JP3148994B2 - Non-A non-B hepatitis-related nucleic acids, antigens, antibodies and methods for detecting them - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to a novel nucleic acid, protein, antigen, antibody, and a method for detecting non-A, non-B hepatitis using the same .

(Prior Art) Pathogens of non-A non-B hepatitis cause disease states such as acute hepatitis, fulminant hepatitis, chronic hepatitis, liver cirrhosis and liver cancer in humans. The major cause of post-transfusion hepatitis, which occurs in about 10%, is a serious problem. Moreover, the pathogenic viruses of hepatitis A and B have already been elucidated and are subject to medical control, whereas the presence of non-A non-B hepatitis has been estimated for the first time. Despite more than a decade, the pathogen remained elusive.

[0003] In 1988, the United States
Chiron's research group has announced that the gene for this pathogen has been successfully cloned, and has created a virus antibody detection system that has been offered to the world as a specific diagnostic method for non-A, non-B hepatitis. In facilities, blood centers, etc.
Its usefulness is being studied. According to Chiron, the genetic structure indicates that the pathogen is Flavi Vi.
rus, and they named it hepatitis C virus (HCV). The data to date confirm that HCV antibodies by their diagnostic method show a good correlation with the disease status of non-A, non-B hepatitis. However, it has been pointed out that it is not sufficient in terms of specificity and sensitivity, and this antibody is also detected in a considerable part of autoimmune hepatitis and a considerable part of hepatitis B.
Unresolved problems remain, and there is no proof that their claimed HCV is a non-A, non-B hepatitis pathogen.

The present inventors have been enthusiastically investigating this unknown pathogen for a long time, but after 10 years of hardship, they have previously published Japanese Patent Application Nos. 2-28191 and 2-15.
The inventions of Japanese Patent Application No. 3887 and Japanese Patent Application No. 2-335806 have been achieved, but the present invention has been achieved subsequently. According to the specific diagnostic method for non-A, non-B hepatitis using the detection system of the present invention, it is possible to diagnose even patients with this disease that cannot be identified by the diagnostic method of Chiron.

(Problems to be Solved by the Invention) The present invention provides nucleic acids and protein peptides required for diagnosis of non-A, non-B hepatitis, which have not been found so far. The nucleic acid, protein and peptide of the present invention, both in their nucleotide sequence and in their amino acid sequence, relate to the HCV gene of Chiron (European Patent Application 883310922.5, filed on Jan. 1988).
(January 18). The nucleic acids and proteins of the present invention have the above-mentioned utility,
Both substances are new substances provided for the first time by the present invention. Therefore, the scope of right of each substance according to the present invention is not limited by its use.

(Means for Solving the Problems) The present invention relates to a deoxyribonucleic acid GORgabDNA having the nucleotide sequence of SEQ ID NO: 1 and a protein GORgabPROTEIN having the amino acid sequence of SEQ ID NO: 2.
And a ribonucleic acid GOR47-1RNA having the nucleotide sequence of SEQ ID NO: 3, a ribonucleic acid GOR47-1RNAC having the nucleotide sequence of SEQ ID NO: 4, and a deoxyribonucleic acid GOR having the nucleotide sequence of SEQ ID NO: 5
47-1 DNA, a deoxyribonucleic acid GOR47-1DANC having the nucleotide sequence of SEQ ID NO: 6, and a peptide GOR47-1P having an amino acid sequence of SEQ ID NO: 7
ROTEIN, oligopeptide spGOR1 having the amino acid sequence of SEQ ID NO: 8, oligopeptide spGOR2 having the amino acid sequence of SEQ ID NO: 9, spGORa-epi having the amino acid sequence of SEQ ID NO: 10, and oligopeptide having the amino acid sequence of SEQ ID NO: 11 is an invention of spGORb-epi, non-a, non-B hepatitis related nucleic acid detection method to further using the nucleic acid as a probe, the inventor of the non-a, non-B hepatitis related nucleic acid detection method using the nucleic acid as a primer, further A method for detecting a non-A non-B hepatitis-related antibody using the protein and the peptide as an antigen; and a method for detecting a non-A non-B hepatitis-related antibody using the protein, a fusion protein of the peptide and an expression protein as an antigen. detection method, the protein, peptidase The id is an invention of the non-A, non-B hepatitis specific monoclonal or mixed Mab obtained using an animal other than a human as an antigen, also the detection of non-A, non-B hepatitis-associated antigen using the above specific antibody It is an invention of a method .

The detection system of the present invention can be used as an excellent diagnostic agent and diagnostic method for non-A non-B hepatitis.

The present inventors have continued the research described below.
Thus, the present invention was completed. (1) Preparation of animal model of non-A non-B hepatitis The study of the present inventors was started by first preparing an animal model of non-A non-B hepatitis. Various animals were inoculated with the serum of a donor known to have caused hepatitis after transfusion in humans to try to reproduce non-A non-B hepatitis. As a result, only chimpanzees showed disease states similar to humans. In experimental non-A non-B hepatitis induced by chimpanzees, specific ultrastructural changes in the cytoplasm of hepatocytes (the ER-like partial thickening: co
nvolved curved membrane,
Tubular structure: tubular structure, sponge-like structure: sponge-like structure
re, microtubules aggregates: microbula
Therefore, the experiment was carried out using this as a marker for infection, and the passaging of the non-A non-B hepatitis pathogen was repeated from human to chimpanzee and from chimpanzee to chimpanzee.

(2) Raw Material for Isolation of Non-A Non-B Hepatitis-Related Genes Acute hepatitis serum of chimpanzee (C37) affected by inoculation of human serum (# 30017) was further added to five chimpanzees (C41, C43). , C45, C46, C47)
To be infected. From the five affected chimpanzees, the above ultrastructured positive phase pooled plasma was taken, and the non-A of chimpanzees (CH19) affected by the inoculation were collected.
The non-hepatitis B acute phase plasma was used as a raw material for isolation of non-A non-B hepatitis-related genes.

(3) Extraction of Nucleic Acid from Chimpanzee Plasma About 6 non-A non-B hepatitis acute phase plasmas of the chimpanzee (CH19) were collected in a J6 rotor (Beckman) for 3,000 hours.
0 ng for 30 minutes and centrifuged at 4 ° C. for 30,000 ng in a Ti-15 rotor (Beckman).
Centrifuged for 5.3 hours. The precipitate is washed with 120 ml of 50 mM
By resuspending in Tris-Cl (PH7.5) / 5 mM EDTA, a virus fraction approximately 50-fold concentrated was obtained. For 6 ml of this fraction (corresponding to 300 ml of raw plasma), 6 ml of SDS / Proteinase K coc
ktail (400 mM NaCl / 20 mM EDT
A / 4% SDS / 100 mM Tris-HCl buffer (pH 8.0) / Proreinase K 2 mg / m
After l) was added and incubated at 37 ° C. overnight, nucleic acids were extracted with phenol and concentrated by ethanol precipitation.

(4) cDNA synthesis One quarter of the nucleic acid fraction obtained in (3) above (75 m
1)). The reaction was divided into two tubes, one for oligo (dT) ₁₂ and the other for random hexanucleotide as a primer to synthesize a primary cDNA strand. Prior to that, template RNA was heated at 65 ° C for 3 minutes. Heated. Secondary cDN
This series of cDNA synthesis reactions, including A-chain synthesis and blunting of double-stranded DNA ends, were performed using a cDNA synthesis kit (Amersham, UK) based on the method of Gubler.

(5) Preparation of cDNA library The EcoRI breakpoint inside the double-stranded cDNA obtained in (4) is protected by EcoRI methylase, and E.
A EcoRI linker was added, and EcoRI of λgt11 DNA was added.
A series of reactions for insertion into the RI break and assembly with the phage protein to produce a recombinant phage were performed using a λgt11 cloning kit (Amersham, UK). The library size was 1.0 × 10 ⁶ PFU, 4.3 for cDNA-derived library primed with oligo (dT) and one for cDNA derived with random hexanucleotide.
× 10 ⁶ PFU.

(6) Screening of cDNA Library Screening for picking up a non-A non-B hepatitis-related cDNA clone from the cDNA library prepared above was performed by the two-antibody method. That is, Escherichia coli Y1090 was infected with the above-mentioned recombinant phage, and LB-
After seeding on an Agar plate and culturing at 43 ° C. for 3 hours, IP
After infiltrating TG, the filter was put on the upper layer. After culturing at 37 ° C. for 3 hours, the filter was peeled off, washed with a buffer, and the first antibody was added thereto. The primary antibodies include chimpanzee plasma confounded with non-A non-B hepatitis, human plasma that caused non-A non-B hepatitis by needle accident, and non-A non-B
A mixture of plasma of a patient with chronic hepatitis C was incubated at 4 ° C. overnight. After washing with a buffer, a second antibody (anti-human 1gG: peroxidase label) was added to this, and reacted at room temperature for 30 minutes. After washing with buffer, add DA
A mixed solution of B, Ni, Co, and H ₂ O ₂ was added to develop a color. c
In the DNA library, there is a fragment of a non-A non-B hepatitis-related gene, and if it is in-frame λgt
If the fusion with the β-galactosidase ORF of the phage DNA was performed, the phage-infected E. Co
By li, the fusion protein is expressed, and the antigenicity of the non-A, non-B hepatitis-related antibody contained in the first antibody solution should be recognized and bound, and a positive signal should be produced. The inventors' experiments were also confirmed as such. Among a plurality of clones that gave a positive signal by screening, G
There is a clone OR47-1, which is the basis of the present invention.

(7) Determination of base sequence of non-A non-B hepatitis-related cDNA clone (GOR47-1) The GOR47-1 phage obtained by the above (6) is purified, and its DNA is cut with EcoRI to obtain cDNA. Take it out and delete it from Phasescript (Stratagene
(Registered trademark: USA) at the EcoRI site, and the nucleotide sequence was determined by the method of Sanger.
At the same time, the nucleotide sequence of the insert arm junction of GOR47-1 phage DNA was similarly determined using phage DNA-derived primers. As a result, GOR47-1 DNA was converted to λg with the orientation shown in FIG.
It turned out that it was inserted into t11 DNA. FIG. 1 shows the nucleotide sequence of GOR47-1 DNA as λgt.
It is shown together with the base sequence of the junction with 11 DNA. GOR47-1 DNA is in the frame, and the base sequence of λ phage is on the left and right of the frame.

(8) Determination of the primary structure of the protein encoded by the gene of the non-A non-B hepatitis-related cDNA clone From the nucleotide sequence of GOR47-1 obtained in (7) above,
The ORF of GOR47-1 linked in frame to the ORF of β-galactosidase of λgt11 was defined as shown in FIG. Λgt11 used to construct the cDNA library from which GOR47-1 is derived
Phage DNA is a so-called "expression vector"
cDN in the operon of IacZ, the t11 gene
A is incorporated so that a fusion protein of β-galactosidase and a cDNA-derived protein is expressed. Of the six possible reading frames of GOR47-1, only one fuses in-frame between λgt11 and the β-galactosidase ORF, and that frame is shown in FIG. There are no stop codons in this frame. This ORF also corresponds to 55 amino acids. In the present invention, an expression protein such as alkaline phosphatase or superoxide dismutase can be used instead of β-galactosidase.

[0016]

FIG.

(9) cDNA having a sequence overlapping with a non-A non-B hepatitis-related cDNA clone (GOR47-1)
Search for clone GOR47-1 phage D obtained by the above (6)
Insert DNA from NA, ie GOR47-1 DNA
And GOR47-1 DNAC, and GOR4 from the recombinant phage script shown in (7) above.
Create a 7-1RNA and GOR47-1RNAC with _{T 3} and _{T 7} promoters, they were as probe labeled with a radioactive isotope, the result of searching the cDNA library I obtained by (5), GOR47-
A plurality of cDNA clones overlapping with 1 were obtained. GORgab DNA was obtained by determining these nucleotide sequences using the same method as in the above (7) and ligating each other. G
The nucleotide sequence of ORgab DNA was as described in SEQ ID NO: 1. The nucleotide sequence of GOR47-1 DNA is GOR
This was a region extending from base number 1703 to base number 1868 in the base sequence of gabDNA.

(10) Non-A non-B hepatitis-related gene GOR
Protein GORgab encoded by gab DNA
PROTEIN By searching for an ORF containing a sequence overlapping with the ORF of GOR47-1 described in (8), from among the six possible open reading frames (ORFs) encoded by the GORgab DNA obtained in (9) above, GORgabPROTEIN consisting of 661 amino acid residues was obtained. The amino acid sequence of GORgabPROTEIN is as described in SEQ ID NO: 2. The ORF encoded by GOR47-1 was a region extending from the amino acid residues 569 to 623 in the amino acid sequence of GORgabPROTEIN.

(11) Determination of non-A non-B hepatitis-related epitope (GORa) on GORgabPROTEIN To identify the non-A non-B hepatitis-related epitope locus on GORgabPROTEIN obtained in (10) above, a fusion protein and The following experiment was performed using the synthetic peptide.

(11-1) Non-A non-B hepatitis-related cDNA
Preparation of fusion protein of clone GOR47-1 and λgt11β-galactosidase Lysogenic infection of Escherichia coli Y1089 with the non-A non-B hepatitis-related cDNA clone (GOR47-1) obtained in the above (6), Lysogen was prepared (for details of lysogen preparation, see “Constructing and
Screening cDNA Libraries
in λgt11 ”Thanh V. Huyuh et al.
DNA Cloning Vol. l, a practice
icalapproach, ed. by D. M. Cl
over, 949-78, IRL Press, Oxf
ord, 1985. After culturing this lysogen and inducing protein expression with IPTG, the cells were disrupted by freeze-thawing and sonication to obtain a lysate, which was then subjected to β-galactosidase antibody solid phase affinity chromatography (Sepharose 4B Immo).
builtIgG Fraction Rabbi
anti-β-glactosidase: Cap
pel U.S. S. The fusion protein was prepared according to A).

[0021]

(11-3) Epitope mapping by enzyme-labeled antibody method (EIA) and spGORa-epi As described in detail in Examples of the present invention,
Antibodies against the fusion protein and the synthetic peptide obtained in (11-1) and (11-2) were detected. As a result, as shown in FIG. 2, the fusion protein (βGal / GO
R) and spGOR1 and spGOR2 shared the same antigenicity, whereas spGOR3 did not. Thus, the antigen epitope is the following orcopeptide spGORa-ep shared by the former three
i (SEQ ID NO: 10). Gly Arg Arg Gly Gln Lys Ala Lys Ser Asn Pro Asn Arg Pro Leu The present inventors have proposed the oligopeptide spGORa-e.
pi was synthesized.

[0023]

FIG. 2

(12) Another epitope locus on GORgabPROTEIN and spGORb-epi GORgabPROTEIN obtained in the above (10) includes:
Two regions with the highest hydrophilicity were found. One of them was spGORa-epi obtained in the above (11-3). Oligopeptide, another hydrophilic region, was synthesized in the same manner as in (11-3), and the reactivity with the antibody was examined. As shown in the Examples, as shown in the Examples, this peptide identified an epitope associated with non-A non-B hepatitis. We were able to confirm that we had. This oligopeptide spGOR
The amino acid sequence of b-epi (SEQ ID NO: 11) was as follows. Val Ala Lys Gln His Val Val Arg Asp Gly Arg Lys Asp Ser Leu Asp Gly Phe Val (Tyr) In addition, (Tyr) at the C-terminus is added for the purpose of peptide modification, and the remaining 18 are amino acids other than the above. The group belongs to GORgabPROTEIN from 346 to 363.

(13) Preparation of Specific Antibody Against Non-A Non-B Hepatitis-Related Antigen GORgabPROTEIN, GOR47-1 of the Present Invention
PROTEIN, spGOR1, spGOR2 and non-A non-B determined by (11) and (12) above
Synthetic peptide spGOR carrying hepatitis B antigen epitope
When a-epi and spGORb-epi were inoculated into mice, guinea pigs, rabbits, goats, horses, etc., monoclonal and mixed clonal specific antibodies could be obtained.

(14) Preparation of an antigen detection system using a specific antibody against a non-A non-B hepatitis-related antigen The non-A non-B hepatitis is prepared by labeling the specific antibody obtained in the above (13) with FITC to form a probe. By staining tissue sections such as livers of affected patients, the intra- and intracellular localization of non-A, non-B hepatitis-specific antigen was examined. On the other hand, by labeling the specific antibody with peroxidase, biotin, or the like, a detection system for non-A, non-B hepatitis-related antigen in serum or plasma of a patient could be prepared. This was based on a sandwich method in which an unlabeled specific antibody was immobilized on a polystyrene microplate or beads, and a test sample was added thereto, followed by reaction with the labeled specific antibody.

(15) Polymer chain
Detection of Non-A Non-B Hepatitis-Related Genes by Reaction (hereinafter PCR) Method According to the method described in the above (3), a virus fraction was obtained from the test plasma or serum by ultracentrifugation, and this was subjected to SDS /
After treatment with Proteinase K cocktail, nucleic acids were extracted with phenol and subjected to PCR.
Primers used in the PCR method were oligonucleotides consisting of 20 bases located at both ends of the non-A non-B hepatitis-related gene cDNA GOR47-1 shown in FIG.
G1 and G2. G1 corresponds to the 5'-end 20 bases of the GOR47-1 DNA and is the 5'-most end on the sense strand, and G2 corresponds to the 5'-end 20 bases of the GOR47-1 DNAC and the 5'-end also on the antisense strand. It is in. As a result, the presence of a PCR product having the same length of 166 bases as GOR47-1 was confirmed in chimpanzee and human plasma in which non-A non-B hepatitis is presumed.

[0028]

FIG. 3

(Examples) Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited to the examples.

Example 1 Deoxyribonucleic acid GOR47-
1 DNA and GORgab DNA The present inventors first determined the nucleotide sequence of a non-A non-B hepatitis-related cDNA clone (GOR47-1 DNA) as follows. That is, the GO obtained by the above (6)
The R47-1 phage was purified and its DNA was EcoRI
And cDNA is taken out, and the
(Stratagene trademark: USA) EcoRI
It was subcloned to the site, and the nucleotide sequence was determined by the method of Sanger. GOR47-1 phage D
The base sequence at the junction of the insert arm of NA
It was determined similarly using primers derived from NA.
As a result, the GOR with the orientation shown in FIG.
It became clear that 47-1 DNA was inserted into λgt11 DNA,
To insert DNA, ie, GOR47-1 DNA
Was cut out. FIG. 1 shows the nucleotide sequence of GOR47-1 DNA together with the nucleotide sequence of the junction with λgt11 DNA. GOR47-1 is in the frame, and the base sequence of the λ phage is on the left and right of the frame. GOR47-1 DNA has the nucleotide sequence of SEQ ID NO: 5. Similarly, GOR47-1D
GOR47-1 DNAC having a complementary strand to NA was excised from GOR47-1 phage DNA. GOR
47-1 DNAC has the nucleotide sequence of SEQ ID NO: 6. Next, GOR was obtained from the above phage script recombinant.
The 47-1RNA and GOR47-1RNAC was created using the _{T 3} and _{T 7} promoter. These have the nucleotide sequences of SEQ ID NOs: 3 and 4. These were labeled with radioisotopes to form probes, and the cDNA library obtained by the above (5) was searched.
GORgab DNA, a cDNA clone overlapping and longer than R47-1, was obtained. GORgabDNA
Was determined in the same manner as described above. GORga
The nucleotide sequence of bDNA was as described in SEQ ID NO: 1.

Example 2 Protein GORgabPROTE
The protein GORgabPROTEIN encoded thereby was obtained from IN GORgab DNA. GORgab
The λgt11 phage DNA used to construct the cDNA library from which the DNA is derived is a so-called “expression vector”, which is a λgt11 gene, Iac
The cDNA is incorporated into the Z operon, and a mechanism for expressing a fusion protein of β-galactosidase and a cDNA-derived protein is provided. Of the possible reading frames of GORgab DNA, only one is λg
It fuses in frame with the β-galactosidase ORF at t11. The amino acid sequence of GORgabPROTEIN was as described in SEQ ID NO: 2. GORgab
In order to obtain PROTEIN, an expression protein such as alkaline phosphatase or superoxide dismutase can be used instead of β-galactosidase.

Example 3 Non-A Non-B Hepatitis-Associated Antigen Epitope spGORa-e Using Synthetic Oligopeptides Based on the Amino Acid Sequence Encoded by GORgab DNA
pi and spGORb-epi GORgab ORFs were prepared to produce synthetic peptides of various lengths and regions, and their reactivity with non-A non-B hepatitis-related antibodies was examined. Non-A non-B hepatitis antigen epitope mapping Was done. The peptide synthesized based on the Merrifield solid phase method was immobilized on a polystyrene microplate, a test serum was added as a first antibody, and a peroxidase-labeled anti-human immunoglobulin IgG or IgM was added as a second antibody to cause a color reaction. . As a result, spGORa-epi and sp
GORb-epi was identified. spGORa-epi
Has the amino acid sequence of SEQ ID NO: 10, and
GORb-epi has the amino acid sequence of SEQ ID NO: 11.

Example 4 Detection System of Non-A Non-B Hepatitis-Related Antibody Using Protein and Peptide of the Present Invention as Antigen Non-A Non-B Hepatitis-Specific Antibody Detection System Using Protein GORgabPROTEIN of the Present Invention and Peptide as Antigen Example of the usefulness of the enzyme ( enzyme antibody method: EIA
Method) . The protein GORgabPROTEI of the present invention
N and the peptide in Tris-HCl buffer (10 m
M, pH 7.5) and adjusted to a concentration of 5 μg / ml, and 50 μm was injected into a well of a Costar vinyl plate (Toyobo) and allowed to stand at room temperature overnight (solid phase of peptide). Well washing (0.1% Twe
en20, 150 mM NaCl;
0 μm blocking solution (0.1% Tween2
(0.150 mM NaCl, 30% fetal bovine serum) was injected into the wells and left at 4 ° C. overnight. After washing the wells, a test serum or plasma previously diluted 30-fold with the above blocking solution was injected into a 50 μm well and shaken at room temperature for 30 minutes. After washing the wells, 50 μl of peroxidase-labeled anti-human IgG or IgM mouse monoclonal antibody solution
m was injected into the well and allowed to react at room temperature with shaking for 30 minutes, then the well was washed, a peroxidase substrate solution was injected to cause a color reaction, and the absorbance at a wavelength of 492 m was measured.

(1) When 100 cases of chronic liver disease (hepatitis B-related and non-A non-B hepatitis-related mixed) were assayed by the EIA method, they were detected by the EIA method using the protein or peptide of the present invention as an antigen. The protein and peptide of the present invention have a high antibody detection rate in chronic hepatitis of non-A non-B hepatitis, cirrhosis, and liver cancer, while in hepatitis B, hepatitis, cirrhosis, and liver cancer, respectively. Low, and low in lupoid hepatitis and primary biliary cirrhosis, where autoimmune mechanisms are supposed, and low in normal individuals, whereby the protein and peptide of the present invention can detect non-A non-B hepatitis-related antibodies. <br/> be needed use useful method was confirmed.

(2) G in normal people and various chronic diseases
ORa antibody: EIA method using spGOR2 As shown in FIG. 4, the detection rate of the GORa antibody detected by the EIA method using spGOR2 is non-A non-B
Of chronic hepatitis, cirrhosis and liver cancer
%, 56% and 56%, respectively, whereas 2%, 7% and 0% in chronic hepatitis B, cirrhosis and liver cancer, respectively.
0%, low in 0%, 0% in lupoid hepatitis and primary hepatic cirrhosis, which are assumed to have a self-immune mechanism, and 2 out of 200 (1%) in normal subjects
It was only. This demonstrated a close relationship with non-A, non-B hepatitis.

[0036]

FIG. 4

(3) G in patients with acute non-A non-B hepatitis
Time-dependent change of the ORa antibody As described above, the positive rate of the GORa antibody in non-B type chronic liver disease (chronic hepatitis, cirrhosis, liver cancer) is high. Appeared very early. Fig. 5 shows a nursing patient who developed acute hepatitis at a later date due to an accident (needle accident) of accidentally inserting a syringe needle under his / her own skin during blood sampling of a non-A non-B chronic hepatitis patient. It is an example of a woman. In this example, the GORa antibody is I
Both the gG class and the IgM class were already positive on day 49 of the needle accident and detected. In contrast, the anti-HCV antibody (by Ortho kit) was positively converted only on day 131. GORa antibodies proved to be useful for early detection. GOR47-1PROTEI
N, spGOR1, spGOR2, spGORa-ep
The antibodies derived from i were collectively referred to as GORa antibodies.

[0038]

FIG. 5

[0039]

[Table 4]

Example 5 Non-A non-B hepatitis epitope spGORa-ep of the present invention
An example of a method for detecting a non-A, non-B hepatitis-specific antibody using a fusion protein containing i as an antigen will be described. In the experiment, GOR47-1ORFλgt1 of the present invention was used.
Although a fusion protein of 1β-galactosidase was used, in the present invention, an expression protein such as alkaline phosphatase or superoxide dismutase may be used instead of β-galactosidase. How to detect
The Western blot method was used as the method, and the method is as follows. That is, the transferred nitrocellulose membrane was washed with water for 10 minutes, air-dried, cut into 3.5 mm widths, and cut with a TBS (50 m) containing 2% Skim milk.
MTris-HCl buffer pH 7.4, 150 mM N
aCl) for 1 hour at room temperature to 4 ° C. overnight (blocking), followed by TBST (Tween 200. aCl).
(TBS containing 0.5% TBS), and dilute the test serum or plasma in the first antibody solution diluted 30-fold with 3% BSA. After washing, the plate was washed with TBST, and then washed with biotin-labeled anti-human IgG or anti-human IgM (Vectas).
tain ABCkit, Vector U.S.A. S. A)
After washing with TBST for 30 minutes at room temperature (second-time antigen-antibody reaction) and then washing with TBST, this was mixed with peroxidase-labeled biotin and avidin (same as above).
(Biotin-Azidine reaction), and then washed with TBST. Finally, a color developing solution was added to the mixture to check for the presence of a reaction product. Coloring liquid is 50m
1 of 50 NaPO ₄ pH 7.4 with DAB (3,
3'-Diaminobenzidine tetra
hydrochloride: Sigma, U.S.A. S.
A) Add 25 mg, dissolve and add 1 ml of 5% C
_{oCl 3 -6H 2 0.5% (NH} 4) 2 Ni (SO 4)
6H 2 O was added and finally 50 μm of 30% H ₂ O ₂ was added to this to make it. About 120K for antibody positive
The Dalton band stained dark blue.

The following was shown by the above detection method . (1) Frequency of appearance of non-A non-B hepatitis-related antibody (spGORa) in patients with chronic liver disease 100 cases of chronic liver disease (40 cases derived from hepatitis B, 60 cases of non-B type)
The GORa antibody was assayed by Western blot for Example). The results are shown in Tables 1 and 2, and FIG. As shown in Tables 1 and 2, the GORa antibody was positive in 52 out of 100 cases, but was higher in non-B than in hepatitis B cases (10%).
Hepatitis hepatitis cases (80%) were highly positive. Particularly in non-B chronic hepatitis, 27 out of 30 cases (90 cases)
%), The GORa antibody was positive. Looking at this in comparison with Chiron's anti-HCV antibody, the GORa antibody appears less frequently than the anti-HCV antibody for hepatitis B (10%, 13%) and the opposite for non-A non-B hepatitis ( 80%, 68%). In addition, as shown in FIG. 6, there were 14 non-hepatitis B cases that could be diagnosed with the GORa antibody but could not be diagnosed with the anti-HCV antibody. On the other hand, there are only six reverse cases, and GORa
This shows that the specificity of the antibody is excellent.

[0042]

FIG. 6

[0043]

[Table 1]

[0044]

[Table 2]

[0045]

[Table 5]

(2) G in blood donors with abnormal liver function
Positive Rate of ORa Antibody Positive rates of GORa antibody and anti-HCV antibody in 41 randomly selected blood donors (ALT value 80 or higher, non-B) were 20% and 17%, respectively, as shown in Table 3. . GO
Ra and anti-HCV antibody were both positive in 5 cases.
The serum ALT value is a marker for liver function and, of course, an abnormal value (high value) in viral hepatitis. However, it is caused by liver damage due to other causes, such as alcohol, drugs, and obesity. It is high even when liver function is abnormal. The 41-donor population is "subjectively" healthy and "objectively" abnormal in ALT levels, but the cause of liver dysfunction is being investigated further. This is different from the population (chronic hepatitis, cirrhosis, liver cancer group) in the above item (1). GOR
It is considered that the fact that the positive rate of the antibody a is significantly different between the above population and this population is related to this point.

[0047]

[Table 3]

(3) Comparison with GORa Antibody Detection Method Using Synthetic Peptide The GORa antibody was determined by Western method and EI for 100 cases of chronic liver disease (hepatitis B-related and non-A non-B hepatitis-related).
When assayed by Method A, as shown in Table 4, EIA
Most of the specimens were positive for the Western method and negative for the EIA method and negative for the Western method.
There were 11 samples which were positive or negative by the Western method or false positive by the Western method. This indicates that the EIA method detects the GORa antibody with higher sensitivity than the Western method.

Example 6 An antibody detection system using the synthetic peptide spGORb-epi containing the non-A non-B hepatitis-related epitope (GORb) of the present invention as an antigen was prepared in the same manner as in Example 1. . 290 healthy subjects, 36 cases of type B liver disease, non-A
The GORb antibody positive rate for 54 non-hepatitis B diseases was
They were 3% (8 cases), 3% (1 case) and 31% (17 cases), respectively (FIG. 7, cut-off value A ₄₉₂ = 0.4), and spG
ORb-epi was shown to be effective as a detection system for non-A non-B liver disease.

[0050]

FIG. 7

Example 7 Preparation of Monoclonal and Mixed-Clone Antibodies GORgabPROTEIN and each of the synthetic peptides bearing the non-A, non-B hepatitis antigen epitope shown in the above Examples were inoculated into mice, guinea pigs, rabbits, goats, horses and the like. Clonal and mixed clonal specific antibodies were obtained.

[0052] Example 8 non-A, non-B hepatitis related antigen detection how the examples obtained specific antibodies labeled with FITC probe and without using a specific antibody to the antigen, non-A, non-B hepatitis patients suffering By staining a tissue section of the liver or the like, the localization of the non-A, non-B hepatitis-specific antigen in the tissue and in the cell was examined. On the other hand, a detection system for non-A, non-B hepatitis-related antigen in the serum or plasma of a patient was prepared by labeling a specific antibody with peroxidase or biotin. That is, a non-labeled specific antibody was immobilized on a polystyrene microplate or beads, a test sample was added thereto, and then a labeling specific action was added thereto for reaction. As a result, Rukoto used usefully confirmed the detection method of the monoclonal antibody and mixed clones antibodies non-A, non-B hepatitis-associated antigen of the present invention.

Example 9 According to the method described in the above (3), a virus fraction was obtained from a test plasma or serum by ultracentrifugation, and this was subjected to SDS /
After treatment with Proteinase K cocktail, nucleic acids were extracted with phenol and subjected to PCR.
Primers used in the PCR method were oligonucleotides consisting of 20 bases located at both ends of the non-A non-B hepatitis-related gene cDNA GOR47-1 shown in FIG.
G1 and G2. As a first step reaction of the PCR method, first, a primer G2 (ant
i Sense), and a reverse transcriptase was added together with the four nucleotides to cause a reaction. After the primary cDNA synthesis, primer G1 was added thereto, and then Cetus (U.S.A.) Taqpolymerera
The PCR reaction was carried out for 36 cycles according to a conventional method using the automatic temperature control device and s. The reaction product (DNA) was subjected to agarose gel electrophoresis and the expected length (GOR4
The presence or absence of a DNA fragment (same 166 pace as 7-1) (marked with * in FIG. 8) was checked. As shown in FIG.
The chimpanzee (CH19) plasma from which the hepatitis C gene clone GOR47-1 is derived clearly has 166
The base PCR product was present (FIG. 8, lane 1). Also, 10 ⁷ CIU / ml (Chippanzeefefec)
A similar band was obtained from the plasma of another chimpanzee (CH413) known to have an infectious titer of (twin units / ml) (lane 2). On the other hand, in the case of human plasma (TS) having an infectious titer of 1/10 ⁶ , that is, 10 CIU / ml, although a band of the same length is present, the amount is 10 ⁷ CIU / ml. m
1/10 compared to that of 1 chimpanzee.
(Lane 3) A molecular weight marker was flowed in the leftmost lane in the figure.

[0054]

FIG. 8

(Effect) The antigen and specific antibody comprising the protein and the peptide of the present invention have a more rapid and broader detection activity than any conventional diagnostic agent as a specific diagnostic agent for non-A non-B hepatitis. Further, it can be effectively used in blood centers, blood product manufacturing institutions, hospital transfusion departments, and the like for the purpose of excluding the blood of non-A, non-B hepatitis pathogen carrier from blood for blood transfusion or blood products. This can achieve complete prevention of post-transfusion hepatitis, which has been a serious problem for many years. In addition, the nucleic acid, protein and peptide of the present invention are industrially produced as important reagents for virological and serological analysis of non-A and non-B hepatitis pathogens and pathological analysis of pathological conditions resulting therefrom. As well as
It is an indispensable raw material for producing the antigen / antibody.

[0056]

[Arrangement table]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of a recombinant λgt11 phage DNA that includes GOR47-1 DNA at the EcoRI breakpoint (GOR47-1 DNA in a frame, λgt11DN in left and right of the frame).
A). ORF and amino acid sequence of the fusion protein produced by the GOR47-1 · λgt11 recombinant (GO
R47-1 PROTEIN ORF and amino acid sequence) are also shown.

FIG. 2 shows the reactivity of GOR47-1 to a partial peptide on the ORF measured by the EIA method. ○ is non-A non-B
Hepatitis B, ●: Hepatitis B specimen. In the figure, βGal / GOR
Are fusion proteins of β-galactosidase and GOR47-1 ORF, spGOR1, spGOR2, spGOR
3 is a synthetic peptide having a sequence listed or a sequence described in the specification.

FIG. 3 shows primers G ₁ and G ₂ used in the PCR method.

FIG. 4 is a histogram of measured values of GORa antibody by EIA using spGOR2 as an antigen in normal humans and chronic liver diseases, and the vertical axis indicates frequency. Cutoff value is A
bs ₄₉₂ : 0.4, indicated by a dotted line. The positive rate of each test serogroup is as follows. (1) Normal person: 1%,
(2) non-A non-B hepatitis: 76%; (3) chronic hepatitis B:
2%, (4) non-A non-B cirrhosis: 56%, (6) B-type cirrhosis: 7%, (6) non-A non-B liver cancer: 56%, (7) B
Hepatitis C: 0%, (8) Lupoid hepatitis: 0%, (9) Primary hepatic cirrhosis: 0%

FIG. 5 shows a time-dependent change in the GORa antibody in acute non-A non-B hepatitis measured by an EIA method using spGOR2. ：: IgG class GORa antibody, □: IgM class NANB-GOR antibody, ● Anti-HC using Otho kit
V antibody, black: serum ALT value

FIG. 6 is a diagram illustrating the distribution of the positivity of GORa antibodies in type B and non-type B chronic liver diseases (for comparison, HC Chiron HC
V antibody positivity is indicated by *).

FIG. 7 shows detection of an antibody against a GORb epitope using spGORb-epi in a healthy subject (1) liver disease B (2)
Comparison between three groups of non-A non-B liver disease (3). The vertical axis is the number of cases. The horizontal axis is absorbance.

FIG. 8 is an agarose gel electrophoresis diagram of a PCR product.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 33/576 G01N 33/576 Z 33/577 33/577 B // C12P 21/02 C12P 21/02 C 21/08 21 / 08 (C12P 21/02 C12R 1:19) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/11-15/62 C07K 14/00-14/825 BIOSIS (DIALOG) WPI (DIALOG) ) GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq

Claims (17)

(57) [Claims] 1. A deoxyribonucleic acid GORgab DNA having the nucleotide sequence of SEQ ID NO: 1. 2. A protein GORgabPROTEIN having the amino acid sequence of SEQ ID NO: 2. 3. A ribonucleic acid GOR47-1 RNA having the nucleotide sequence of SEQ ID NO: 3. A ribonucleic acid GOR47-1RNAC having the nucleotide sequence of SEQ ID NO: 4. 5. Deoxyribonucleic acid GOR47-1 DNA having the nucleotide sequence of SEQ ID NO: 5. 6. A deoxyribonucleic acid GOR47-1DANC having the nucleotide sequence of SEQ ID NO: 6. 7. A peptide GOR47-1PROTEIN having the amino acid sequence of SEQ ID NO: 7. 8. An oligopeptide spGOR1 having the amino acid sequence of SEQ ID NO: 8. 9. An oligopeptide spGOR2 having the amino acid sequence of SEQ ID NO: 9. 10. An oligopeptide spGORa-epi having the amino acid sequence of SEQ ID NO: 10. An oligopeptide spGORb-epi having the amino acid sequence of SEQ ID NO: 11. 12. A method for detecting a non-A non-B hepatitis-related nucleic acid, wherein the nucleic acid according to claim 1 or 3 to 6 is used as a probe. 13. A method for detecting a non-A non-B hepatitis-related nucleic acid, wherein the nucleic acid according to claim 1 or 3 to 6 is used as a primer. 14. A second or seventh to eleventh aspect.
A method for detecting a non-A, non-B hepatitis-related antibody using the protein or peptide described in the above section as an antigen. 15. The second or seventh to eleventh aspects of the present invention.
A method for detecting a non-A non-B hepatitis-related antibody, wherein the fusion protein of the protein or the peptide and the expression protein is used as an antigen. 16. The second or seventh to eleventh aspects of the present invention.
A non-A non-B hepatitis-specific monoclonal or mixed-clonal antibody obtained by using the protein or peptide described in the above item as an antigen for animals other than humans . 17. A method for detecting a non-A, non-B hepatitis-related antigen using the specific antibody according to claim 16.