CN101221185B - Biologic sensor for detecting human beta interferon and its special polypeptide - Google Patents
Biologic sensor for detecting human beta interferon and its special polypeptide Download PDFInfo
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Abstract
The present invention discloses a biological sensor for detecting the human interferon Beta and the dedicated polypeptide thereof. The amino acid sequence of the polypeptide provided by the invention is SEQ N1, for example. The biological sensor for detecting the human interferon Beta of the invention includes an identifying element and a transducer, wherein, the identifying element is the polypeptide which at least contains the SEQ N1 amino acid sequence or the derivative thereof. The invention designs and synthesizes an antisense peptide which has the specific interaction with the human interferon Beta, and a biological sensor is prepared by using the antisense peptide, thus realizing the detection of the human interferon Beta. The biological sensor of the present invention shows the obvious special combination of the human interferon Beta and has good selectivity; the signal response and the sample size of the human interferon Beta show a linear relation, the reproducibility is good, and the invention can be used for the quantitative detection of human interferon Beta.
Description
Technical field
The present invention relates to a kind of biology sensor and special polypeptide thereof that detects human beta interferon.
Background technology
Human beta interferon (hIFN-β) is that a kind of molecular weight of being made up of 166 amino acid is the glycoprotein of 20kD, is induced by virus and bifilar RNA mainly in the body that secretion produces behind the fibroblast, also can be by protokaryon or the generation of eukaryotic culture expression.As a kind of Bio-engineering Products, hIFN-β belongs to I type interferon, has antiviral, the antitumor and immunoregulation effect of wide spectrum, is mainly used in the treatment of multiple sclerosis, hepatitis B, hepatitis C and kinds of tumors clinically.At present, the method for detection human beta interferon mainly contains the antiviral activity detection method, ultraviolet spectrophotometry, isoelectric focussing, SDS-PAGE, HPLC method [Liu Changnuan, Zhang Yi, Rao Chunming, Lin Jianwei, Gao Kai, Zhang Mingfang, Wang Junzhi: recombinant human interferon alpha 2-β Products Quality research on standard, China's tumor biotherapy magazine, 2003,7 (3), 212-215 ］ and enzyme-linked immunosorbent assay (ELISA) ［ Staehelin, T.,, C., Hobbs, D.S., andPestka, S. ＂ A Rapid Quantitative Assay of High Sensitivity for Human Leukocyte Interferonwith Monoclonal Antibodies ＂ in Methods in Enzymology, Vol.79 (S.Pestka, ed.), Academic Press, New York, 1981,589-595.] etc.
Biology sensor system is according to biological chemistry and electrochemical principle, the signal of biochemical reaction is converted into light or electric signal and by processing, the processing of light, electric signal being monitored or measured biochemical reaction or participating in the kind of material of biochemical reaction or the device of concentration.Biology sensor generally is made of recognition component and transducer.Recognition component is used to discern the signal with the biochemical reaction of perception measured target and generation thereof, is the basis that biology sensor carries out selective determination; Transducer is then carried out the function of conversion of signals.At present, the comparatively ripe biology sensor of development mainly comprises surface plasma body resonant vibration (SPR), QCM (Quartz Crystal Microbalance) (QCM) and electrochemical sensor etc.The change of QCM quartz crystal oscillation frequency and the change of SPR resonance angle or frequency shifts are proportional to the mass change of adsorbed material.Based on the biology sensor of QCM and SPR have that equipment is simple, easy to operate, sample need not mark etc. characteristics, both can carry out concentration determination, can carry out dynamic, real-time transactional analysis again, be widely used in the analysis and the mensuration of various biological system in recent years.
Common immunosensor system makes up based on the specific recognition effect between antigen and antibody molecule, has good biospecificity and very high detection sensitivity.But sensing element is antigen or the antibody molecule that is fixed on the sensing chip surface, and most protein such as antigen or antibody are very responsive to environment, and its immobilization process and preservation condition subsequently cause its bioactive decline and even sex change inactivation probably.In addition, most protein such as antigen or Antibody Preparation are very difficult, and cost an arm and a leg.Therefore, development has higher affinity and than Johnson ﹠ Johnson's thing selectivity, but the micromolecule recognition component of stable performance, convenient sources is a new research focus of immunosensor.
Summary of the invention
The purpose of this invention is to provide a kind of biology sensor and special polypeptide thereof that detects human beta interferon.
The biology sensor of detection human beta interferon of the present invention comprises recognition component and transducer, and wherein, recognition component is for containing the polypeptide or derivatives thereof of SEQ № 1 amino acid sequence at least.
In the present invention, the amino acid residue in the polypeptide be for can being L-type, also D-type, or L-, D-type mixed type; And the amino end group of polypeptide, carboxyl end group or its side-chain radical can be the original shapes of this group, or its ether type, ester type, acidamide type derivative.Preferably, recognition component is the polypeptide of sequence such as SEQ № 2 or SEQ № 3 amino acid sequences.In biology sensor of the present invention, the charge capacity of recognition component on transducer is 2.0 * 10
-12-5.0 * 10
-12Mol/mm
2
In above-mentioned biology sensor, transducer commonly used is gold-plated quartz wafer.Connexon with formula I structure between recognition component and the quartz wafer is connected:
The preparation method of biology sensor of the present invention comprises the steps:
1) substrate is modified: by 1, the 2-dithioglycol makes sulfydryl on the electrode surface band in the self assembly of wafer gold electrode surfaces; In weak basic condition order sulfydryl and bis-epoxy 1, the reaction of 4-butanediol diglycidyl ether, the wafer that obtains modifying;
2) polypeptide solution of connection recognition component: SEQ № 2 or SEQ № 3 amino acid sequences joins on the wafer of modification, by the reaction of epoxide group on amino and the wafer, connects recognition component to wafer, obtains biology sensor.
Preferably, step 1) is carried out according to the following procedure:
After two-sided gold-plated quartz wafer surface clean, immerse in the ethanolic solution of dithioglycol, immerse 1 again, the Na of 4-butanediol diglycidyl ether and pH9.5
2CO
3In the mixed solution that damping fluid is formed, the water flushing of reaction back, the wafer that obtains modifying.
Step 2) carry out according to the following procedure:
The polypeptide solution of SEQ № 2 or SEQ № 3 amino acid sequences is dripped in the chip electrode surface of having modified, after the reaction wafer is immersed the remaining epoxy radicals of sealing in the monoethanolamine HCl aqueous solution, obtain described biology sensor.
In the preparation process,, before and after connecting, all measure its resonant frequency, according to the amount of Sauerbray Equation for Calculating bound substances in order to control the charge capacity of polypeptide.For the quartz wafer of 10MHz, the mass loading of 1 μ g produces the frequency shift of 457Hz approximately.The charge capacity of general may command polypeptide is 2.0-5.0 * 10
-12Mol/mm
2
This biology sensor and human beta interferon have the specific adsorption effect, can be installed in the QCM (Quartz Crystal Microbalance) (QCM) to be used to detect human beta interferon; Or prepare sensing chip with identical method of attachment and be used for surface plasma body resonant vibration instrument (SPR) and detect human beta interferon.
The polypeptide of amino acid sequence provided by the present invention such as SEQ № 1 also belongs to protection scope of the present invention.
The present invention design, synthesize with human beta interferon and have the interactional antisense peptide of specificity, and be prepared as biology sensor, and then realize detection human beta interferon.Biology sensor of the present invention all demonstrates tangible selectivity combination to human beta interferon, has good selectivity; The sample size of signal response and human beta interferon is linear, and favorable reproducibility can be used for the detection by quantitative of human beta interferon.
Description of drawings
Fig. 1 is three kinds of gold electrode surfaces structural representations of modifying wafer;
Fig. 2 is the non-specific adsorption of different proteins on blank wafer;
Fig. 3 is the interaction that different proteins and antisense peptide are modified wafer;
Fig. 4 is that human beta interferon is three kinds of combinations and regeneration sensing curve of modifying on the wafer;
Fig. 5 is the sensing curve of human beta interferon on immobilization AS-IFN1 wafer of variable concentrations;
Fig. 6 is the sensing curve of human beta interferon on immobilization AS-IFN3 wafer of variable concentrations.
Embodiment
Biologically, genetic code has degeneracy, that is to say that in most of the cases an amino acid may have two or more codon, this just means that the some amino acid residues on the just peptide chain might match mutually with a different set of antisense amino acid residue.Therefore, given corresponding many antisense peptides of just peptide possibility, and have different interactions, we are referred to as this phenomenon the degeneracy of antisense peptide.Based on this, at the leucine residue on the polypeptide fragment of human beta interferon 1-14 position, two degeneracy antisense peptides of this polypeptide have been designed, respectively called after AS-IFN1 and AS-IFN3 (table 1).Investigated they and human beta interferon 1-14 position polypeptide, interactional intensity between the promptly just peptide, AS-IFN1 compares with the direct-reading antisense peptide, has significantly through the affinity interaction power of the antisense peptide 3 of degeneracy aminoacid replacement and just peptide and improves.On this basis, be recognition component with the antisense peptide of human beta interferon (hIFN-β), made up a kind of biology sensor, be used to measure human beta interferon.
The positive and negative adopted peptide sequence of table 1 human beta interferon
Above-mentioned two kinds of antisense peptide AS-IFN1 and AS-IFN3 can synthesize (W.C.Chan, P.D.White, " FMOC Solid phase Peptide Synthesis " .Oxford University Press, 2000) according to the polypeptide solid phase synthesis process.
The preparation of this biology sensor is carried out as follows:
By 1, the 2-dithioglycol makes sulfydryl on the electrode surface band in the self assembly of wafer gold electrode surfaces; In weak basic condition order sulfydryl and bis-epoxy 1, the reaction of 4-butanediol diglycidyl ether, gold electrode surfaces is epoxide group and modifies; Micromolecule recognition component antisense peptide AS-IFN1 or AS-IFN3 can modify the surface of gold electrode by its amino reaction with epoxide group, promptly obtain biology sensor, its structure as shown in Figure 1, (a) AS-IFN1 immobilization wafer among the figure; (b) AS-IFN3 immobilization wafer; (c) blank wafer.
In order to control the charge capacity of polypeptide, before and after fixing, all measure its resonant frequency, according to the amount of Sauerbray Equation for Calculating bound substances.For the quartz wafer of 10MHz, the mass loading of 1 μ g produces the frequency shift of 457Hz approximately.The charge capacity of general may command polypeptide is 2.0 * 10
-12-5.0 * 10
-12Mol/mm
2
With rubber seal biology sensor is fixed on the (QCM-FIA) or in the flow cell of SPR of QCM (Quartz Crystal Microbalance)-Flow Injection Analysis system, inject the protein solution of variable concentrations by sampling valve, record oscillation frequency or refractive index are the sensing spectrum over time, to carry out the mensuration of human beta interferon.Utilize special software, can write down and calculate the dynamic interaction parameter of human beta interferon and immobilization antisense peptide.
Two kinds of QCM immunosensors of prepared immobilization AS-IFN1 and immobilization AS-IFN3, in the scope of 0.12mg/mL to 0.96mg/mL, the oscillation frequency drop-out value all presents good linear relationship with the sample size of human beta interferon, and favorable reproducibility can be used for the detection by quantitative of human beta interferon.
Two kinds of QCM immunosensors of prepared immobilization AS-IFN1 and immobilization AS-IFN3, irrelevant albumen bovine serum albumin(BSA) (BSA), human serum albumins (HSA) and chicken ovalbumin (CEA) there is not non-specific adsorption, but human beta interferon is all demonstrated tangible selectivity combination, have good selectivity.
Two kinds of QCM biology sensors of prepared immobilization AS-IFN1 and immobilization AS-IFN3, the affinity interaction power of they and human beta interferon shows evident difference, and its frequency descends and is respectively 150Hz and 300Hz.
Utilize two kinds of QCM immunosensors of prepared immobilization AS-IFN1 and immobilization AS-IFN3, can in QCM-FIA system, measure the interaction of human beta interferon and immobilization antisense peptide.After measured as can be known, the affinity interaction force rate direct-reading antisense peptide AS-IFN1 of human beta interferon and immobilization AS-IFN3 has improved 15 times.
Below with specific embodiment the present invention is described.
The preparation of embodiment 1, biology sensor
Get 10MHz AT and hob calcium carbide English wafer, diameter 12.0mm, two-sided gold-plated electrode, electrode diameter 6.0mm.With the Piranha solution (concentrated sulphuric acid: 30%H
2O
2=3:1 v/v) handles, and carefully cleans, dries up standby with ethanol, tri-distilled water respectively then.Clean wafer immerses in the ethanolic solution of 5% dithioglycol, after lucifuge leaves standstill 2 hours, cleans with ethanol and tri-distilled water, immerses by 1mL1 the 0.1mol/LNa of 4-butanediol diglycidyl ether and 4mL pH9.5 again
2CO
3In the mixed solution that damping fluid is formed, 40 ℃ of reactions stop reaction after 4 hours, water flushing, the wafer that obtains modifying.Antisense peptide AS-IFNl is dissolved in the solution of making 2mg/ml in the damping fluid of pH10.0, drips in the chip electrode surface of having modified, 4 ℃ of reactions 20 hours with micro syringe.At last, wafer is immersed 30min in the 1.0mol/L monoethanolamine .HCl aqueous solution, to seal remaining epoxy radicals, promptly making with antisense peptide AS-IFN1 is the human beta interferon biology sensor of aglucon.Carry out above-mentioned steps if replace AS-IFN1 with antisense peptide AS-IFN3, then can make with antisense peptide AS-IFN3 is the human beta interferon biology sensor of aglucon.If adopt above-mentioned treatment step but do not add antisense peptide, then can prepare blank wafer.
With O shape rubber seal biology sensor is fixed in the QCM flow cell, places gas phase with the one side contact solution another side of having fixed antisense peptide.Moving phase is the phosphate buffer of 20mmol/L, and pH7.4, flow velocity are 30-90 μ L/min.Inject the protein solution of 100 μ L variable concentrations with six-way injection valve, record sensing spectrum.According to the Sauerbray Equation for Calculating as can be known, 1 μ g mass loading has caused the frequency shift of about 457Hz.With antisense peptide AS-IFN1 and AS-IFN3 is that the human beta interferon biology sensor of aglucon is after the coupling of finishing antisense peptide, frequency reduces and to be respectively 69.6Hz and 44.2Hz (n=5), calculate in view of the above gold electrode surfaces fixedly the amount of antisense peptide be respectively: 3.64 * 10
-12Mol/mm
2(AS-IFN1) and 2.31 * 10
-12Mol/mm
2(AS-IFN3).After once measuring, (100mmol/L pH20) regenerates, and carries out subsequent measurements wafer with the glycine hydrochloride salt solusion.
The specificity experiment of embodiment 2, biology sensor
With O shape rubber seal blank wafer is fixed in the QCM flow cell, places gas phase with the one side contact solution another side of modifying.Moving phase is the phosphate buffer of 20mmol/L, and pH7.4, flow velocity are 30-90 μ L/min.Inject human beta interferon, bovine serum albumin(BSA) (BSA), human serum albumins (HSA) and chicken ovalbumin (CEA) protein solution of 100 μ L variable concentrations respectively with six-way injection valve, record sensing spectrum, (moving phase: 20mmol/L phosphate buffer (pH7.4) as shown in Figure 2; Flow velocity: 60 μ L/min; Protein concentration: 1.0mg/mL; Sample size: 100 μ L).The result shows that human beta interferon and other 3 kinds of albumen all do not have the non-specific adsorption effect on blank wafer.
With O shape rubber seal will be that the human beta interferon biology sensor of aglucon is fixed in the QCM flow cell with AS-IFN1 and AS-IFN3, place gas phase with the one side contact solution another side of having fixed antisense peptide.Moving phase is the phosphate buffer of 20mmol/L, and pH7.4, flow velocity are 30-90 μ L/min.Inject bovine serum albumin(BSA) (BSA), human serum albumins (HSA) and chicken ovalbumin (CEA) protein solution of 100 μ L variable concentrations respectively with six-way injection valve, record sensing spectrum, as shown in Figure 3, the wafer that (a) AS-IFN1 modifies among the figure; (b) wafer of AS-IFN3 modification.The result shows that they all do not show tangible frequency change on the human beta interferon biology sensor that with AS-IFN1 and AS-IFN3 is aglucon.
With O shape rubber seal will be that the human beta interferon biology sensor of aglucon is fixed in the QCM flow cell with AS-IFN1 and AS-IFN3, place gas phase with the one side contact solution another side of having fixed antisense peptide.Moving phase is the phosphate buffer of 20mmol/L, and pH7.4, flow velocity are 30-90 μ L/min.Inject 100 μ L variable concentrations human beta interferon solution respectively with six-way injection valve, record sensing spectrum, as shown in Figure 4, and among the figure, (a) blank wafer; (b) wafer of AS-IFN1 modification; (c) wafer of AS-IFN3 modification; Protein solution: 0.96mg/mL; Sample size: 100 μ L; Flow velocity: 60 μ L/min.The result shows that human beta interferon can't cause the frequency change of blank wafer, the wafer frequency that AS-IFN1 modifies about 150Hz that descended; The wafer frequency that AS-IFN3 modifies about 300Hz that descended.
Above result shows, is between the human beta interferon biology sensor of aglucon and the human beta interferon specific interaction to be arranged with AS-IFN1 and AS-IFN3, and other albumen can not disturb the mensuration of this biology sensor to human beta interferon.
With O shape rubber seal will be that the human beta interferon biology sensor of aglucon is fixed in the QCM flow cell with AS-IFN1 and AS-IFN3, place gas phase with the one side contact solution another side of having fixed antisense peptide.Moving phase is the phosphate buffer of 20mmol/L, and pH7.4, flow velocity are 30-90 μ L/min.Inject 100 μ L variable concentrations human beta interferon solution respectively with six-way injection valve, investigated the relation of human beta interferon concentration and frequency response on the wafer that antisense peptide AS-IFN1 and AS-IFN3 modify.In the concentration range of 0.12mg/mL to 0.96mg/mL, show good linear relationship between the frequency response values (Y) on concentration of human beta interferon (X) and AS-IFN1 and the AS-IFN3 modification wafer, Y=228.54X+7.545, r=0.994 (AS-IFN1); Y=380.52X+19.453, r=0.9897 (AS-IFN3); Typical curve carries out the quantitative test of human beta interferon in view of the above.
With O shape rubber seal will be that the human beta interferon biology sensor of aglucon is fixed in the QCM flow cell with AS-IFN1 and AS-IFN3, place gas phase with the one side contact solution another side of having fixed antisense peptide.Moving phase is the phosphate buffer of 20mmol/L, and pH7.4, flow velocity are 30-90 μ L/min.Inject 100 μ L variable concentrations human beta interferon solution respectively with six-way injection valve, the human beta interferon of having investigated variable concentrations (0.12,0.24,0..48 and 0.96mg/mL) is modified cohesive process on the wafer at antisense peptide AS-IFN1 and AS-IFN3, as Fig. 5 and shown in Figure 6.Can draw by calculating, the equilibrium dissociation constant of human beta interferon and AS-IFN1 is (1.89 ± 0.101) * 10
-4Mol/L; With the equilibrium dissociation constant of AS-IFN3 be (1.22 ± 0.0479) * 10
-5Mol/L.
Sequence table
<160>3
<210>1
<211>14
<212>PRT
<213>1
<220>
<221>misc-feature
<222>(6，10)
＜223〉Xaa=Gln or Glu
<400>1
<210>2
<211>14
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
<210>3
<211>14
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Claims (8)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005068497A1 (en) * | 2004-01-06 | 2005-07-28 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Iis: a novel ifn-gamma-inducible protein, member of the secretoglobin family, that modulates cellular invasion and migration |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005068497A1 (en) * | 2004-01-06 | 2005-07-28 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Iis: a novel ifn-gamma-inducible protein, member of the secretoglobin family, that modulates cellular invasion and migration |
Non-Patent Citations (1)
| Title |
|---|
| 赵睿等.基因工程β型干扰素的分离纯化.《中华微生物学和免疫学杂志》.1996,第16卷(第6期),452-456. * |
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