprocessed substrate or through the SS of the substrate of processing) will be retained in described film the bottom (by with the affinity of described antisense DNA isolation).The TS (do not contain and separate the ssDNA chain) that passes through the substrate of processing is only arranged, and (for example, A1ssDNA-antisense A1ssDNA, A2ssDNA-antisense A2ssDNA etc.) can move up and combine with its predetermined area along described film.
Because the different protease of each place representative, therefore may determine which kind of protease is present in the described clinical sample by the existence of described substrate through processing.The existence of respective specific virus has been confirmed in the existence of described protease.But only can adopt single test section to assign to detect several viruses according to the addressable location of the described assay products on the described solid phase.
Being to be understood that some feature of the present invention of for clarity sake describing in independent embodiment content also can make up in single embodiment provides.On the contrary, also can provide respectively for the various features of in single embodiment content, describing for purpose of brevity or close with any suitable subgroup and to provide.
Though invention has been described in conjunction with the specific embodiment of the present invention, obvious many changes, modification and variant are conspicuous to those skilled in the art.Therefore, be intended to contain spirit and interior these changes, modification and the variant of broad range that drops on claims.Publication in this manual, patent and patent application this by reference integral body be incorporated in the description, as every piece independently publication, patent and patent application this with special and independent shown in introduce the same scope reached by reference.In addition, any list of references in this application quotes or identifies and should not be construed as and admit that so list of references can be used as prior art of the present invention and obtains.
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124.Nestorowicz, A., T.J.Chambers and C.M.Rice.1994.Mutagenesisof the yellow fever virus NS2A/2B cleavage site:effects on proteolyticprocessing, viral replication and evidence for alternative processing of theNS2A protein.Virology 199:114-123.
125.Yusof R, Clum S, Wetzel M, Krishna Murthy HM and Padmanabhan R.Purified NS2B/NS3 serine protease of dengue virus type 2exhibits cofactor NS2B dependence for cleavage of substrates with dibasicamino acids.J Biol Chem, 2000,275:9963-9969.
126.Khumthong R, Niyomrattanakit P, Chanprapaph S, Angsuthanasombat C, Panyim S and Katzenmeier G.Steady-state cleavagekinetics for dengue vir us type 2 NS2B-NS3 (pro) serine protease with syntheticpeptides.Prot Pept Lett, 2003,10:19-26.
127.Li，J.，Lim，S.P.，Beer，D.，Patel，V.，Wen，D.，Tumanut，C.，Tully，D.C.，Williams，J.A.，Jiricek，J.，Priestle，J.P.，Harris，J.L.，Vasudevan，S.G.，2005.Functional?profiling?of?recombinant?NS3?proteases?from?all?fourserotypes?of?dengue?virus?using?tetrapeptide?and?octapeptide?substratelibraries.J.Biol.Chem.280(31)，28766-28774
128.Yusof R, Clum S, Wetzel M, Krishna Murthy HM and Padmanabhan R.Purified NS2B/NS3 serine protease of dengue virus type 2exhibits cofactor NS2B dependence for cleavage of substrates with dibasicamino acids.J Biol Chem, 2000,275:9963-9969.
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Sequence table
＜110〉MND Diagnostic Ltd.
＜120〉method that is used to detect the compositions of virus and uses said composition detection virus
<130>FCI08IL0658C
<160>151
＜170〉Patentln version 3 .3
<210>1
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the adenovirus protein zymolyte is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be M, I or L
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be any aminoacid
<220>
<221>misc_feature
>222>(4)..(5)
＜223〉protease cleavage site
<400>1
<210>2
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the adenovirus protein zymolyte is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be M, L, I, V, N or Q
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be A or G
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>2
Figure A200680038972D01011
<210>3
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉α virus protein zymolyte is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be A or V
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be A, G or any alkaline amino acid residue
<220>
<221>misc_feature
<222>(3)..(4)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be A, G or Y
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be any hydrophobic amino acid residues
<400>3
<210>4
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the rubella virus protein zymolyte is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>4
Figure A200680038972D01013
<210>5
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the hiv protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be S or G
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be Q, G, R or K
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be N, C or D
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be Y, any hydrophobicity or aromatic amino acid residue)-/-
P (I/V/ hydrophobicity) (V/Q
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be I, V or any hydrophobic amino acid residues
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be V or Q
<400>5
Figure A200680038972D01021
<210>6
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the HTLV protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be V, L, T or P
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be F or L
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be any hydrophobic amino acid
<400>6
Figure A200680038972D01031
<210>7
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the HTLV protease substrate is total
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be F or L
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<400>7
Figure A200680038972D01032
<210>8
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the HTLV protease substrate is total
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<400>8
<210>9
<211>3
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the arteritis virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be L or any aromatic series or alkaline amino acid residue
<220>
<221>misc_feature
<222>(2)..(3)
<223>
<400>9
Figure A200680038972D01034
<210>10
<211>8
<212>PRT
<213>
<220>
＜223〉coronavirus 3C protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be S or any hydrophobic amino acid
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be S or A
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉can be any hydrophobicity or basic amino acid
<400>10
Figure A200680038972D01041
<210>11
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉sars coronavirus PLpro protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be K, R or N
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be A or K
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be any hydrophobic amino acid
<400>11
Figure A200680038972D01051
<210>12
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉sars coronavirus 3CL protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be A or S
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>12
Figure A200680038972D01052
<210>13
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Orbivirus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be P or any alkalescence or aromatic amino acid
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be S, A or G
<400>13
Figure A200680038972D01053
<210>14
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the CMV protease substrate is total
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be any aminoacid except that K
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>14
Figure A200680038972D01061
<210>15
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the CMV protease substrate is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>15
Figure A200680038972D01062
<210>16
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the hsv protein zymolyte is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be any hydrophobicity or aromatic amino acid
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be Q, N, E or D
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be S, T, D or E
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be any hydrophobic amino acid or HB donor
<400>16
Figure A200680038972D01063
<210>17
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the banzi virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be G or any basic amino acid
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(3)..(4)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be S, G or A
<400>17
Figure A200680038972D01071
<210>18
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the dengue virus protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(3)..(4)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be S, T or A
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be the HD donor
<400>18
Figure A200680038972D01072
<210>19
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the dengue virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be S, T or A
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be any hydrophobic amino acid
<400>19
Figure A200680038972D01081
<210>20
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the dengue virus protease substrate is total
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<400>20
Figure A200680038972D01082
<210>21
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the west nile virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be A or HB donor
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(3)..(4)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be S or G
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be any hydrophobic amino acid
<400>21
Figure A200680038972D01091
<210>22
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the west nile virus protease substrate is total
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<400>22
Figure A200680038972D01092
<210>23
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the west nile virus protease substrate is total
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>23
Figure A200680038972D01093
<210>24
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the yellow fever virus protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(3)..(4)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be S or G
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be any hydrophobic amino acid
<400>24
Figure A200680038972D01101
<210>25
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the yellow fever virus protease substrate is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>25
Figure A200680038972D01102
<210>26
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the yellow fever virus protease substrate is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>26
Figure A200680038972D01103
<210>27
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Japanese encephalitis virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(2)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be S or G
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉can be any hydrophobic amino acid
<400>27
Figure A200680038972D01111
<210>28
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Japanese encephalitis virus protease substrate is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>28
Figure A200680038972D01112
<210>29
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Japanese encephalitis virus protease substrate is total
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉can be any hydrophobic amino acid
<400>29
Figure A200680038972D01113
<210>30
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉Ticks biography virus protein zymolyte is total
<220>
<221>misc_feature
<222>(1)..(2)
＜223〉can be any basic amino acid
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be any acidic amino acid
<400>30
<210>31
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉Ticks biography virus protein zymolyte is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>31
Figure A200680038972D01122
<210>32
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉Ticks biography virus protein zymolyte is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>32
Figure A200680038972D01123
<210>33
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be T, S or any acidic amino acid
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be E, T, I, N, K, D or V
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be C or F
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be M or Nleu
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉can be S or D
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉can be Y or any hydrophobic amino acid
<400>33
Figure A200680038972D01131
<210>34
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS2-3 protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(2)..(4)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be any aminoacid except that P
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉can be any hydrophobicity or basic amino acid
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉can be any aminoacid except that P
<400>34
Figure A200680038972D01132
<210>35
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS2-3 protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(5)..(6)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be A, V or R
<400>35
<210>36
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Pestivirus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉can be G, N or Q
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉can be S or A
<220>
<221>misc_feature
<222>(6)..(5)
＜223〉can be G, A or HB donor
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉can be N or A
<400>36
Figure A200680038972D01142
<210>37
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis A 3C protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be L or I
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉can be D or E
<400>37
Figure A200680038972D01151
<210>38
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis A 3C protease substrate is total
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉protease cleavage site
<400>38
Figure A200680038972D01152
<210>39
<211>4
<212>PRT
＜213〉protease cleavage site
<220>
＜223〉HRV 3C protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<400>39
Figure A200680038972D01153
<210>40
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉HRV 2A protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be S or T
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any hydrophobic amino acid
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be any hydrophobic amino acid
<400>40
Figure A200680038972D01161
<210>41
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉enterovirus 3C protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<400>41
Figure A200680038972D01162
<210>42
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Coxsackie virus protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(5)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be G or S
<400>42
Figure A200680038972D01163
<210>43
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the Coxsackie virus protease substrate is total
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aromatic amino acid
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be H or Q
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be G or S
<400>43
<210>44
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the ECHO virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be T or N
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉any aminoacid
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉any aromatic amino acid
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be G or S
<400>44
<210>45
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the ECHO virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be T or N
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be Y or H
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be G or S
<400>45
Figure A200680038972D01181
<210>46
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the ECHO virus protease substrate is total
>220>
<221>misc_feature
<222>(1)..(1)
＜223〉Xaa can be any naturally occurring aminoacid
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be G or S
<400>46
Figure A200680038972D01182
<210>47
<211>9
<212>PRT
＜213〉artificial sequence
<220>
<223>
The ECHO virus protease substrate is total
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉can be T or E
<220>
<221>misc_feature
<222>(2)..(2)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(2)..(3)
＜223〉protease cleavage site
<220>
<221>misc_feature
<222>(4)..(4)
＜223〉can be any aminoacid
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉can be G or S
<400>47
<210>48
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉single strand dna oligonucleotide
<400>48
<210>49
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉single strand dna oligonucleotide
<400>49
Figure A200680038972D01193
<210>50
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉single strand dna oligonucleotide
<400>50
Figure A200680038972D01194
<210>51
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉single strand dna oligonucleotide
<400>51
Figure A200680038972D01195
<210>52
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>52
Figure A200680038972D01196
<210>53
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>53
Figure A200680038972D01201
<210>54
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>54
Figure A200680038972D01202
<210>55
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>55
Figure A200680038972D01203
<210>56
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>56
Figure A200680038972D01204
<210>57
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>57
Figure A200680038972D01211
<210>58
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>58
Figure A200680038972D01212
<210>59
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>59
Figure A200680038972D01213
<210>60
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>60
Figure A200680038972D01214
<210>61
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>61
Figure A200680038972D01221
<210>62
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>62
Figure A200680038972D01222
<210>63
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>63
Figure A200680038972D01223
<210>64
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>64
<210>65
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>65
Figure A200680038972D01231
<210>66
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>66
Figure A200680038972D01232
<210>67
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>67
Figure A200680038972D01233
<210>68
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>68
Figure A200680038972D01234
<210>69
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>69
<210>70
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>70
Figure A200680038972D01242
<210>71
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>71
Figure A200680038972D01243
<210>72
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>72
Figure A200680038972D01244
<210>73
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>73
Figure A200680038972D01251
<210>74
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>74
Figure A200680038972D01252
<210>75
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>75
<210>76
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>76
Figure A200680038972D01254
<210>77
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>77
Figure A200680038972D01261
<210>78
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>78
Figure A200680038972D01262
<210>79
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>79
Figure A200680038972D01263
<210>80
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>80
Figure A200680038972D01264
<210>81
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>81
Figure A200680038972D01271
<210>82
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>82
Figure A200680038972D01272
<210>83
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<400>83
Figure A200680038972D01273
<210>84
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>84
Figure A200680038972D01274
<210>85
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>85
<210>86
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>86
<210>87
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>87
Figure A200680038972D01283
<210>88
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>88
Figure A200680038972D01284
<210>89
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>89
Figure A200680038972D01291
<210>90
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>90
Figure A200680038972D01292
<210>91
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>91
Figure A200680038972D01293
<210>92
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>92
Figure A200680038972D01294
<210>93
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>93
<210>94
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>94
Figure A200680038972D01302
<210>95
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>95
Figure A200680038972D01303
<210>96
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>96
Figure A200680038972D01304
<210>97
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>97
Figure A200680038972D01305
<210>98
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>98
Figure A200680038972D01311
<210>99
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>99
<210>100
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>100
Figure A200680038972D01313
<210>101
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>101
Figure A200680038972D01314
<210>102
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>102
<210>103
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>103
<210>104
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>104
Figure A200680038972D01323
<210>105
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>105
Figure A200680038972D01324
<210>106
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>106
Figure A200680038972D01331
<210>107
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>107
Figure A200680038972D01332
<210>108
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>108
Figure A200680038972D01333
<210>109
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>109
Figure A200680038972D01334
<210>110
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>110
Figure A200680038972D01341
<210>111
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>111
Figure A200680038972D01342
<210>112
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>112
Figure A200680038972D01343
<210>113
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>113
Figure A200680038972D01344
<210>114
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>114
Figure A200680038972D01351
<210>115
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>115
Figure A200680038972D01352
<210>116
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>116
<210>117
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>117
Figure A200680038972D01354
<210>118
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>118
Figure A200680038972D01361
<210>119
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>119
Figure A200680038972D01362
<210>120
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>120
Figure A200680038972D01363
<210>121
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>121
Figure A200680038972D01364
<210>122
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>122
Figure A200680038972D01371
<210>123
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>123
<210>124
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<400>124
<210>125
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>125
Figure A200680038972D01374
<210>126
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>126
Figure A200680038972D01381
<210>127
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>127
<210>128
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>128
Figure A200680038972D01383
<210>129
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>129
<210>130
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>130
Figure A200680038972D01391
<210>131
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>131
Figure A200680038972D01392
<210>132
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>132
Figure A200680038972D01393
<210>133
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>133
Figure A200680038972D01394
<210>134
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>134
Figure A200680038972D01401
<210>135
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>135
<210>136
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>136
Figure A200680038972D01403
<210>137
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>137
Figure A200680038972D01404
<210>138
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>138
Figure A200680038972D01411
<210>139
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>139
Figure A200680038972D01412
<210>140
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>140
Figure A200680038972D01413
<210>141
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>141
Figure A200680038972D01414
<210>142
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hepatitis C NS3 protease cutting sequence
<220>
<221>misc_feature
<222>(6)..(7)
＜223〉protease cleavage site
<400>142
Figure A200680038972D01421
<210>143
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV3C protease substrate
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉DABCYL is link coupled
<220>
<221>misc_feature
<222>(11)..(11)
＜223〉EDANS is link coupled
<400>143
Figure A200680038972D01422
<210>144
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV3C protease substrate
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉DABCYL is link coupled
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉EDANS is link coupled
<400>144
Figure A200680038972D01423
<210>145
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV3C protease substrate
<220>
<221>misc_feature
＜223〉the link coupled peptide of C ' pNA
<400>145
Figure A200680038972D01424
<210>146
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV 3C protease substrate
<220>
<221>misc_feature
＜223〉the link coupled peptide of C ' pNA
<400>146
Figure A200680038972D01431
<210>147
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV3C protease substrate
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉DABCYL is link coupled
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉EDANS is link coupled
<400>147
Figure A200680038972D01432
<210>148
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV 3C protease substrate
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉DABCYL is link coupled
<220>
<221>misc_feature
<222>(11)..(11)
＜223〉EDANS is link coupled
<400>148
Figure A200680038972D01433
<210>149
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV 3C protease substrate
<220>
<221>misc_feature
＜223〉the link coupled peptide of C ' pNA
<400>149
Figure A200680038972D01434
<210>150
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV 3C protease substrate
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉DABCYL is link coupled
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉EDANS is link coupled
<220>
<221>MOD_RES
<222>(11)..(11)
＜223〉amidatioon
<400>150
Figure A200680038972D01441
<210>151
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic HRV 3C protease substrate
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉DABCYL is link coupled
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉EDANS is link coupled
<220>
<221>MOD_RES
<222>(11)..(11)
＜223〉amidatioon
<400>151
Figure A200680038972D01442

Claims (70)

1. isolated peptides, this isolated peptides contains the aminoacid sequence that is selected from by SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46 and 47 groups of forming, and the length of described aminoacid sequence is no more than 14 aminoacid.
2. the compositions that contains the substrate of virus protease, but described substrate links to each other with at least one test section and contains the described aminoacid sequence of claim 1.
3. compositions as claimed in claim 2, but wherein said at least one test section is a proenzyme, and the cutting of described substrate activates described proenzyme.
4. compositions as claimed in claim 2, but wherein said at least one test section is that FRET is right, and the cutting of described substrate generation comes from the right signal of described FRET.
5. compositions as claimed in claim 4, wherein said compositions also contains separating part.
6. general formula is the compositions of X-Y-Z,
Wherein:
Y contains the substrate of virus protease, and described substrate contains the described aminoacid sequence of claim 1, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
But X contains the test section; With
Z contain can with the separating part that combines mutually separating of two phase-separated system;
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease.
7. compositions as claimed in claim 6, but wherein said test section X contains the marking agent that is selected from the group of being made up of enzyme, fluorogen, chromophore, albumen, proenzyme, chemiluminescent substance and radiosiotope.
8. as claim 5 or 6 described compositionss, wherein said separating part Z is selected from by immunoconjugator, magnetic knot and closes the group that part, peptide bound fraction, affinity bound fraction, nucleic acid moiety are formed.
9. general formula is the compositions of X-Y-Z,
Wherein:
Y contains the substrate of virus protease, and described substrate contains the described aminoacid sequence of claim 1, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
X or Z contain underlined, but perhaps test section and/or can separate through separating part cutting or uncut compositions in suitable mode;
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease.
10. compositions as claimed in claim 9, wherein said labelling, X or Z partly contain and are selected from the marking agent that, pearl, peptide, proenzyme and radiosiotope, immunoconjugator, magnetic knot is closed the group that part, peptide bound fraction, affinity bound fraction, nucleic acid moiety form by enzyme, fluorogen, chromophore, albumen, chemiluminescent substance, quencher, FRET.
11. the method for at least a virus in the test sample, described method comprises
(a) at least a in sample and claim 2,4,5,6,7,8, the 9 or 10 described compositionss contacted under the condition of the cutting that can carry out described substrate; With
(b) cutting of the described substrate of monitoring, the described cutting of wherein said substrate are the indications that at least a virus exists described in the described sample.
12. method as claimed in claim 11, wherein step (a) comprises described sample is contacted with at least two kinds of substrates of different virus protease, and any described cutting do not occur and then indicates in the described sample and do not have virus in wherein said at least two kinds of substrates.
13. method as claimed in claim 11, wherein said sample are selected from by mucus, saliva, throat cleanout fluid, nose cleanout fluid, spinal fluid, expectorant, urine, seminal fluid, perspiration, feces, blood plasma, blood, bronchovesicular liquid, vaginal secretion, tear and biopsy thing.
14. method as claimed in claim 11, the detection of cleavage activity described in the wherein said sample is the diagnosis to medical condition.
15. method as claimed in claim 11, wherein said monitoring are to adopt homogeneous determination to realize.
16. method as claimed in claim 11, wherein said monitoring are to adopt out-phase to measure to realize.
17. be used for the diagnostic kit of at least a virus of test sample, described test kit contains claim 2,4,5,6,7,8,9 or 10 described at least a compositionss and is used to detect the reagent of the cutting of described substrate.
18. contain packaging material and the diagnostic kit that is used to detect the multiple viral multiple compositions that exists, wherein the general formula of every kind of described compositions is
X-Y-Z
Wherein:
Y contains the substrate of virus protease, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
X or Z contain underlined, but perhaps test section and/or can separate through separating part cutting or uncut compositions in suitable mode,
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease,
But each among wherein said X or the Z comprises at least one special test section, and described packaging material comprise that label or package insert are used for the multiple virus of test sample to indicate described test kit.
19. contain packaging material and the diagnostic kit that is used to detect the multiple viral multiple compositions that exists, wherein the general formula of every kind of described compositions is
X-Y-Z
Wherein:
Y contains the substrate of virus protease, and X-Y-Z forms cleaved products X-Y ' and Y ＂-Z through described virus protease cutting, and wherein Y ' is first cleaved products of Y, and Y ＂ is second cleaved products of Y;
But X contains the test section; With
Z contain can with the separating part that combines mutually separating of two phase-separated system;
Wherein said X-Y-Z does not form the adjacent part of the natural substrate of described virus protease,
Among the wherein said X each is special detectable, and described packaging material comprise that label or package insert are used for the multiple virus of test sample to indicate described test kit.
20. as claim 18 or 19 described diagnostic kits, wherein said multiple compositions links to each other with single solid support.
21. diagnostic kit as claimed in claim 20, wherein said special detection are to locate by the addressable on described single solid support to realize.
22. diagnostic kit as claimed in claim 20, but wherein said special detection is to assign to realize by different test sections.
23. as claim 18,19 or 20 described diagnostic kits, each in the wherein said multiple compositions links to each other with solid support.
24. diagnostic kit as claimed in claim 23, wherein said solid support is shaped as pearl.
25. diagnostic kit as claimed in claim 24, wherein said pearl are selected from by molecular group of coloured pearl, magnetic pearl, label pearl and fluorescent bead.
26. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is to breathe test kit, contains at least two kinds of viruses that are selected from the group of being made up of coronavirus, SARS, HMPV (human stroma lung virus), influenza virus A+B, bird flu virus, adenovirus, RSV (respiratory syncytial virus), rhinovirus, parainfluenza virus.
27. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is the breathing test kit that contains Hantaan and LaCrosse's encephalitis.
28. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is the gastrointestinal test kit, contains at least two kinds of viruses that are selected from the group of being made up of rotavirus, adenovirus 40/41, hepatitis A virus, hepatitis C virus, hepatitis E virus, Calicivirus and CMV (cytomegalovirus).
29. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is the meningitis test kit, contains at least two kinds of viruses that are selected from by enterovirus (1-～80), west nile virus, herpes simplex virus 1,2 and 6 groups of forming.
30. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is the meningitis test kit, contains at least two kinds of viruses that are selected from the group of being made up of togavirus, banzi virus and rabies virus.
31. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is the sexually transmitted disease (STD) test kit, contains at least two kinds of viruses that are selected from the group of being made up of HI1V Strain, herpes simplex virus 1, herpes simplex virus 2, HSV-1, HSV-2, HPV (human papillomavirus) and HTLV-1.
32. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is traveller's test kit, contains at least two kinds of viruses that are selected from the group of being made up of hepatitis A virus, hepatitis B virus, hepatitis C virus, HIV, herpesvirus 1 and 2.
33. as claim 17,18 or 19 described diagnostic kits, described diagnostic kit is veterinary's test kit, contains at least two kinds of viruses that are selected from the group of being made up of rabies virus and canine distemper virus.
34. diagnostic kit as claimed in claim 17, wherein said at least a sample comprises several samples.
35. diagnostic kit as claimed in claim 17, wherein said at least a virus comprises multiple virus.
36. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that adenovirus and described substrate contain SEQ ID NO:1 or 2.
37. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that α virus and described substrate contain SEQ ID NO:3.
38. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that rubella virus and described substrate contain SEQ ID NO:4.
39. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that HIV and described substrate contain SEQ ID NO:5.
40. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that HTLV and described substrate contain SEQ ID NO:6,7 or 8.
41. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that arteritis virus and described substrate contain SEQ ID NO:9.
42. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that coronavirus and described substrate contain SEQ ID NO:10.
43. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that sars coronavirus and described substrate contain SEQ ID NO:11,12,148 or 149.
44. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that Orbivirus and described substrate contain SEQ ID NO:13.
45. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that CMV virus and described substrate contain SEQ ID NO:14 or 15.
46. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that herpesvirus and described substrate contain SEQ ID NO:16.
47. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that banzi virus and described substrate contain SEQ ID NO:17.
48. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that dengue virus and described substrate contain SEQ ID NO:18,19 or 20.
49. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that west nile virus and described substrate contain SEQ ID NO:21,22 or 23.
50. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that yellow fever virus and described substrate contain SEQ ID NO:24,25 or 26.
51. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that Japanese encephalitis virus and described substrate contain SEQ ID NO:27,28 or 29.
52. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that Ticks passes virus and described substrate contains SEQ ID NO:30,31 or 32.
53. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that hepatitis C virus and described substrate contain SEQ ID NO:33,34 or 35.
54. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that Pestivirus and described substrate contain SEQ ID NO:36.
55. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that hepatitis A virus and described substrate contain SEQ ID NO:37 or 38.
56. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that HRV and described substrate contain SEQ ID NO:39 or 40.
57. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that enterovirus and described substrate contain SEQ ID NO:41,42,43,44,45,46 or 47.
58. as claim 11,17,18 or 19 described method or test kits, wherein said virus is that HRV virus and described substrate contain SEQ ID NO:143～147,150～151.
59. a design motivation is learned the method for optimized virus protein zymolyte, described method comprises:
(a) in a plurality of cutting sequences of the polyprotein of at least a Strain of described virus, differentiate demonstrate cutting sequence that the fastest cutting power that cut by described protease learns and
(b) differentiate the total cutting of the family's scope sequence that demonstrates the fastest cutting power, the total cutting of described family scope sequence is used for design motivation and learns optimized described virus protein zymolyte.
60. method as claimed in claim 59, wherein said virus protease are the protease of encoding viral.
61. method as claimed in claim 59, wherein said virus is selected from the group of being made up of DNA viruses and RNA viruses.
62. method as claimed in claim 61, wherein said virus are selected from by tectivirus section, papovaviridae, porcine circovirus section, Parvoviridae and Hepadnaviridae, Cystovirus section, birnavirus section, Reoviridae, coronaviridae, flaviviridae, Togaviridae, arteritis virus, Astroviridae, Caliciviridae, Picornaviridae, marmor upsilon section, Retroviridae, orthomyxovirus section, filamentous virus section, Paramyxoviridae, Rhabdoviridae and Bunyan Viraceae, Adenoviridae, herpetoviridae, the group that Picornaviridae is formed.
63. method as claimed in claim 60, wherein said virus protease is selected from by serine protease, metalloproteases, aspartic protease, cysteine proteinase, 3C protease, the PA transcriptase, adenine protease, 2A protease, chymase or trypsin, NS3 for example, NS2, the NS-pro cysteine proteinase, the nsP2 cysteine proteinase, nsP23pro, C protein enzyme, SFV NS, the HIV aspartic protease, nsp4 arteritis virus protease, HCMV protease, NS2-3, NS3-4Ap protease, the group that HTLV-1PR forms.
64. method as claimed in claim 59, described method is further comprising the steps of:
(c) design has a plurality of cutting sequences of the total cutting of described family scope sequence; With
(d) in described a plurality of cutting sequences, differentiate to have the cutting sequence that the fastest cutting power of described protease is learned.
65. as the described method of claim 64, wherein said design comprises that design has the described cutting sequence of optimal dissolution degree, temperature sensitivity and/or pH sensitivity.
66. method as claimed in claim 59, wherein the described discriminating of step (a) comprises empirical experiment.
67. method as claimed in claim 59, wherein the described discriminating of step (a) comprises data mining.
68. isolated peptides, described isolated peptides contains the aminoacid sequence that is selected from by SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46 and 47 groups of forming, the length of described aminoacid sequence is no more than 14 aminoacid, and contains the active analogies of the corresponding virus protease of inhibition.
69. the peptide of claim 68 is used for the treatment of purposes in the medicine for treating viral infections in preparation through discriminating.
70. contain the pharmaceutical composition of the isolated peptides of claim 68 as active component.
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CN103088007A (en) * 2013-01-28 2013-05-08 厦门大学 Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design
CN104020287A (en) * 2014-06-24 2014-09-03 朱高红 A serum specific activity protease detection nano kit containing fluorescent and/or radionuclide and its preparation method and application
CN105021819A (en) * 2014-04-28 2015-11-04 中国科学院苏州纳米技术与纳米仿生研究所 Fluorescence nanometer magnetic particle-polypeptide substrate complex and preparing method thereof
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CN103088007A (en) * 2013-01-28 2013-05-08 厦门大学 Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design
CN103088007B (en) * 2013-01-28 2015-05-13 厦门大学 Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design
CN105021819A (en) * 2014-04-28 2015-11-04 中国科学院苏州纳米技术与纳米仿生研究所 Fluorescence nanometer magnetic particle-polypeptide substrate complex and preparing method thereof
CN104020287A (en) * 2014-06-24 2014-09-03 朱高红 A serum specific activity protease detection nano kit containing fluorescent and/or radionuclide and its preparation method and application
CN104020287B (en) * 2014-06-24 2015-09-16 朱高红 A kind of blood serum special active protease containing radioactive nuclide detects nanometer kit
CN111225679A (en) * 2017-08-19 2020-06-02 俄亥俄州创新基金会 Novel peptide-based cancer imaging agents
CN111225679B (en) * 2017-08-19 2024-03-29 俄亥俄州创新基金会 Novel peptide-based cancer imaging agents
CN110863065A (en) * 2019-10-21 2020-03-06 珞可为科技(武汉)有限公司 Kit for detecting four enteroviruses and detection method thereof
CN115335532A (en) * 2020-02-09 2022-11-11 Nlc制药有限公司 SARS-COV-2 quick detection test
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US11603552B2 (en) 2020-07-20 2023-03-14 Mesa Photonics, LLC Method for pathogen identification
US11851698B2 (en) 2020-07-20 2023-12-26 Mesa Photonics, LLC Method for pathogen identification
CN113388632A (en) * 2021-06-29 2021-09-14 广西大学 Primer and kit for detecting bovine astrovirus ELISA antibody and application

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