CN103145807B - Hepatitis C virus (HCV) B-cell epitope peptide PUHI37 and application thereof - Google Patents
Hepatitis C virus (HCV) B-cell epitope peptide PUHI37 and application thereof Download PDFInfo
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Abstract
The invention provides a novel hepatitis C virus (HCV) B-cell epitope peptide PUHI37. An overlapping peptide method is utilized to find out the new linear protective B-cell epitope peptide of HCV envelope protein and obtain protective antibodies capable of neutralizing HCV 1b genotype/subtype HCVpp (HCV pseudotyped particle). The amino acid composition of the epitope peptide recognized by the antibodies is TDVLLLNNTRPPQGN, and the site is 534-548 (using HCV H77 as the reference standard, Accession No.NC_004102). The determination of the protective B-cell epitope peptide provides a new solution for research and development of HCV therapeutic antibodies and HCV preventive vaccines.
Description
Technical field
The invention belongs to field of immunology, specifically, relate to hepatitis C virus B cell epitope peptide PUHI37 and application thereof.
Background technology
Hepatitis C virus (Hepatitis C virus, HCV) is single strand plus RNA virus, is under the jurisdiction of flaviviridae.HCV genome comprises the single long open reading frame polymeric protein being approximately made up of 3000 amino-acid residues of encoding.Polymeric protein, under the effect of cell and virus protease, is cracked into three kinds of major structural proteins and the necessary Nonstructural Protein of several virus replication.
The variation of HCV genome height, highly between the strain isolated of variation, only having 60% nucleotide sequence is homology.HCV can be divided into 6 genotype and different subtype at present, and HCV1b and 2a genotype are comparatively common in China, wherein taking 1b type as main.There is la, 2b and 3b type report certain areas, and 6 types are mainly seen in Hong Kong and Macao Area, also visible this genotype in province, southern border.
The whole world approximately has 1.8 hundred million people to infect HCV at present, and wherein the infected of approximately 80% understands chronicity, and then develops into liver cirrhosis and hepatocellular carcinoma.In China, HCV chronic infection person is about 6,500,000.For HCV chronic infection, topmost therapeutic modality is the stdn treatment of Interferon, rabbit associating ribavirin, but poor for gene 1 type and 4 type results for the treatment of, and because toxic side effect is obvious, medical expense is higher, and makes a large amount of HCV the infecteds yield treatment.Being infected the liver transplantation causing by HCV has become the main reason of during orthotopic liver transplantation in western countries, also be one of major reason in China, and stdn treatment plan infects to blocking-up HCV the liver transplantation causing to infect curative effect poor again, in the urgent need to new prevention, treatment plan.
At present, identify the more protectiveness B cell epitope for HCV envelope protein, such as 313-327,396-407,412-423 and 613-621 etc., it is active that the corresponding antibody of these epi-positions has good neutralization for HCV.Study the more linear B cell epitope peptide of protectiveness 412-423(QLINTNGSWHIN); its corresponding antibody, called after HCV1, has completed clinical second phase experiment; in chimpanzee animal model, 250mg/kg HCV1 can block HCV representative strains H77(1a gene hypotype completely) infection.This shows to infect at HCV the liver transplantation field causing, and this antibody has a extensive future aspect the infecting again of blocking-up liver transplantation HCV, aspect the development of HCV vaccine, also has comparatively wide application prospect.Series of experiments shows, protective epitope's qualification, and prevention and the treatment field of infecting at HCV, all have very wide application prospect.
Summary of the invention
The object of this invention is to provide a kind of novel hepatitis C virus B cell epitope peptide PUHI37 and application thereof.
In order to realize the object of the invention, the invention provides a kind of hepatitis C virus B cell epitope peptide PUHI37, its amino acid consists of TDVLLLNNTRPPQGN.
The Chinese 1b gene hypotype HCV strain isolated envelope protein sequence of having reported is carried out sequence alignment by the present invention, finds out the common recognition sequence of envelope protein total length.The method of application overlapping peptide, by HCV envelope protein total length (common recognition sequence), from the 192nd amino acids to the 746 amino acids (taking hepatitis C virus H77 as reference standard, Accession No.NC – 004102), be split as 57 different polypeptide (not comprising cross-film district), wherein, in sequence 192-35,384-413,444-523,544-613 and 624-717, every polypeptide length is 20 amino acid, overlapping 10 amino acid of adjacent polypeptide; In sequence 404-453,514-553 and 604-633, every polypeptide length is 15 amino acid, overlapping 10 amino acid of adjacent polypeptide.By synthetic these polypeptide of synthetic method, respectively by these polypeptide immunes Balb/c mouse, to obtain polyclonal antibody (antiserum(antisera)), immunity 3 times altogether, every minor tick 2 weeks, 50 μ g/ time.The polyclonal antibody of acquisition is carried out to bioactivity, and method comprises indirect ELISA (reference: Ndongo N, Berthillon P, Pradat P; Vieux C, Bordes I, Berby F, Maynard M; Zoulim F, Trepo C, Petit MA.Hepatology2010; 52:1531-1542.).Serum is diluted in proportion for 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and eight concentration of 1:128000, under 1:128000 Dilution ratio, OD value >=0.25,490nm place, and polyclonal antibody OD value and negative control hole OD value ratio >=2 when 1:128000 weaker concn, be judged as polyclonal antibody tire qualified.By the HCV polyclonal antibody obtaining, carry out HCV pseudovirion (HCV-pseudotyped particles, HCVpp) neutralization experiment; method comprises that packaging has 1a, 1b, 2a, 3a, 4a, the 5 and 6 HCVpp(reference of totally seven kinds of different genotype/hypotypes of luciferase reporter gene: Tong Y, Zhu Y, Xia X; Liu Y, Feng Y, Hua X; Chen Z, Ding H, Gao L; Wang Y, Feitelson MA, Zhao P; Qi ZT.J Virol, 2011; 85:2793-2802.); then by 57 kinds of HCV polyclonal antibodies; respectively by different Dilution ratios; mix respectively with the HCVpp of seven kinds of different genotype/hypotypes; then be added in 96 orifice plates of pre-vaccination Huh7.5 cell; after 72 hours, detect uciferase activity; and compare with control wells; to determine in different polyclonal antibodies and the activity (reference: Tarr AW, Urbanowicz RA, Jayaraj D of HCVpp; Brown RJ; McKeating JA, Irving WL, Ball JK.J Virol2012; 86:2739-2749.), and then definite protectiveness B cell epitope peptide.
Experimental result shows; sequence 534-548(is taking hepatitis C virus H77 as reference standard; Accession No.NC_004102) be protectiveness B cell epitope peptide; corresponding amino acid consists of TDVLLLNNTRPPQGN; by its called after PUHI37, in its corresponding Anti-TNF-α physical efficiency and 1b gene hypotype HCVpp.
The present invention also provides described hepatitis C virus B cell epitope peptide PUHI37 in the application of preparing in anti-HCV medicament and HCV preventative vaccine.Using PUHI37 epitope peptide as immunogen, be aided with adjuvant immunity laboratory animal (as Balb/c mouse), prepare polyclonal antibody.Or, using PUHI37 epitope peptide as immunogen, be aided with adjuvant immunity laboratory animal, adopt hybridoma technology and DNA recombinant technology, prepare the Humanized monoclonal antibodies of identification PUHI37 epitope peptide antigen.The polyclonal antibody making above or monoclonal antibody are infected for blocking 1b genotype liver of patients with chronic HCV infection transplantation and postoperative HCV alone or in combination again.
The present invention further provides HCV1b gene hypotype neutrality antibody, it is taking described hepatitis C virus B cell epitope peptide PUHI37 as immunogen, is aided with adjuvant immunity laboratory animal (as Balb/c mouse), the polyclonal antibody of preparation.
Aforesaid neutrality antibody; by PUHI37 epitope peptide respectively with carrier proteins KLH and BSA coupling (wherein; after BSA coupling epitope peptide completes for immunity, Balb/c mice serum antibody titers detects) (reference: Darwish IA; Alzoman NZ; Abuhejail RM, El-Samani TE.Chem Cent J2012; 6:125.), KLH coupling epitope peptide antigen and adjuvant are pressed equal-volume and are mixed, and after emulsification, adopt hypodermic injection to divide immune Balb/c mouse 3 times, after immunity completes, collect the antibody in serum, to obtain final product.
Particularly, by PUHI37 epitope peptide respectively with carrier proteins KLH and BSA coupling (wherein, after BSA coupling epitope peptide completes for immunity, Balb/c mice serum antibody titers detects), KLH coupling epitope peptide antigen and adjuvant are pressed equal-volume and are mixed, after emulsification completely, respectively at the 1st day, 15 days, 29 days with the multiple spot hypodermic injection immunity 8-12 of the nape portion Balb/c mouse in age in week, immunity 3 times altogether, use for the first time Fu Shi Freund's complete adjuvant, the incomplete Fu Shi adjuvant of rear twice use, antigen after every mouse per injection 500 μ l emulsifications, antigen amount is 50 μ g, after immunity completes, antibody titers in serum adopts indirect ELISA method to detect.Collect the antibody in serum, to obtain final product.
The present invention is by the method for overlapping peptide; find the linear protection B cell epitope peptide that hepatitis C virus envelope protein is new; and obtained can in and the protection antibody of hepatitis C virus 1b gene hypotype HCVpp; the epitope peptide of these antibody recognition is protectiveness B cell epitope peptide; its amino acid consists of TDVLLLNNTRPPQGN; position be 534-548(taking hepatitis C virus H77 as reference standard, Accession No.NC_004102).Determining of this protectiveness B cell epitope peptide, for the research and development of HCV therapeutic antibodies and the research and development of HCV preventative vaccine provide new solution.
Brief description of the drawings
Fig. 1 is the schematic flow sheet that screens HCV envelope protein protectiveness B cell epitope peptide in the embodiment of the present invention 1.
Fig. 2 is protectiveness B cell epitope peptide 534-548(PUHI37 in the embodiment of the present invention 2) corresponding antibodies bioactivity result.
Fig. 3 is protectiveness B cell epitope peptide 534-548(PUHI37 in the embodiment of the present invention 3) in the 1b gene hypotype HCVpp of corresponding antibodies and experimental result.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The acquisition of embodiment 1 hepatitis C virus B cell epitope peptide PUHI37
The preparation of 1.1 polypeptide antigens and polyclonal antibody
In ncbi database, search the Chinese 1b gene hypotype HCV strain isolated HCV full length protein sequence data of having reported, the sequence of collecting is compared, find out envelope protein total length (taking hepatitis C virus H77 as reference standard, Accession No.NC_004102, amino acid residue position is 192-747) common recognition sequence.Since the 192nd being amino-acid residue, synthetic length is 20 amino acid whose polypeptide, and (for example Article 1 sequence is 192-211 to overlapping 10 amino acid of adjacent polypeptide, and Article 2 is 202-221, by that analogy).Sequence 404-453,514-553 and 604-633 comprise the possible binding site of CD81 (HCV invades one of acceptor of cell), and every synthetic polypeptide length within the scope of this sequence is 15 amino acid, overlapping 10 amino acid of adjacent polypeptide.Envelope protein cross-film region sequence, not within synthetic polypeptide scope, synthesizes 57 polypeptide altogether.By every polypeptide respectively with carrier proteins KLH and BSA coupling (reference: Darwish IA, Alzoman NZ, Abuhejail RM, El-Samani TE.Chem Cent J 2012; 6:125.), wherein, after BSA coupling polypeptide completes for immunity, Balb/c mice serum antibody titers detects, KLH coupling polypeptide antigen and adjuvant are pressed equal-volume and are mixed, after emulsification completely respectively at the 1st day, 15 days, 29 days with the hypodermic method immunity of nape portion multiple spot Balb/c mouse (8-12 age in week), totally 3 times.Fu Shi Freund's complete adjuvant for the first time, rear is for twice incomplete Fu Shi adjuvant, antigen after every mouse per injection 500 μ l emulsifications, antigen amount is 50 μ g.After immunity completes, Serum Antibody titre adopts indirect ELISA method to detect, and result shows that 46 polypeptide obtain corresponding polyclonal antibody (antiserum(antisera)), and 11 polypeptide do not produce corresponding antibodies.
The preparation of 1.2HCVpp
HCVpp is obtained by cotransfection 293T cell together with the pLenti6 plasmid of HCV envelope protein E1 E2 expression plasmid and HIV gag/pol (pLP1), HIV rev (pLP2), coding fluorescence element enzyme.HCV envelope protein E1 E2 expression plasmid is HCV envelope protein E1 E2 encoding sequence to be cloned into pCR3.1 vector construction form, and after transfection 293T cell, can give expression to HCV envelope protein E1 E2.In the present invention, 7 kinds of different genotype/hypotype HCV envelope protein E1 E2DNA encoding sequences used come from different genotype HCV the infected, and these DNA encoding sequences are cloned into respectively to pCR3.1(Invitrogen) carrier, build HCV envelope protein E1 E2 expression plasmid; concrete grammar is with reference to Lavillette D; Tarr AW, Voisset C, Donot P; Bartosch B; Bain C, Patel AH, Dubuisson J; Ball JK, Cosset FL.Hepatology 2005; 41:265-274..The pLenti6 plasmid of HIV gag/pol (pLP1), HIV rev (pLP2), coding fluorescence element enzyme is purchased from invitrogen company.The preparation process of HCVpp comprises: respectively by 7 kinds of different genotype/hypotype (gene 1a, 1b, 2a, 3a, 4a, 5 and 6 types) HCV envelope protein E1 E2 expression plasmid and HIV gag/pol (pLP1), HIV rev (pLP2), the pLenti6 plasmid cotransfection 293T cell together of coding fluorescence element enzyme, transfection is placed in 37 DEG C of cell culture incubators, hatch the cells and supernatant that after 48 hours, collection contains HCVpp, can obtain the HCVpp(reference of 7 kinds of different genotype/hypotypes: Tong Y, Zhu Y, Xia X, Liu Y, Feng Y, Hua X, Chen Z, Ding H, Gao L, Wang Y, Feitelson MA, Zhao P, Qi ZT.J Virol, 2011, 85:2793-2802.).After 0.45 μ m filter filters, concentrated 20 times of the application U.S. 100K of PALL company ultrafiltration and concentration centrifuge tube, can be used in HCVpp and experiment.
In 1.3HCVpp, survey with experiment and luciferase biopsy
By Huh7.5 cell pre-vaccination, in containing 96 orifice plates of DMEM perfect medium, inoculum density is 1 × 10
4/ hole.Being placed in 37 DEG C of cell culture incubators hatches.Second day, by containing the supernatant (20 μ l/ hole) of HCVpp and different polyclonal antibody and negative control sera, with different Dilution ratio mixing, (polyclonal antibody and negative control sera Dilution ratio are respectively 1:50, 1:100, 1:200, 1:400, 1:800), do not add the HCVpp supernatant of polyclonal antibody and negative serum also in contrast, and to add final concentration be the polybrene (polybrene) of 4 μ g/ml, cumulative volume is that (less than 100 μ l DMEM perfect mediums complement to 100 μ l) in 100 μ l/ holes, hatch 1 hour for 37 DEG C, then add in 96 orifice plates, each weaker concn is all done 3 parallel holes.Hatch after 5 hours for 37 DEG C, discard cells and supernatant, be replaced by fresh complete DMEM substratum, hatch 72 hours for 37 DEG C.Detect by luciferase detection system (Promega), method comprises and discards cells and supernatant, and PBS washes 1 time, adds cell pyrolysis liquid 20 μ l/ holes, hatch after 10 minutes and add substrate fluorescein 100 μ l/ holes, put into fluorescence radiation detector reading numerical values.
1.4 determining of neutrality polyclonal antibody and protectiveness B cell epitope peptide
Compare with the control wells that does not add polyclonal antibody and negative control sera, add after polyclonal antibody, uciferase activity value reduce by 50% and more than, think that to have neutralization active.PUHI37 corresponding antibodies is in the time of 1:50 Dilution ratio; can reduce 1b gene hypotype HCVpp cells infected uciferase activity approximately 50%; can regard as this polyclonal antibody is neutrality polyclonal antibody; the epitope peptide of its identification; be that PUHI37(amino acid consists of TDVLLLNNTRPPQGN), be protectiveness B cell epitope peptide.
The schematic flow sheet of screening HCV envelope protein protectiveness B cell epitope peptide as shown in Figure 1.
Embodiment 2 protectiveness B cell epitope peptide 534-548(PUHI37) corresponding antibodies preparation and bioactivity
1.1 protectiveness B cell epitope peptide 534-548(PUHI37) preparation of corresponding antibodies
By the hepatitis C virus B cell epitope peptide PUHI37 epitope peptide of synthetic respectively with KLH and BSA coupling (reference: Darwish IA, Alzoman NZ, Abuhejail RM, El-Samani TE.Chem Cent J2012; 6:125.), wherein, after BSA coupling epitope peptide completes for immunity, Balb/c mice serum antibody titers detects, KLH coupling epitope peptide antigen and adjuvant are pressed equal-volume and are mixed, after emulsification completely respectively at the 1st day, 15 days, 29 days with the hypodermic method immunity of nape portion multiple spot Balb/c mouse (8-12 age in week), totally 3 times.Fu Shi Freund's complete adjuvant for the first time, rear is for twice incomplete Fu Shi adjuvant, antigen after every each 500 μ l emulsifications of mouse, antigen amount is 50 μ g.
1.2 protectiveness B cell epitope peptide 534-548(PUHI37) corresponding antibodies bioactivity
After immunity completes, serum antibody titer adopts indirect ELISA method to detect.Concrete grammar comprises:, in 96 orifice plates, use containing 1 × PBS of 10% lowlenthal serum and seal antigen coated the corresponding epitope peptide of BSA coupling, 200 μ l/ holes, hatch 1 hour for 37 DEG C.Wash after 3 times with PBS, add the polyclonal antibody (antiserum(antisera)) of different Dilution ratios, Dilution ratio is respectively 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000, hatches 2 hours for 37 DEG C.1 × PBS with PBST(containing 0.05%Tween20) wash after 4 times, add the anti-mouse IgG antibody of coupling horseradish peroxidase, hatch 1 hour for 37 DEG C.PBST washes after 4 times, adds substrate incubated at room 30 minutes, 2N HCL termination reaction, the OD value at mensuration 490nm place.Each weaker concn of polyclonal antibody is all done 3 parallel holes.The mapping taking Dilution ratio and OD value as transverse and longitudinal coordinate respectively, if linear relationship is good and polyclonal antibody OD value >=0.25 in the time of 1:128000 weaker concn, and polyclonal antibody OD value and negative control hole OD value ratio >=2 when 1:128000 weaker concn, illustrate that this polyclonal antibody tires well, can be used in HCVpp and experiment.
Protectiveness B cell epitope peptide 534-548(PUHI37) corresponding antibodies bioactivity result is as shown in Figure 2.
Embodiment 3B cell epitope peptide 534-548(PUHI37) in corresponding antibodies and 1b gene hypotype HCVpp infect the detection of Huh7.5 cell
Obtained polyclonal antibody is carried out in HCVpp and experiment.Method comprises that by Huh7.5 cell pre-vaccination inoculum density is 1 × 10 in containing 96 orifice plates of DMEM perfect medium
4/ hole.Being placed in 37 DEG C of cell culture incubators hatches.Second day, by containing the supernatant (20 μ l/ hole) of HCVpp and different polyclonal antibody and negative control sera, with different Dilution ratio mixing, (polyclonal antibody and negative control sera Dilution ratio are respectively 1:50, 1:100, 1:200, 1:400, 1:800), do not add the HCVpp supernatant of antibody and negative serum also in contrast, and to add final concentration be the polybrene (polybrene) of 4 μ g/ml, cumulative volume be 100 μ l/ holes (less than 100 μ l DMEM perfect mediums mend to 100 μ l), hatch 1 hour for 37 DEG C, add in 96 orifice plates, each weaker concn is all done 3 parallel holes.Hatch after 5 hours for 37 DEG C, discard cells and supernatant, be replaced by fresh complete DMEM substratum, hatch 72 hours for 37 DEG C.Detect by luciferase detection system (Promega), method comprises and discards cells and supernatant, and PBS washes 1 time, adds cell pyrolysis liquid 20 μ l/ holes, hatch after 10 minutes and add substrate fluorescein 100 μ l/ holes, put into fluorescence radiation detector reading numerical values.Detected result is carried out to statistical study, compares with the control wells that does not add polyclonal antibody and negative control sera, add after polyclonal antibody uciferase activity value reduce by 50% and more than, think that to have neutralization active.Thereby it is active further to verify whether this antibody has neutralization to the HCVpp of a certain or several genotype/hypotypes.
Protectiveness B cell epitope peptide 534-548(PUHI37) in the 1b gene hypotype HCVpp of corresponding antibodies and experimental result as shown in Figure 3.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Reference
Ndongo?N,Berthillon?P,Pradat?P,Vieux?C,Bordes?I,Berby?F,Maynard?M,et?al.Association?of?Anti-E1E2Antibodies?with?Spontaneous?Recovery?or?Sustained?Viral?Response?to?Therapy?in?Patients?Infected?with?Hepatitis?C?Virus.Hepatology?2010;52:1531-1542.
Tong?Y,Zhu?Y,Xia?X,Liu?Y,Feng?Y,Hua?X,Chen?Z,et?al.Tupaia?CD81,SR-BI,claudin-1,and?occludin?support?hepatitis?C?virus?infection.J?Virol?2011;85:2793-2802.
Tarr?AW,Urbanowicz?RA,Jayaraj?D,Brown?RJ,McKeating?JA,Irving?WL,Ball?JK.Naturally?occurring?antibodies?that?recognize?linear?epitopes?in?the?amino?terminus?of?the?hepatitis?C?virus?E2protein?confer?noninterfering,additive?neutralization.J?Virol?2012;86:2739-2749.
Darwish?IA,Alzoman?NZ,Abuhejail?RM,El-Samani?TE.Synthesis?of?hapten?and?preparation?of?specific?polyclonal?antibody?with?high?affinity?for?lenalidomide,the?potent?drug?for?treatment?of?multiple?myeloma.Chem?Cent?J2012;6:125.
Lavillette?D,Tarr?AW,Voisset?C,Donot?P,Bartosch?B,Bain?C,Patel?AH,et?al.Characterization?of?host-range?and?cell?entry?properties?of?the?major?genotypes?and?subtypes?of?hepatitis?C?virus.Hepatology2005;41:265-274.
Claims (8)
1. hepatitis C virus B cell epitope peptide PUHI37, is characterized in that, its amino acid consists of TDVLLLNNTRPPQGN.
2. the application of hepatitis C virus B cell epitope peptide PUHI37 in the anti-1b gene hypotype HCVpp medicine of preparation described in claim 1.
3. the application of hepatitis C virus B cell epitope peptide PUHI37 in preparation 1b gene hypotype HCVpp preventative vaccine described in claim 1.
4. application according to claim 2, is characterized in that, using PUHI37 epitope peptide as immunogen, is aided with adjuvant immunity laboratory animal, prepares polyclonal antibody.
5. application according to claim 2, is characterized in that, using PUHI37 epitope peptide as immunogen, is aided with adjuvant immunity laboratory animal, adopts hybridoma technology and DNA recombinant technology, prepares the Humanized monoclonal antibodies of identification PUHI37 epitope peptide antigen.
6.HCV1b hypotype neutrality antibody, it is taking hepatitis C virus B cell epitope peptide PUHI37 claimed in claim 1 as immunogen, is aided with adjuvant immunity laboratory animal, the polyclonal antibody of preparation.
7. neutrality antibody according to claim 6, it is characterized in that, by PUHI37 epitope peptide and carrier proteins KLH coupling, KLH coupling epitope peptide antigen and adjuvant are pressed equal-volume and are mixed, after emulsification, adopt hypodermic injection to divide immune Balb/c mouse 3 times, after immunity completes, collect the antibody in serum, to obtain final product.
8. neutrality antibody according to claim 7, it is characterized in that, by PUHI37 epitope peptide and carrier proteins KLH coupling, KLH coupling epitope peptide antigen and adjuvant are pressed equal-volume and are mixed, after emulsification completely, respectively at the 1st day, 15 days, 29 days with the nape portion multiple spot hypodermic injection immunity 8-12 Balb/c mouse in age in week, immunity 3 times altogether, use for the first time Fu Shi Freund's complete adjuvant, the incomplete Fu Shi adjuvant of rear twice use, antigen after every mouse per injection 500 μ l emulsifications, antigen amount is 50 μ g, after immunity completes, collect the antibody in serum, to obtain final product.
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| CN1609213A (en) * | 2004-09-17 | 2005-04-27 | 中国人民解放军第二军医大学 | Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence |
| WO2008079890A1 (en) * | 2006-12-20 | 2008-07-03 | Progenics Pharmaceuticals (Nevada), Inc. | Hepatitis c virus (hcv) inhibitory monoclonal antibody and bindable ligand thereof |
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