CN102206256A

CN102206256A - HCV envelope protein E2 with deleting hypervariable region 1 and use thereof

Info

Publication number: CN102206256A
Application number: CN2011100572844A
Authority: CN
Inventors: 赵平; 戚中田
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2011-03-10
Filing date: 2011-03-10
Publication date: 2011-10-05
Anticipated expiration: 2031-03-10
Also published as: CN102206256B

Abstract

The invention relates to the technical field of biological engineering. At present, there is no effective vaccine for preventing hepatitis C virus (HCV) infection. The amino terminal of HCV envelope protein E2 has 27 amino acid residues with highest variability, known as hypervariable region 1(HVR1). The invention provides an HCV envelope protein E2 without HVR1, also provides an application of the HCV envelope protein E2 without HVR1 on HCV vaccines and HCV infection immunity diagnostic reagents. Animal immunization tests have discovered that HVR1 in 1-6 genotype HCV envelope protein E2 has substantial inhibition effect against immunogenicity of conservative neutralizing epitope in the envelope protein E2, the effectiveness of inducting broad spectrum neutralizing antibody by the E2 protein is significantly enhanced by deleting HVR1, the HCV envelope protein E2 with deleting HVR1 possibly can be used as an effective target antigen of HCV vaccines. The invention has proved that the HCV envelope protein E2 with deleting HVR1 has strong reaction with E2 antibody in serums of HCV infections and can be used as an immunity diagnostic antigen of HCV infections.

Description

Deleted HCV coating E2 albumen of hypervariable region 1 and uses thereof

Technical field

The present invention relates to bio-pharmaceutical engineer technology domain, is a kind of hepatitis C virus of deleting hypervariable region 1 (hepatitis C virus, HCV) coating E2 albumen and application in hcv vaccine and HCV infection immunity diagnostic reagent thereof.

Background technology

(hepatitis C virus HCV) belongs to flaviviridae to hepatitis C virus, is one of main virulence factor of the acute and chronic hepatitis propagated through blood.HCV is the sub-thread positive chain RNA virus, has only an open reading frame, about 3010 the amino acid whose amyloid protein precursors of encoding, comprise three kinds of structural protein (core protein Core, envelope protein E1, envelope protein E 2) and seven kinds of Nonstructural Protein (p7 albumen, NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Fig. 1) (referring to document: Reed KE, Rice CM.Overviewof hepatitis C virus genome structure, polyprotein processing, and protein properties.Curr Top Microbiol Immunol.2000; 242:55-84.).NS5B is the RNA polymerase that RNA relies on, and does not have correct functioning in guide RNA synthetic process, makes the HCV genome mutation very high, according to the difference of its genome nucleotide sequence, at present HCV is divided into six genotype.Present global HCV the infected surpasses 1.7 hundred million, and about 80% acute HCV infection can develop into chronic infection.The HCV chronic infection can cause hepatopathy in whole latter stage such as liver cirrhosis, hepatocellular carcinoma, and is also with the outer disease of multiple liver, closely related as lymphoma, Combination cryoglobulinemia, glomerulonephritis, porphyria cutanea tarda, diabetes etc.Though immunology and nucleic acid detection method have been arranged, these methods all can not be got rid of false negative fully, and in addition, " window phase " of infection be because antibody horizontal is low and the virus replication level is low, cause omission easilier.Treatment aspect, present standard treatment are to unite to use ribavirin and polyethyleneglycol modified Interferon, rabbit (IFN-α), and different genotype HCV has very big-difference to the susceptibility of this therapy, and the lasting response rate that I type HCV infects this therapy only is 50%.Though the hepatitis C case that infects through blood and blood product approach in the global range with last century the nineties compared in the past and declined to a great extent, remain the important channel of propagating HCV at developing country's blood and blood product.In addition, annual hepatitis C new cases sporadic and that the route of transmission is not clear still can be in any more in the world.The key measure that control HCV infects still depends on develops effective vaccine, and since being cloned from HCV genome in 1989, the research of hcv vaccine is the research focus in developed countries such as American-European and Japan always, but does not have effective vaccine to come out so far.

The surface protein of virus is the key protein of inducing neutralizing antibody, and the surface protein of HCV is an envelope protein.The HCV envelope protein comprises two kinds of E1 and E2, and the two forms heterodimer by non covalent bond, the receptors bind on mediation HCV and target cell surface, and intrusion target cell inside.The HCV acceptor that identifies at present has four kinds at least, comprises the plain family member CD81 of tetratransmembrane, category-B I type scavenger receptor (SR-BI) and tight junction protein claudin 1 and occludin.HCV coating E1 albumen is equivalent to 192-383 amino acids in the HCV amyloid protein precursor (referring to document: Reed KE, Rice CM.Overview of hepatitis C virusgenome structure, polyprotein processing, and protein properties.Curr Top MicrobiolImmunol.2000; 242:55-84.; Op De Beeck A, Cocquerel L, Dubuisson J.Biogenesisof hepatitis C virus envelope glycoproteins.J Gen Virol.2001; 82 (Pt 11): 2589-95.), its function is not studied clearly as yet at present, merges relevant but may enter the later film of cell with HCV.HCV coating E2 albumen is equivalent to 384-746 amino acids in the HCV amyloid protein precursor (referring to document: Reed KE, Rice CM.Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties.Curr Top Microbiol Immunol.2000; 242:55-84.; Op DeBeeck A, Cocquerel L, Dubuisson J.Biogenesis of hepatitis C virus envelopeglycoproteins.J Gen Virol.2001; 82 (Pt 11): 2589-95.), function is relatively clear, and E2 albumen can combine with HCV acceptor CD81 and SR-BI, and blocking-up E2 albumen combines with the CD81 or the SR-BI on target cell surface, all can block the infection of HCV.In E2 albumen, identified a large amount of linearities or space conformation dependency neutralizing antibody epi-position, all can effectively suppress HCV in vitro and in vivo at the antibody of these epi-positions and infect.Therefore, E2 albumen can be used as the important candidate antigens of hcv vaccine, and still, E2 albumen is the highest albumen of variability in the whole albumen of HCV, thereby the proteic height variation of E2 is that the HCV immune evasion forms one of chronically infected major reason.

The proteic variation of E2 is not a stochastic distribution, being arranged in aminoterminal 27 amino-acid residues of E2 albumen (the 384-410 amino acids that is equivalent to the HCV amyloid protein precursor) is the highest zone of E2 albumen variability, be called as hypervariable region 1 (hypervariable region 1, HVR1) (Fig. 1) (referring to document: Markedsequence diversity in the putative envelope proteins of hepatitis C viruses.Kato N, Ootsuyama Y, Tanaka T, Nakagawa M, Nakazawa T, Muraiso K, Ohkoshi S, HijikataM, Shimotohno K.Virus Res.1992; 22 (2): 107-23.; Kato N, Ootsuyama Y, Ohkoshi S, Nakazawa T, Sekiya H, Hijikata M, Shimotohno K.Characterization of hypervariableregions in the putative envelope protein of hepatitis C virus.Biochem Biophys ResCommun.1992; 189 (1): 119-27.).Variation frequency that this is regional and the infection chronicity behind the acute HCV infection and liver cirrhosis process are closely related (referring to document: Weiner AJ, Geysen HM, ChristophersonC, Hall JE, Mason TJ, Saracco G, Bonino F, Crawford K, Marion CD, Crawford KA, et al.Evidence for immune selection of hepatitis C virus (HCV) putative envelopeglycoprotein variants:potential role in chronic HCV infections.Proc Natl Acad Sci US is A.1992; 89 (8): 3468-72.; Farci P, Shimoda A, Coiana A, Diaz G, Peddis G, Melpolder JC, Strazzera A, Chien DY, Munoz SJ, Balestrieri A, Purcell RH, Alter HJ.The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.Science.2000; 288 (5464): 339-44.).HVR1 participation E2 albumen combines with the SR-BI acceptor, and deletion HVR1 can make the infectious of HCV significantly reduce.Contain one or more neutralizing epitopes among the HVR1, have hyperimmunization originality.HVR1 antibody in external energy is efficient and HCV infect, but the neutralizing effect of HVR1 antibody has strict strain specificity, promptly at can only the neutralize infection of same strain virus of the antibody of a certain strain HCV HVR1, the infection of other strains HCV is not had neutralizing effect.Generally acknowledged that at present thereby HCV utilizes HVR1 to escape immunne response and realizes that persistent infection is (referring to document: Weiner AJ, Geysen HM, Christopherson C, Hall JE, Mason TJ, Saracco G, Bonino F, Crawford K, Marion CD, Crawford KA, et al.Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants:potential role in chronic HCV infections.Proc Natl Acad Sci USA.1992; 89 (8): 3468-72.; Farci P, Shimoda A, Coiana A, Diaz G, Peddis G, Melpolder JC, Strazzera A, Chien DY, Munoz SJ, Balestrieri A, Purcell RH, Alter HJ.The outcome of acute hepatitis C predicted by the evolution ofthe viral quasispecies.Science.2000; 288 (5464): 339-44.).

Summary of the invention

The purpose of this invention is to provide a kind of modified hepatitis C virus (hepatitis C virus, HCV) coating E2 albumen and encoding gene thereof, and described albumen and the application of encoding gene in preparing prevention and therapeutic hcv vaccine, preparation HCV infection immunity diagnostic reagent thereof.

The inventor thinks, although the proteic variability height of E2, the function that mediation HCV infects must have specific constraint to its variability, makes its variation have certain limit or certain specific feature, thereby keeps the basic functions structure.Big quantity research proves that also the HVR1 in E2 albumen contains conservative linearity or space conformation dependence antigen epi-position with exterior domain.In order to inquire into HVR1 and these conservative antigen epi-positions in the difference aspect the immunogenicity, we have compared E2 albumen and the E2 albumen of having deleted HVR1 in the neutralizing effect to HCV of mouse and rabbit inductive antibody response and antibody, we find that HVR1 has significant inhibitory effect to the immunogenicity of these conservative antigen epi-positions, with E2 albumen that contains HVR1 or respective coding genetic immunization animalcule, in the E2 antibody that induces mainly be antibody at HVR1, very low at HVR1 with the antibody proportion of exterior domain epitope.It is protein induced at the antibody of HVR1 with the exterior domain epitope that deletion HVR1 then significantly strengthens E2, and immune serum is to also corresponding the significantly improving of neutralization activity of other strains HCV.We find that also deletion HVR1 can obviously improve E2 albumen and HCV infected person anteserum's reaction.These results suggest, deletion HVR1 can significantly strengthen the ability of protein induced cross-reacting antibody of HCV coating E2 (with different strain HCV envelope protein reaction) and cross neutralization antibody (the different strain HCV that neutralizes infects), represent a kind of brand-new strategy of HCV vaccine research and development, this albumen also can be used for the immunodiagnosis that HCV infects.

The present invention at first uses the method for genetic immunization (claiming nucleic acid immunization, dna immunization again) will contain HVR1 and has deleted the raq gene difference immune mouse of HVR1, analyze the influence of HVR1 to the genotypic E2 protein immunization of 1-6 originality, and with HCV pseudovirus (HCV pseudoparticles, HCVpp) and the HCV that produces of cell cultures (cell culture produced HCV HCVcc) is the neutralization activity of model analysis mouse immune serum to HCV.The result shows: HVR1 has significant inhibitory effect to the proteic immunogenicity of coating E2, and when having HVR1, the conservative antigen epi-position in the E2 albumen is difficult to induce corresponding antibody, and immune serum is very low to the neutralization activity of different strain HCV; After deletion HVR1, the conservative antigen epi-position can efficiently be induced corresponding antibody in the E2 albumen, and immune serum significantly improves the neutralization activity of different strain HCV; It is active with combining of HCV infected person anteserum that deletion HVR1 can also improve E2 albumen.In 6 HCV genotype coating E2 albumen, all there is similar phenomenon.

The 1b HCV genotype coating E2 albumen of having used expressing cho cell subsequently comprises two kinds of forms that contain HVR1 and deleted HVR1.Expression product is an adjuvant with ISA720 with affinity chromatography method purifying then, immunizing rabbit, and antibody in the detection rabbit anteserum and serum analysis are to the neutralization activity of HCV, and HCV infected person anteserum and two kinds of proteic activity that combine of form E2.The result shows: deletion HVR1 can significantly strengthen protein induced cross-reacting antibody of E2 and cross neutralization antibody, and the E2 albumen of deletion HVR1 has important use in the hcv vaccine research and development, also can be used as the immunodiagnosis antigen that HCV infects.

The particular content of technical solution of the present invention is as follows:

1.1-6 genotype is the amplification of totally seven strain HCV coating E2 protein genes: according to the proteic gene order design of corresponding coating E2, synthetic primer, the HCV coating E2 protein gene that contains HVR1 and deletion HVR1 with polymerase chain reaction (PCR) amplification, pcr amplification product inserts mammalian cell expression vector with restriction endonuclease digestion back, then raq gene is carried out dna sequencing.

2.HCV the evaluation of coating E2 protein expressioning product: with expression plasmid transfection human embryo kidney (HEK) (HEK) the 293T cell that makes up, with the E2 albumen in enzyme-linked immunosorbent assay (ELISA) and western blot analysis of cells culture supernatant and the cell pyrolysis liquid, be to detect antibody with the proteic polyclonal antibody of anti-E2.

3. mouse genetic immunization and Serum Antibody Detection: with above-mentioned E2 protein expressing plasmid immune Balb/C mouse respectively, serum is regularly gathered in immunity three times, detects E2 antibody and HVR1 antibody in the mice serum.

Mouse immune serum in and activation analysis: with HCVpp and HCVcc is the neutralization activity of model analysis mouse immune serum to HCV.

5. make up the proteic expressing cho cell plasmid of 1b type HCV coating E2: the 1b type HCV coating raq gene that will contain HVR1 and deletion HVR1 respectively inserts the expressing cho cell plasmid pCIDA-GS-neo with independent intellectual property right and (sees the Chinese patent ZL 200610024202.5 that the applicant has authorized, denomination of invention is: a kind of method with mammal cell with high efficient secretion expression hepatitis C virus envelope protein E 2, invention is artificial: Zhao Ping, Liao Xiaoling, Cao Jie, Qi Zhongtian.), dna sequencing is identified then.

6. structure and the cell cultures and the protein purification of the proteic Chinese hamster ovary celI strain of stably express 1b type HCV coating E2: with liposome with above-mentioned plasmid transfection CHO-K1 cell, obtain the CHO-K1 cell clone of stable transfection with first sulfonyl sulfuric acid amine (MSX) screening, carry out the domestication of serum-free suspension culture then, make cell be adapted to the serum-free culture basal growth fully.The collecting cell culture supernatant, centrifugal, nickel post affinitive layer purification E2 albumen is used in ultrafiltration then.

7. the E2 protein immunization rabbit that contains HVR and deleted HVR1: will contain HVR and E2 albumen and the abundant mixing of immunological adjuvant ISA720 of having deleted HVR1 respectively, immunizing rabbit, rabbit anteserum is regularly gathered in immunity three times.

The rabbit anteserum antibody test with in and activation analysis: detecting E2 antibody and HVR1 antibody in the rabbit immune serum, is the neutralization activity of model analysis rabbit anteserum with HCVpp and HCVcc.

9 usefulness contain HVR and have deleted E2 antibody among the Protein Detection HCV infected person anteserum of HVR1: with the E2t and the E2t Δ albumen coated elisa plate of equivalent, be used for detecting HCV infected person anteserum's E2 antibody.

The invention provides a kind of modified hepatitis C virus (hepatitis C virus, HCV) coating E2 albumen and encoding gene thereof, described modified hepatitis C virus coating E2 albumen is meant the peptide section of having deleted the hypervariable region 1 in the hepatitis C virus coating E2 albumen.

The peptide section (HVR1) of hypervariable region 1 is aminoterminal 27 amino acid of E2 albumen in the hepatitis C virus coating E2 albumen, is equivalent to the 384-410 amino acids residue of HCV amyloid protein precursor.

The present invention also provides described albumen and the application of encoding gene in preparation prevention and therapeutic hcv vaccine thereof.

The present invention also provides described albumen and the application of encoding gene in the immunological diagnostic reagent of preparation infection with hepatitis C virus thereof.

The present invention has deleted the hypervariable region 1 in the HCV coating E2 albumen, and the proteic immunogenicity of this HCV coating E2 significantly is better than the HCV coating E2 albumen that contains hypervariable region 1, induces the ability of antibody response significantly to strengthen; Induce the ability of cross reacting antibody (with the antibody of other genotype, the reaction of other strains HCV envelope protein) significantly to be better than the HCV coating E2 albumen that contains hypervariable region 1; Induce the ability of cross neutralization antibody (other genotype that neutralize, the infective antibody of other strains HCV) significantly to be better than the HCV coating E2 albumen that contains hypervariable region 1; Significantly be better than the HCV coating E2 albumen that contains hypervariable region 1 with the reaction of envelope protein antibody among the HCV infected person anteserum.

The present invention has proved that HVR1 has remarkable restraining effect to the immunogenicity of conservative antigen epi-position in the HCV coating E2 albumen, deletion HVR1 can effectively strengthen protein induced cross-reacting antibody of HCV coating E2 and cross neutralization antibody, for the research and development hcv vaccine provides a kind of effective antigens layout strategy, and the immunology diagnosis that infects for HCV provides a kind of new detection antigen.

Description of drawings

Fig. 1: the amyloid protein precursor of HCV genome encoding and the proteic structure of coating E2.The HCV amyloid protein precursor comprises three kinds of structural protein (core protein Core, envelope protein E1, envelope protein E 2) and seven kinds of Nonstructural Proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B).About 30 amino acid composition of coating E2 protein carboxyl terminal is striden the film district, and all the other are extracellular fragment, and 27 amino acid of aminoterminal are formed hypervariable region 1 (HVR1), and HVR1 is the part of extracellular fragment.Numeral is an amino acid position.

The structure of Fig. 2: pCI-E2t and pCI-E2t Δ plasmid.PCI-E2t coding proteic signal peptide of E2 and extracellular fragment contain HVR1; PCI-E2t Δ coding has been deleted the E2 albumen of HVR1, and all the other sequences are identical with pCI-E2t.PCMV is a cytomegalovirus promoter, and polyA is a SV40 virus polyadenylic acid sequence.

Fig. 3:, detect in the cell pyrolysis liquid and E2t (containing HVR1) and E2t Δ albumen (not containing HVR1) in the culture supernatant with enzyme-linked immunosorbent assay respectively with 1-6 genotype totally 7 proteic expression plasmid pCI-E2t of strain HCV coating E2 and pCI-E2t Δ transfection HEK293T cell.

Fig. 4: the HCV (HCVcc) that produces with cell cultures is the neutralizing effect to JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1 of model evaluation 1a, 1b and 2a type raq gene mice immunized serum, figure is the mean value (every group of 10 mouse, serum dilution in 1: 100) of each group mice serum neutralization ratio (%).

Fig. 5: the structure that is used for the E2 protein expressing plasmid of transfection CHO-K1 cell.PCMV is a cytomegalovirus CMV promotor, introD is the intron donor sequences, and GS is the glutaminase synthase gene, and introA is the intron receptor sequence, E2 is HCV coating raq gene E2t (containing HVR1) or E2t Δ (not containing HVR1), and polyA is a SV40 virus polyadenylic acid sequence.

Fig. 6: 1b type HCV coating E2t and the E2t Δ proteic SDS-PAGE recombinant expressed with the CHO-K1 cell analyze.

Fig. 7: use HCV coating E2t and E2t Δ albumen and the abundant mixing of adjuvant ISA720 respectively, immunizing rabbit, immunity three times, each dosage 50 μ g are with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and the homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) the detection mice serum.Figure is that different time points is respectively organized the antibody positive rate (every group of 10 mouse, serum dilution in 1: 100) in the rabbit anteserum.

Fig. 8: with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and the homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) the detection rabbit anteserum.Figure is the mean value (every group of 10 rabbit, serum dilution in 1: 100) that different time points is respectively organized the absorbance value of rabbit anteserum antibody test.

Fig. 9: analyze the neutralizing effect of immune for the third time back rabbit anteserum to HCVpp, figure is mean value (every group of 10 rabbit, serum dilution in 1: 100) of each group rabbit anteserum neutralization ratio (%).

Figure 10: analyze the neutralizing effect of immune for the third time back rabbit anteserum to JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1, figure is mean value (every group of 10 rabbit, serum dilution in 1: 100) of each group rabbit anteserum neutralization ratio (%).

Figure 11: marked microwell plate by enzyme with equivalent E2t and E2t Δ albumen bag, with the corresponding antibodies in enzyme-linked immunosorbent assay (ELISA) detection 30 routine HCV the infecteds and the 10 routine healthy human serums (serum dilution in 1: 100), numerical value is the mean value of the parallel three hole absorbance values of same sample.

Table 1:1a, 1b, 2a, 3,4,5,6 types are the nucleotide sequence of totally 7 strain HCV coating E1E2 genes

Table 2:1a, 1b, 2a, 3,4,5,6 types are the amplimer of totally 7 strain HCV coating raq genes

Table 3: respectively with the 1-6 genotype proteic expression plasmid pCI-E2t of totally 7 strain HCV coating E2 intramuscular injection immune mouse, immunity three times, each dosage 20 μ g are with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and the homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) the detection mice serum.Figure is the antibody positive rate (every group of 10 mouse, serum dilution in 1: 100) of each group mice serum different time points.

Table 4: respectively with the 1-6 genotype proteic expression plasmid pCI-E2t of totally 7 strain HCV coating E2 intramuscular injection immune mouse, immunity three times, each dosage 20 μ g are with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and the homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) the detection mice serum.Figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group mice serum at the absorbance value of different time antibody test.

Table 5: respectively with the 1-6 genotype proteic expression plasmid pCI-E2t of totally 7 strain HCV coating E2 Δ intramuscular injection immune mouse, immunity three times, each dosage 20 μ g are with homophyletic E2t (containing HVR1) and homophyletic E2t Δ (the having deleted HVR1) antibody in enzyme-linked immunosorbent assay (ELISA) the detection mice serum.Figure is the antibody positive rate (every group of 10 mouse, serum dilution in 1: 100) of each group mice serum different time points.

Table 6: respectively with the 1-6 genotype proteic expression plasmid pCI-E2t of totally 7 strain HCV coating E2 Δ intramuscular injection immune mouse, immunity three times, each dosage 20 μ g are with homophyletic E2t (containing HVR1) and homophyletic E2t Δ (the having deleted HVR1) antibody in enzyme-linked immunosorbent assay (ELISA) the detection mice serum.Figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group mice serum at the absorbance value of different time antibody test.

Table 7: detect in 1a type pCI-E2t and the pCI-E2t Δ plasmid immune serum at the proteic antibody of other genotype E2 with enzyme-linked immunosorbent assay (ELISA), figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group mice serum at the absorbance value of different time antibody test.

Table 8: detect in 1b type pCI-E2t and the pCI-E2t Δ plasmid immune serum at the proteic antibody of other genotype E2 with enzyme-linked immunosorbent assay (ELISA), figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group mice serum at the absorbance value of different time antibody test.

Table 9: detect in 2a type pCI-E2t and the pCI-E2t Δ plasmid immune serum at the proteic antibody of other genotype E2 with enzyme-linked immunosorbent assay (ELISA), figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group mice serum at the absorbance value of different time antibody test.

Table 10: 1a, 1b and 2a type raq gene mice immunized serum are to homophyletic and different strain HCVpp neutralizing effect after using HCV pseudovirus (HCVpp) for model analysis immunity for the third time, figure is the mean value (every group of 10 mouse, serum dilution in 1: 100) of each group mice serum neutralization ratio (%).

Table 11: different time points is respectively organized the anti-proteic antibody positive rate of different strain HCV coating E2 in the rabbit anteserum (every group of 10 rabbit, serum dilution in 1: 100).

Embodiment

Below in conjunction with embodiments of the invention and accompanying drawing enforcement of the present invention is elaborated; following examples are to be to implement under the prerequisite with the technical solution of the present invention; provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

Embodiment 1: contain the amplification of the 1-6 HCV genotype coating E2 protein gene of HVR1 and deletion HVR1:

1a, 1b, 2a type HCV envelope protein E1E2 gene are presented by professor C.M.Rice of U.S. Rockefeller university; 3a, 4,5,6 type HCV envelope protein E1E2 genes are presented by professor J.K.Ball of Britain Nottingham university.Gene order sees Table 1.According to 1-6 genotype (1a, 1b, 2a, 3a, 4,5,6 types) corresponding sequence design, the synthetic primer of totally 7 strain HCV coating raq genes, with polymerase chain reaction (PCR) amplification E2 albumen extracellular fragment gene (be equivalent to HCV polyprotein 364-661 amino acids residue, 364-383 amino acids residue is as the proteic secretion signal peptide of E2).The sequence of amplimer (upstream primer FP1, downstream primer RP) sees Table 2.Pcr amplification reagent is U.S. Promega company product.

Set up following reaction system at 0.5 milliliter of centrifuge tube:

Replenish two aqua sterilisas that steam to total reaction volume 50 μ l, carry out thermal cycle reaction: 94 ℃ of sex change 2 minutes, 94 ℃ of sex change 40 seconds with MJ PTC-100 type PCR instrument, annealed 40 seconds for 60 ℃, 72 ℃ were extended 1 minute 20 seconds, and carried out 30 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.Reaction product is carried out agarose gel electrophoresis, the purpose band is reclaimed, 1a, 1b, 2a, 3a, 4,6 type PCR products are cut with the NheI/XbaI enzyme, 5 type PCR products are cut with the XhoI/XbaI enzyme, and enzyme is cut product and inserted mammalian cell expression plasmid vector pCI-neo (U.S. Promega company product).The gene that inserts is carried out dna sequencing.The raq gene called after E2t of amplification, the E2 expression plasmid called after pCI-E2t of structure, structure is as shown in Figure 2.

Deleted the E2 albumen extracellular fragment gene (be equivalent to HCV polyprotein 364-661 amino acids residue, but lack 384-411 amino acids residue) of HVR1 fully with the two-step pcr legal system.The sequence of the amplimer of each pnca gene (upstream primer FP1, upstream primer FP2, downstream primer RP) sees Table 2.The primer FP2 and the RP of PCR reaction, other components are the same, and the thermal cycle reaction condition is the same, reaction product is carried out agarose gel electrophoresis, the purpose band is reclaimed, carry out the PCR reaction again, primer FP1 and RP, other components are the same, the thermal cycle reaction condition is the same, and reaction product is carried out agarose gel electrophoresis, and the purpose band is reclaimed, cut with the NheI/NotI enzyme, enzyme is cut product and is inserted mammalian cell expression plasmid vector pCI-neo (U.S. Promega company product).The gene that inserts is carried out dna sequencing.The raq gene called after E2t Δ of amplification, the E2 expression plasmid called after pCI-E2t Δ that makes up, its expression regulation structure is as shown in Figure 2: PCMV is a cytomegalovirus promoter, and E2 is HCV coating raq gene E2t or E2t Δ, and polyA is a SV40 virus polyadenylic acid sequence.

Table 1:HCV envelope protein E1E2 gene order

1a type H77 strain	Genbank?Accession：AF009606.1GI：2316097	Shown in SEQ ID NO:1
			1b type Con1 strain	Genbank?Accession：AJ238799.1GI：5420376	Shown in SEQ ID NO:2
2a type J6 strain	Genbank?Accession：D00944.1GI：221650	Shown in SEQ ID NO:3
			3a type UKN3A1.28C strain		Shown in SEQ ID NO:4
4 type UKN4.21.16 strains		Shown in SEQ ID NO:5
			5 type UKN5.15.7 strains		Shown in SEQ ID NO:6
6 type UKN6.5.8 strains		Shown in SEQ ID NO:7

Table 2: the primer sequence of amplification HCV coating raq gene

The evaluation of embodiment 2:1-6 HCV genotype coating E2 protein expressioning product:

With the DMEM nutrient solution that contains 10% foetal calf serum cultivator embryonic kidney (HEK) the 293T cell that goes down to posterity, when cell 80% merged, with 0.25% trypsin digestion and cell, going down to posterity was inoculated in 35mm Tissue Culture Dish, overnight incubation.With the 1-6 genotype of the above-mentioned structure of 4 μ g plasmids totally 7 strain HCV coating E2 protein expressing plasmid pCI-E2t (containing HVR1), pCI-E2t Δ (having deleted HVR1) and empty carrier pCI-neo respectively with 10 μ l transfection reagent liposome Lipofectamine (American I nvitrogen company product) mixings, transfection HEK 293T cell, operation is carried out according to operation instruction.After the plasmid-lipidosome mixture added Tissue Culture Dish, Tissue Culture Dish is placed 37 ℃, the cell culture incubator of 5% carbonic acid gas, saturated humidity, add the complete DMEM nutrient solution that 1ml contains 10% foetal calf serum after 8 hours, continue to cultivate 48 hours.Sucking-off culture supernatant then contains the cell of phosphate buffered saline buffer (PBS) the cracking adherent growth of 2%SDS and proteinase inhibitor (U.S. Roche company product) with 400 μ l.Cell culture supernatant and cell pyrolysis liquid are carried out sds polyacrylamide gel electrophoresis respectively, carrying out western blot again analyzes, detecting antibody is goat-anti HCV coating E2 protein polyclone antibody (U.S. BioDesign company product), enzyme mark second antibody is the anti-sheep IgG of rabbit (U.S. Sigma company product) of horseradish peroxidase (HRP) mark, with chemoluminescence method detection reaction band.Operation is all carried out with reference to " molecular cloning experiment guide second edition " (Science Press, 1995).The result shows: 1-6 genotype totally 7 strain HCV coating E2 albumen all can be at the HEK of plasmid transfection 293 cell inner expressions, and can secrete to cells and supernatant, and molecular weight is between 50,000 to 60,000 dalton; For same strain HCV, it is similar with secretion level with the proteic expression of E2 of deletion HVR1 to contain HVR1.

Further analyze HVR1 to proteic expression of E2 and excretory influence with enzyme-linked immunosorbent assay (ELISA).The HCV envelope protein is a high glycosylation albumen, lectin can be special N-glucosides on the absorption E2 albumen, thereby can be used for the proteic detection of E2.Lectin GNA (U.S. Sigma company product) is dissolved into concentration 20 μ g/ml with PBS, add enzyme mark microwell plate (U.S. Corning company product), every hole 0.1ml, place 4 ℃ of refrigerator overnight, absorb GNA next day, every hole is washed once with 0.2ml PBS damping fluid, every then hole adds PBS damping fluid (confining liquid) 0.1ml that contains 3% bovine serum albumin bletilla 0.05%Tween 20, room temperature was placed 2 hours, absorb confining liquid then, every hole adds the lysate or the culture supernatant 0.1ml (cell pyrolysis liquid and culture supernatant are all with 50 times of confining liquid dilutions) of the HEK 293T cell of plasmid pCI-E2t or the transfection of pCI-E2t Δ, room temperature was placed 2 hours, absorb cell pyrolysis liquid or culture supernatant then, every hole adds the goat-anti HCV coating E2 protein polyclone antibody 0.1ml with 1000 times of dilutions of confining liquid, room temperature was placed 40 minutes, absorb serum then, with PBS damping fluid (washing lotion) hole flushing that contains 0.05%Tween 20 5 times, every then hole adds goat anti-mouse igg (the U.S. Sigma company product) 0.1ml with the HRP mark of confining liquid dilution, room temperature was placed 40 minutes, absorb antibody then, with washing lotion hole flushing 5 times, every hole adds the substrate solution 0.1ml that contains TMB, the room temperature lucifuge was placed after 10 minutes, add 2M sulfuric acid and each 0.05ml of 30% superoxol, mixing is with the absorbance value of microplate reader detection 450nm wavelength, reference wavelength 630nm.The result all can detect HCV coating E2 albumen in shown in Figure 3 in cell pyrolysis liquid and cells and supernatant, whether HVR1 exists does not influence proteic expression of E2 and secretion.

Embodiment 3: mouse genetic immunization and antibody test:

With a large amount of preparations of plasmid extraction kit (German Qiagen company product) and the above-mentioned 1-6 genotype of purifying totally 7 strain HCV coating E2 protein expressing plasmid pCI-E2t, pCI-E2t Δ and empty carrier pCI-neo, be dissolved in PBS damping fluid (pH 7.4), adjust concentration to 100 μ g/ml, immunity Balb/C mouse, 10 mouse of every kind of plasmid immunity, every side tibialis anterior muscle injection plasmid solution 0.1ml, amounting to dosage is every mouse 20 μ g plasmids.Immunity is three times altogether, two weeks of pitch time.Each immune the day before yesterday, (time was respectively for 0,2,4 weeks, blood sampling time was decided to be for 0 week first) and the last immunity after every two weeks (totally 7 times, time was respectively for 6,8,10,12,14,16,18 weeks) get blood from mouse orbit, with E2t (containing HVR1), E2t Δ (having deleted HVR1) and the HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) the detection mice serum.E2t, the antibody test of E2t Δ: with plasmid pCI-E2t and pCI-E2t Δ respectively transfection be inoculated in the HEK 293T cell of 10cm plate, collecting cell culture supernatant after 72 hours.Lectin GNA (U.S. Sigma company product) is dissolved into concentration 20 μ g/ml with PBS, add enzyme mark microwell plate (U.S. Corning company product), every hole 0.1ml, place 4 ℃ of refrigerator overnight, absorb GNA next day, every hole is washed once with 0.2ml PBS damping fluid, every then hole adds PBS damping fluid (confining liquid) 0.1ml that contains 3% bovine serum albumin bletilla 0.05%Tween 20, room temperature was placed 2 hours, absorb confining liquid then, every hole adds the HEK 293T cells and supernatant 0.1ml of plasmid pCI-E2t or the transfection of pCI-E2t Δ, room temperature was placed 2 hours, absorb cells and supernatant then, every hole adds the mouse immune serum 0.1ml with the confining liquid dilution, room temperature was placed 40 minutes, absorb serum then, with PBS damping fluid (washing lotion) hole flushing that contains 0.05%Tween 20 5 times, every then hole adds goat anti-mouse igg (the U.S. Sigma company product) 0.1ml with the HRP mark of confining liquid dilution, room temperature was placed 40 minutes, absorb antibody then, with washing lotion hole flushing 5 times, every hole adds the substrate solution 0.1ml that contains TMB, the room temperature lucifuge was placed after 10 minutes, add 2M sulfuric acid and each 0.05ml of 30% superoxol, mixing is with the absorbance value of microplate reader detection 450nm wavelength, reference wavelength 630nm.Under the identical extent of dilution, it is antibody positive that pCI-E2t and pCI-E2t Δ immune serum absorbance value surpass 2.1 times of empty carrier pCI-neo immune serum average light absorption value.

The HVR1 antibody test: the 1-6 genotype synthetic peptide of totally 7 strain HCV coating E2 albumen HVR1 (gill biochemical company limited in Shanghai is synthetic) is dissolved into concentration 0.5mg/ml with DMSO, be diluted to concentration 0.05mg/ml with PBS, add enzyme mark microwell plate, every hole 0.1ml, place 4 ℃ of refrigerator overnight, absorb the synthetic peptide solution of HVR1 next day, every hole is washed once with 0.2ml PBS damping fluid, every then hole adds confining liquid 0.1ml, room temperature was placed after 2 hours, and with washing lotion hole flushing 5 times, every then hole adds the mice serum of dilution, detect HVR1 antibody, follow-up working method is the same.

The result: the 1-6 genotype is (totally 14 kinds of plasmids in the totally 7 strain HCV raq gene mice immunized, 10 mouse of every kind of plasmid immunity), after the first immunisation, surpass 80% in the pCI-E2t plasmid mice immunized serum and can detect homophyletic HVR1 antibody and homophyletic E2t (containing HVR1) antibody, but only have 1a, 2a, 3 and 5 genotype can detect homophyletic E2t Δ antibody, positive rate is respectively 10%, 30%, 20% and 20% (table 3).After the immunity for the second time, homophyletic HVR1 in the mice serum and homophyletic E2t antibody horizontal obviously raise by (table 4), and positive rate all reaches 100% (table 3), and homophyletic E2t Δ antibody horizontal also has rising, but positive rate all is lower than 50% (table 3).After the immunity, the E2t Δ antibody horizontal in the mice serum further raises by (table 4) for the third time, but positive rate all is no more than 60% (table 3).

After the first immunisation, homophyletic E2t Δ antibody positive rate promptly meets or exceeds 90% (table 5) in the plasmid pCI-E2t Δ mice immunized serum, after the immunity for the second time, each is organized the anti-homophyletic E2t of mice serum Δ antibody horizontal and obviously raises by (table 6), and positive rate reaches 100% (table 5).Homophyletic E2t antibody and homophyletic E2t Δ antibody horizontal similar (table 6) do not detect HVR1 antibody in all mice serums, it is consistent not contain HVR1 in the E2 albumen that this and plasmid pCI-E2t Δ are encoded.

With the corresponding antibodies in the different strain HCV coating E2 Protein Detection mice serum, the antibody horizontal basically identical in its trend and the mice serum at homophyletic E2t Δ.Table 7,8,9 shows with the corresponding antibodies in other 6 strain coatings E2t Protein Detection 1a, 1b and 2a type pCI-E2t and the pCI-E2t Δ plasmid mice immunized serum, significantly is lower than pCI-E2t Δ plasmid mice immunized serum at the proteic antibody horizontal of different strain E2 in the pCI-E2t plasmid immune serum.3, also significantly be lower than pCI-E2t Δ plasmid mice immunized serum at the proteic antibody horizontal of different strain E2 in 4, the 5 and 6 type pCI-E2t plasmid immune serums.

The result shows: HVR1 can efficiently induce antibody response, when having HVR1 among the E2, induce the effectiveness of antibody response significantly to reduce at the epitope beyond the HVR1, deletion HVR1 can significantly strengthen the immunogenicity of other epi-positions of E2 albumen, improves the protein induced cross-reacting antibody level of E2.

Show the antibody positive rate (%) of 3:6 genotype 7 strain pCI-E2t plasmid immune group mouse different times

Show the absorbance value of 4:6 genotype 7 plant type pCI-E2t plasmid immune group mouse different time antibody tests

Show the antibody positive rate (%) of 5:6 genotype 7 strain pCI-E2t Δ immune group mouse different times

Show the absorbance value of 6:6 genotype 7 plant type pCI-E2t Δ immune group mouse different time antibody tests

The absorbance value that detects at other genotype E2 protein antibodies in table 7:1a type pCI-E2t and the pCI-E2t Δ plasmid immune serum

The absorbance value that detects at other genotype E2 protein antibodies in table 8:1b type pCI-E2t and the pCI-E2t Δ plasmid immune serum

The absorbance value that detects at other genotype E2 protein antibodies in table 9:2a type pCI-E2t and the pCI-E2t Δ plasmid immune serum

Embodiment 4: in the virus of mouse immune serum and activation analysis:

With HCVpp and HCVcc is the neutralization activity of model analysis mouse immune serum to HCV.

HCV pseudovirion (HCV pseudoparticles, HCVpp) be a kind of embedded virus, inside is retroviral nucleocapsid, genome is a double-stranded RNA, coding has the reporter gene of being convenient to detect (green fluorescence protein gene or luciferase gene), the surface is a peplos, is inlaid with HCV coating E1, E2 albumen in the coating.The infection characterization of HCVpp is similar to HCV, and Bel7402 Huh7 commonly used or its deutero-clone such as Huh7.5 are the important models of estimating the antibody neutralizing effect as the target cell of its infection.HCV envelope protein expression plasmid and retrovirus packaging plasmid cotransfection HEK 293T cell can be obtained HCVpp.

Analyze the neutralization activity of antibody with HCVpp: with infectivity a certain amount of HCVpp of linearity range respectively with certain dilution mice serum (the control group mice serum and the raq gene mice immunized serum that comprise the empty plasmid immunity) mixing, placed 45 minutes for 37 ℃, add then and inoculate (elder generation absorbs former substratum before adding virus) in 96 orifice plates that the Huh7.5 target cell is arranged, cultivate after 5 hours for 37 ℃ and change complete DMEM substratum, continue to cultivate 48 hours, absorb nutrient solution, lysing cell, detect the uciferase activity in the cell pyrolysis liquid, uciferase activity is the quantitative target that HCVpp infects, and plain unit of enzyme activity represents with relative fluorescence.Cell pyrolysis liquid and luciferase detection reagent are U.S. Promega company product.In and percentage=(the control group serum plain unit of enzyme activity of relative fluorescence-raq gene immune serum of handling the hole is handled the plain unit of enzyme activity of relative fluorescence in hole) ÷ control group serum handle the plain unit of enzyme activity of relative fluorescence * 100% in hole.

HCV (the cell culture produced HCV that cell cultures produces, HCVcc) be real HCV, 2a genotype JFH-1 strain HCV full length genomic rna transfection Bel7402 Huh7 or its deutero-clone such as Huh7.5 with in-vitro transcription obtains can obtain HCVcc.Only have the JFH-1 strain can prepare HCVcc at present, and other strains HCV can not prepare HCVcc.The structure gene (comprising envelope protein gene in the structure gene) of other strains HCV is recombinated with the nonstructural gene of JFH-1 strain, may obtain chimeric HCVcc, this also is to be used for estimating antibody and active truer model.

Analyze the neutralization activity of antibody with HCVcc: with infectivity 1000FFU (foci forming unit)/mlHCVcc 0.4ml respectively with certain dilution mice serum (the control group mice serum and the raq gene mice immunized serum that comprise the empty plasmid immunity) mixing, final volume 0.8ml, placed 45 minutes for 37 ℃, add then and inoculate (elder generation absorbs former substratum before adding virus) in 96 orifice plates that the Huh7.5 target cell is arranged, cultivate after 5 hours for 37 ℃ and change complete DMEM substratum, continue to cultivate 48 hours, absorb nutrient solution then, use the formaldehyde fixed cell, detect the proteic Huh7.5 cell of expression of HCV with immunofluorescence method, detection antibody is the monoclonal antibody at HCV Nonstructural Protein NS5A, tracer antibody is fluorescein-labeled anti-mouse IgG, counts the fluorescence positive cells behind the EO under fluorescent microscope.In and percentage=(control group serum is handled the fluorescencepositive cell number-raq gene immune serum in hole and handled the fluorescencepositive cell number in hole) ÷ control group serum handle fluorescencepositive cell number * 100% in hole.

The result: table 10 shows that immune for the third time back 1a, 1b and 2a type raq gene immune serum are to homophyletic and different strain HCVpp neutralizing effect, as seen plasmid pCI-E2t immune serum and pCI-E2t Δ immune serum all can be effectively in and the infection of homophyletic HCVpp, its effect is similar; But pCI-E2t Δ immune serum significantly is better than the pCI-E2t immune serum to the neutralizing effect of different strain HCVpp.Other 4 genotype E2 immune serums to the neutralizing effect of HCVpp similarly, the be significantly increased neutralization activity of mouse immune serum of deletion HVR1.

With JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1 is the neutralization activity of model evaluation mouse immune serum, Fig. 4 shows the neutralizing effect to this three strain HCVcc of 1a, 1b and 2a genotype E2 immune serum, and visible pCI-E2t Δ immune serum all significantly is better than the neutralizing effect of pCI-E2t immune serum to the neutralizing effect of three strain HCVcc.

Table 10:1a, 1b and 2a type raq gene mice immunized serum are in homophyletic and the different strain pseudovirus (pp) and percentage (%)

Embodiment 5: the expressing cho cell plasmid that makes up 1b type HCV coating E2 albumen (contain HVR1 and do not contain HVR1):

Be template with plasmid pCI-E2t, pCI-E2t Δ respectively, pcr amplification 1b type Con1 strain HCV coating raq gene E2t and E2t Δ gene, upstream primer FP1, downstream primer RP-C, the sequence of FP1 and RP-C primer sees Table 2.3 ends of downstream primer RP-C contain the encoding sequence of 6 Histidines, and 6 histidine residues are as the label of protein purification.The PCR product is cut with NheI and Sa1I enzyme, inserts the expressing cho cell plasmid pCIDA-GS-neo with independent intellectual property right that this laboratory makes up then.PCR reaction system and thermal cycle conditions are with reference to described in the embodiment 1.Operation is all carried out with reference to " molecular cloning experiment guide second edition " (Science Press, 1995).The plasmid structure that obtains is as shown in Figure 5: PCMV is a cytomegalovirus CMV promotor, introD is the intron donor sequences, GS is the glutaminase synthase gene, introA is the intron receptor sequence, E2 is HCV coating raq gene E2t (containing HVR1) or E2t Δ (not containing HVR1), and polyA is a SV40 virus polyadenylic acid sequence.

Embodiment 6: structure and the proteic expression of E2 and the purifying of the suspension growth CHO-K1 cell strain of stably express HCV coating E2 albumen (contain HVR1 and do not contain HVR1):

Go down to posterity with the DMEM nutrient solution that contains 10% foetal calf serum and to cultivate the CHO-K1 cell, when cell 80% merges, with 0.25% trypsin digestion and cell, be inoculated in the 35mm Tissue Culture Dish, next day is with above-mentioned E2 expression plasmid of 4 μ g and 10 μ l lipofectamine Lipofectamine (American I nvitrogen product) mixings, transfection CHO-K1 cell, operation is carried out according to operation instruction.After the plasmid-lipidosome mixture added Tissue Culture Dish, Tissue Culture Dish is placed 37 ℃, the cell culture incubator of 5% carbonic acid gas, saturated humidity, add the DMEM nutrient solution that 1ml contains 10% foetal calf serum after 8 hours, continue to cultivate 48 hours.Then with cell with 0.25% tryptic digestion, be inoculated in 5 100mm Tissue Culture Dishs, GMEM-S nutrient solution (U.S. Sigma company product) with the foetal calf serum that contains 10% dialysis is cultivated, add first sulfonyl sulfuric acid amine (MSX) (U.S. Sigma company product) to final concentration 25 μ mol/L, cell begins death after 3-4 days, the clone that the back culture dish formation of two weeks is formed by individual cells propagation, choose single clone and be inoculated in 96 orifice plates, continue to cultivate with the GMEM-S nutrient solution that contains 25 μ mol/L MSX, the back culture supernatant proteic expression of westernblot method detection E2 of one week, to the highest clone of expression level with tryptic digestion, limiting dilution is inoculated 96 orifice plates, continue to cultivate with the GMEM-S nutrient solution of 25 μ mol/L MSX, after two weeks, there is monoclonal hole inner cell to be transferred to 24 orifice plates length, continue to cultivate, cell is paved with orifice plate and is transferred to amplification culture in the little square vase later on, use serum free medium (JRH product) to substitute the GMEM-S substratum gradually subsequently, make cell adapt to complete serum free medium gradually, change suspension growth into from adherent growth.Use suspension cell instead shake-flask culture, volume of culture increases to 100 milliliters gradually.The cells and supernatant of results concentrates with ultrafiltration with centrifugal removal cell again, and the method for closing chromatography with the nickel ion metal-chelate is carried out purifying (German Qiagen company product) subsequently, can obtain 30 milligrams in E2 albumen in each hundred milliliters of nutrient solution.Fig. 6 is that the proteic SDS-PAGE of purified product E2t and E2t Δ analyzes.

Embodiment 7: use E2t and E2t Δ protein immunization rabbit:

Respectively recombinant expressed 1b type E2t and E2t Δ albumen are adjusted concentration to 0.25mg/ml with the PBS damping fluid, with the abundant mixing of equal-volume oil/water and milk formulation adjuvant ISA720 (French Seppic company product), room temperature was placed 30 minutes, then in 2 subcutaneous injection immunizing rabbits of back part, every some injection 0.2ml, 0.4ml contains E2t or E2t Δ albumen 50 μ g altogether, two weeks, once immunity was three times altogether.With same dosage bovine serum albumin (U.S. Sigma company product) associating ISA720 adjuvant immunity rabbit in contrast.Every group of 10 rabbit.

Embodiment 8: in antibody test in the rabbit anteserum and the virus and activation analysis

Regularly E2t antibody, E2t Δ antibody, HVR1 antibody and the serum in the detection rabbit anteserum is to the neutralization activity of HCV.In the antibody test, the enzyme labelled antibody goat anti-rabbit igg (U.S. Sigma company product) of HRP mark, the consumptive material of other operations and use, reagent are all with described in the embodiment 3.

The result: the rabbit of E2t protein immunization after first immunisation in the serum the positive rate of rotation of homophyletic E2t and HVR1 antibody be 100%, homophyletic E2t Δ antibody male rotary rate is 30%.After the immunity for the second time, homophyletic E2t Δ antibody male rotary rate is 50%.After the immunity, homophyletic E2t Δ antibody male rotary rate is 60% (Fig. 7) for the third time.Antibody horizontal changes sees Fig. 8, and the growth of visible HVR1 antibody horizontal and decay are all very fast, and the decay of E2t Δ antibody horizontal is slower.The rabbit of E2t Δ protein immunization after first immunisation in the serum the positive rate of rotation of homophyletic E2t and E2t Δ antibody be 90%, after the immunity for the second time, homophyletic E2t and E2t Δ antibody male rotary rate are 100%.Do not detect HVR1 antibody always, this with E2t Δ albumen in do not contain HVR1 and conform to.

With the corresponding antibodies in the different strain HCV coating E2 Protein Detection mice serum, the antibody horizontal basically identical in its trend and the mice serum at homophyletic E2t Δ.Anti-other the 6 strains HCV coating E2 protein antibodies positive rate of E2t protein immunization inductive significantly is lower than E2t Δ protein immunization inductive E2 antibody horizontal (table 11).Anti-other the 6 strains HCV coating E2 protein antibodies level of E2t protein immunization inductive also significantly is lower than E2t Δ protein immunization inductive E2 antibody horizontal.

The result shows: the feature similarity of recombinant protein immunity and genetic immunization inductive antibody response, be that HVR1 significantly suppresses conserved epitope inductive antibody response in the E2 albumen, deletion HVR1 can significantly strengthen the immunogenicity of conserved epitope in the E2 albumen, improves the protein induced cross-reacting antibody level of E2.

With the neutralization activity of HCVpp model evaluation rabbit anteserum, method is with described in the embodiment 4.The result shows: the rabbit anteserum of E2t and E2t Δ protein immunization is similar to the neutralization activity of homophyletic HCVpp, and E2t Δ protein immunization serum significantly is better than E2t protein immunization serum (Fig. 9) to the neutralizing effect of different strain HCVpp.With JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1 is the neutralization activity of model evaluation mouse immune serum, and method is with described in the embodiment 4.The result shows: the rabbit anteserum of E2t Δ protein immunization all significantly is better than the rabbit anteserum (Figure 10) of E2t protein immunization to the neutralizing effect of three strain HCVcc.

Table 11:1b type E2 protein immunization rabbit anteserum is in the anti-different proteic antibody positive rate of strain HCV coating E2 (%) of different time points

Embodiment 9: with the E2 antibody among E2t and the E2t Δ Protein Detection HCV infected person anteserum

E2t and E2t Δ albumen are diluted to concentration 20 μ g/ml with the PBS damping fluid, add enzyme mark microwell plate (U.S. Corning company product), every hole 0.1ml, place 4 ℃ of refrigerator overnight, absorb E2 albumen next day, every hole is washed once with 0.2ml PBS damping fluid, every then hole adds PBS damping fluid (confining liquid) 0.1ml that contains 3% bovine serum albumin bletilla 0.05%Tween20, room temperature was placed 2 hours, every hole adds HCV infected person anteserum or the HCV with confining liquid dilution in 1: 100, the serum 0.1ml of health adult that HBV antibody and detection of nucleic acids are all negative, room temperature was placed 40 minutes, absorb serum then, with PBS damping fluid (washing lotion) hole flushing that contains 0.05%Tween 20 5 times, every then hole adds goat anti-human igg's (U.S. Sigma company product) 0.1ml with the HRP mark of confining liquid dilution, room temperature was placed after 40 minutes, absorb antibody, with washing lotion hole flushing 5 times, every hole adds the substrate solution 0.1ml that contains TMB, the room temperature lucifuge was placed 10 minutes, add 2M sulfuric acid and each 0.05ml of 30% superoxol then, mixing is with the absorbance value of microplate reader detection 450nm wavelength, reference wavelength 630nm.

Detected 30 parts of HCV infected person anteserums and 10 parts of health adult's serum that HCV, HBV antibody and detection of nucleic acids are all negative, the result as shown in figure 11, the antibody horizontal of E2t Δ Protein Detection is apparently higher than the antibody of E2t Protein Detection.

Claims

1. a modified hepatitis C virus coating E2 albumen and an encoding gene thereof is characterized in that described modified hepatitis C virus coating E2 albumen is meant the peptide section of having deleted the hypervariable region 1 in the hepatitis C virus coating E2 albumen.

2. a kind of modified hepatitis C virus coating E2 albumen according to claim 1 and the application of encoding gene in preparation prevention and therapeutic hcv vaccine thereof.

3. a kind of modified hepatitis C virus coating E2 albumen according to claim 1 and the application of encoding gene in the immunological diagnostic reagent of preparation infection with hepatitis C virus thereof.