CN102206256B - HCV envelope protein E2 with deleting hypervariable region 1 and use thereof - Google Patents
HCV envelope protein E2 with deleting hypervariable region 1 and use thereof Download PDFInfo
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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
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) Envelope 2 protein and the application in hcv vaccine and HCV infection immunity diagnostic reagent thereof.
Background technology
Hepatitis C virus (hepatitis C virus, HCV) belongs to flaviviridae, is one of the main virulence factor of the acute hepatitis, chronic hepatitis propagated through blood.HCV is single strand plus RNA virus, only has an open reading frame, approximately 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 in the synthetic process of guide RNA, without correct functioning, makes HCV genome mutation very high, according to the difference of its genome nucleotide sequence, at present HCV is divided into six genotype.Current global HCV the infected exceedes 1.7 hundred million, and approximately 80% acute HCV infection can develop into chronic infection.HCV chronic infection can cause the hepatopathy in whole latter stage such as liver cirrhosis, hepatocellular carcinoma, also with multiple Extrahepatic diseases, as closely related in lymphoma, Combination cryoglobulinemia, glomerulonephritis, porphyria cutanea tarda, diabetes etc.Although had immunology and nucleic acid detection method, these methods all can not be got rid of false negative completely, and in addition, " window phase " of infection, because antibody horizontal is low and virus replication level is low, more easily causes undetected.Treatment aspect, current standard treatment is to combine to use ribavirin and polyethyleneglycol modified Interferon, rabbit (IFN-α), different genotype HCV has very big-difference to the susceptibility of this therapy, and it is only 50% to the lasting response rate of this therapy that I type HCV infects.Although the hepatitis C case infecting through blood and blood product approach in global range with last century the nineties decline to a great extent compared with in the past, 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 route of transmission is not clear still can be in any more in the world.The key measure of controlling HCV infection still depends on develops effective vaccine, and since being cloned from HCV genome in 1989, the research of hcv vaccine is study hotspot in developed countries such as American-European and Japan always, but does not have so far effective vaccine to come out.
The surface protein of virus is the key protein of induction neutralizing antibody, and the surface protein of HCV is envelope protein.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 invade target cell inside.The HCV acceptor identifying at present has four kinds at least, comprises tetratransmembrane element family member CD81, 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 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 yet studied clearly at present, merges relevant but may enter the later film of cell with HCV.HCV Envelope 2 protein is equivalent to 384-746 amino acids in 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, E2 albumen can be combined with HCV acceptor CD81 and SR-BI, and blocking-up E2 albumen is combined with CD81 or the SR-BI on target cell surface, all can block the infection of HCV.In E2 albumen, identify a large amount of linearities or space conformation dependency neutralizing antibody epi-position, all can effectively suppress in vitro and in vivo HCV for 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 variation of the height of E2 albumen is that HCV immune evasion forms one of chronically infected major reason.
The variation of E2 albumen Non-random distribution, being arranged in aminoterminal 27 amino-acid residues of E2 albumen (being equivalent to the 384-410 amino acids of HCV amyloid protein precursor) is the highest region 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.).Infection chronicity and liver cirrhosis process after variation frequency and the acute HCV infection in this region 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 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 participates in the combination of E2 albumen and SR-BI acceptor, deletes HVR1 and can make the infectivity of HCV significantly reduce.In HVR1, contain one or more neutralizing epitopes, there is hyperimmunization originality.HVR1 antibody in vitro can be efficiently in and HCV infect, but the neutralizing effect of HVR1 antibody has strict strain specificity, can only neutralize the infection of same strain virus for the antibody of a certain strain HCV HVR1, to the infection of other strains HCV without neutralizing effect.Generally acknowledge that at present thereby HCV utilizes HVR1 escape from immune to reply to realize persistent infection (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 object of this invention is to provide a kind of modified hepatitis C virus (hepatitis C virus, HCV) Envelope 2 protein and encoding gene thereof, and the application in preparation prevention and therapeutic hcv vaccine, preparation HCV infection immunity diagnostic reagent of described albumen and encoding gene thereof.
The inventor thinks, although the variability of E2 albumen is high, 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 basic functional structure.Large quantity research also proves that 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 immunogenicity, we have compared E2 albumen and have deleted the E2 albumen of HVR1 in antibody response and the neutralizing effect of antibody to HCV of mouse and rabbit induction, we find that HVR1 has significant restraining effect to the immunogenicity of these conservative antigen epi-positions, with the E2 albumen that contains HVR1 or corresponding encoding gene immunity animalcule, in the E2 antibody inducing, be mainly the antibody for HVR1, very low with the antibody proportion of exterior domain epitope for HVR1.Delete HVR1 significantly strengthen E2 protein induced for HVR1 the antibody with exterior domain epitope, immune serum is to the neutralization activity of other strains HCV also corresponding significantly improving.We also find, delete HVR1 and can obviously improve reacting of E2 albumen and HCV infected person anteserum.These results suggest, delete HVR1 and can significantly strengthen the ability of HCV Envelope 2 protein induction cross-reacting antibody (reacting with different strain HCV envelope protein) and cross neutralization antibody (neutralizing different strain HCV infection), the completely newly strategy of one that represents the research and development of HCV vaccine, this albumen also can be used for the immunodiagnosis that HCV infects.
First the present invention uses the method for genetic immunization (claiming again nucleic acid immunization, DNA immunization) to contain HVR1 and the raq gene immune mouse respectively of having deleted HVR1, analyze the impact of HVR1 on the genotypic E2 protein immunization of 1-6 originality, and with HCV pseudovirus (HCV pseudoparticles, HCVpp) and cell cultures produce HCV (cell culture produced HCV, HCVcc) be the neutralization activity of model analysis mouse immune serum to HCV.Result shows: HVR1 has significant restraining effect to the immunogenicity of Envelope 2 protein, and in the time there is HVR1, the conservative antigen epi-position in E2 albumen is difficult to induce corresponding antibody, and immune serum is very low to the neutralization activity of different strain HCV; After deleting HVR1, in E2 albumen, conservative antigen epi-position can efficiently be induced corresponding antibody, and immune serum significantly improves the neutralization activity of different strain HCV; Delete HVR1 and can also improve E2 albumen and HCV infected person anteserum's combination activity.In 6 HCV genotype Envelope 2 proteins, all there is similar phenomenon.
The 1b HCV genotype Envelope 2 protein of having used expressing cho cell subsequently, comprises two kinds of forms that contain HVR1 and deleted HVR1.Expression product is purified by affinity chromatography method, is then adjuvant with ISA720, and immunizing rabbit detects antibody the neutralization activity of serum analysis to HCV in rabbit anteserum, and the combination activity of HCV infected person anteserum and two kinds of form E2 albumen.Result shows: delete HVR1 and can significantly strengthen the protein induced cross-reacting antibody of E2 and cross neutralization antibody, the E2 albumen of deleting HVR1 has important use in 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 Envelope 2 protein genes: according to gene order design, the synthetic primer of corresponding Envelope 2 protein, the HCV Envelope 2 protein gene that contains HVR1 and deletion HVR1 with polymerase chain reaction (PCR) amplification, pcr amplification product, with inserting mammalian cell expression vector after restriction endonuclease digestion, then carries out DNA sequencing to raq gene.
The evaluation of 2.HCV Envelope 2 protein expression product: by expression plasmid transfected with human embryonic kidney (HEK) the 293T cell building, with the E2 albumen in enzyme-linked immunosorbent assay (ELISA) and western blot analysis of cells culture supernatant and cell pyrolysis liquid, be to detect antibody with the polyclonal antibody of anti-E2 albumen.
3. mouse genetic immunization and Serum Antibody Detection: by above-mentioned E2 protein expressing plasmid immune Balb/C mouse respectively, immunity three times, regularly gathers serum, detects E2 antibody and HVR1 antibody in mice serum.
Mouse immune serum in and activation analysis: with HCVpp and HCVcc be the neutralization activity of model analysis mouse immune serum to HCV.
5. build the expressing cho cell plasmid of 1b type HCV Envelope 2 protein: the expressing cho cell plasmid pCIDA-GS-neo respectively the 1b type HCV coating raq gene insertion that contains HVR1 and deletion HVR1 to independent intellectual property right (is shown in the Chinese patent ZL 200610024202.5 that the applicant has authorized, denomination of invention is: a kind of method of expressing hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion, invention is artificial: Zhao Ping, Liao little Ling, Cao Jie, Qi Zhongtian.), then DNA sequencing is identified.
6. the structure of the Chinese hamster ovary celI strain of stably express 1b type HCV Envelope 2 protein and cell cultures and protein purification: with liposome by above-mentioned plasmid transfection CHO-K1 cell, obtain the CHO-K1 cell clone of stable transfection with first sulfonyl sulfuric acid amine (MSX) screening, then carry out the domestication of serum-free suspension culture, make cell be adapted to serum-free culture basal growth completely.Collecting cell culture supernatant, centrifugal, ultrafiltration, then purifies E2 albumen with affinity chromatography.
7. the E2 protein immunization rabbit that contains HVR and deleted HVR1: fully mix with the E2 albumen and the immunological adjuvant ISA720 that have deleted HVR1 containing HVR respectively, immunizing rabbit, immunity three times, regularly gathers rabbit anteserum.
Serum Antibodies of Rabbits detect with in and activation analysis: detecting E2 antibody and HVR1 antibody in Rabbit Sera, is the neutralization activity of model analysis rabbit anteserum with HCVpp and HCVcc.
9 use contain HVR and have deleted the E2 antibody in the Protein Detection HCV infected person anteserum of HVR1: by the E2t of equivalent and E2t Δ albumen coated elisa plate, for detection of the E2 antibody in HCV infected person anteserum.
The invention provides a kind of modified hepatitis C virus (hepatitis C virus, HCV) Envelope 2 protein and encoding gene thereof, described modified hepatitis C virus Envelope 2 protein refers to the peptide section of having deleted the hypervariable region 1 in hepatitis C virus Envelope 2 protein.
In hepatitis C virus Envelope 2 protein, the peptide section (HVR1) of hypervariable region 1 is aminoterminal 27 amino acid of 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 preparing infection with hepatitis C virus thereof.
The present invention has deleted the hypervariable region 1 in HCV Envelope 2 protein, and the immunogenicity of this HCV Envelope 2 protein is significantly better than the HCV Envelope 2 protein that contains hypervariable region 1, and the ability of induction antibody response significantly strengthens; The ability of induction cross reacting antibody (antibody reacting with other genotype, other strains HCV envelope protein) is significantly better than the HCV Envelope 2 protein that contains hypervariable region 1; The ability of induction cross neutralization antibody (neutralizing other genotype, the infective antibody of other strains HCV) is significantly better than the HCV Envelope 2 protein that contains hypervariable region 1; Significantly be better than with reacting of envelope protein antibody in HCV infected person anteserum the HCV Envelope 2 protein that contains hypervariable region 1.
The present invention has proved that HVR1 has remarkable restraining effect to the immunogenicity of conservative antigen epi-position in HCV Envelope 2 protein, delete HVR1 and can effectively strengthen HCV Envelope 2 protein induction cross-reacting antibody and cross neutralization antibody, for research and development hcv vaccine provides a kind of effective antigen layout strategy, and the immunology diagnosis infecting for HCV provides a kind of new detectable antigens.
Accompanying drawing explanation
Fig. 1: the amyloid protein precursor of HCV genome encoding and the structure of Envelope 2 protein.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).Approximately 30 amino acid composition cross-film districts of Envelope 2 protein carboxyl terminal, all the other are extracellular fragment, 27 amino acid composition hypervariable regions 1 of aminoterminal (HVR1), the part that HVR1 is extracellular fragment.Numeral is amino acid position.
The structure of Fig. 2: pCI-E2t and pCI-E2t Δ plasmid.Signal peptide and the extracellular fragment of pCI-E2t coding E2 albumen, 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 cytomegalovirus promoter, and polyA is SV40 virus polyadenylic acid sequence.
Fig. 3: respectively by 1-6 genotype expression plasmid pCI-E2t and the pCI-E2t Δ transfection HEK293T cell of totally 7 strain HCV Envelope 2 proteins, with the E2t (containing HVR1) in enzyme-linked immunosorbent assay detection cell pyrolysis liquid neutral incubation supernatant and E2t Δ albumen (containing HVR1).
Fig. 4: the neutralizing effect of the mice serum that the HCV (HCVcc) producing by cell cultures is model evaluation 1a, 1b and the immunity of 2a type raq gene to JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1, figure is the mean value (every group of 10 mouse, serum dilution in 1: 100) of each group of mice serum neutralization ratio (%).
Fig. 5: for the structure of the E2 protein expressing plasmid of transfection CHO-K1 cell.PCMV is cytomegalovirus CMV promotor, introD is intron donor sequences, GS is glutaminase synthase gene, introA is intron receptor sequence, E2 is HCV coating raq gene E2t (containing HVR1) or E2t Δ (not containing HVR1), and polyA is SV40 virus polyadenylic acid sequence.
Fig. 6: with the SDS-PAGE analysis of the recombinant expressed 1b type HCV coating E2t of CHO-K1 cell and E2t Δ albumen.
Fig. 7: fully mix with HCV coating E2t and E2t Δ albumen and adjuvant ISA720 respectively, immunizing rabbit, immunity three times, each dosage 50 μ g, with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) 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 rabbit anteserum.
Fig. 8: with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) 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 Serum Antibodies of Rabbits detection.
Fig. 9: analyze the neutralizing effect of the rear rabbit anteserum of immunity for the third time to HCVpp, figure is the mean value (every group of 10 rabbit, serum dilution in 1: 100) of each group of rabbit anteserum neutralization ratio (%).
Figure 10: analyze the neutralizing effect of the rear rabbit anteserum of immunity for the third time to JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1, figure is the mean value (every group of 10 rabbit, serum dilution in 1: 100) of each group of rabbit anteserum neutralization ratio (%).
Figure 11: with equivalent E2t and the coated enzyme mark microwell plate of E2t Δ albumen, with the corresponding antibodies in enzyme-linked immunosorbent assay (ELISA) detection 30 routine HCV the infecteds and 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, the 6 types nucleotide sequence of totally 7 strain HCV coating E1E2 genes
Table 2:1a, 1b, 2a, 3,4,5, the 6 types amplimer of totally 7 strain HCV coating raq genes
Table 3: respectively by the 1-6 genotype expression plasmid pCI-E2t intramuscular injection immune mouse of totally 7 strain HCV Envelope 2 proteins, immunity three times, each dosage 20 μ g, with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) detection mice serum.Figure is the antibody positive rate (every group of 10 mouse, serum dilution in 1: 100) of each group of mice serum different time points.
Table 4: respectively by the 1-6 genotype expression plasmid pCI-E2t intramuscular injection immune mouse of totally 7 strain HCV Envelope 2 proteins, immunity three times, each dosage 20 μ g, with homophyletic E2t (containing HVR1), homophyletic E2t Δ (having deleted HVR1) and homophyletic HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) detection mice serum.Figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group of mice serum at the absorbance value of different time antibody test.
Table 5: respectively by the 1-6 genotype expression plasmid pCI-E2t Δ intramuscular injection immune mouse of totally 7 strain HCV Envelope 2 proteins, immunity three times, each dosage 20 μ g, with homophyletic E2t (containing HVR1) and homophyletic E2t Δ (having deleted HVR1) antibody in enzyme-linked immunosorbent assay (ELISA) detection mice serum.Figure is the antibody positive rate (every group of 10 mouse, serum dilution in 1: 100) of each group of mice serum different time points.
Table 6: respectively by the 1-6 genotype expression plasmid pCI-E2t Δ intramuscular injection immune mouse of totally 7 strain HCV Envelope 2 proteins, immunity three times, each dosage 20 μ g, with homophyletic E2t (containing HVR1) and homophyletic E2t Δ (having deleted HVR1) antibody in enzyme-linked immunosorbent assay (ELISA) detection mice serum.Figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group of mice serum at the absorbance value of different time antibody test.
Table 7: with the antibody for other genotype E2 albumen in enzyme-linked immunosorbent assay (ELISA) detection 1a type pCI-E2t and pCI-E2t Δ plasmid immune serum, figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group of mice serum at the absorbance value of different time antibody test.
Table 8: with the antibody for other genotype E2 albumen in enzyme-linked immunosorbent assay (ELISA) detection 1b type pCI-E2t and pCI-E2t Δ plasmid immune serum, figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group of mice serum at the absorbance value of different time antibody test.
Table 9: with the antibody for other genotype E2 albumen in enzyme-linked immunosorbent assay (ELISA) detection 2a type pCI-E2t and pCI-E2t Δ plasmid immune serum, figure is the mean value (every group 10 mouse, serum 1: 100 dilution) of each group of mice serum at the absorbance value of different time antibody test.
Table 10: the mice serum that is rear 1a, 1b of model analysis immunity for the third time and the immunity of 2a type raq gene with HCV pseudovirus (HCVpp) is to homophyletic and different strain HCVpp neutralizing effect, figure is the mean value (every group of 10 mouse, serum dilution in 1: 100) of each group of mice serum neutralization ratio (%).
Table 11: different time points is respectively organized the antibody positive rate (every group of 10 rabbit, serum dilution in 1: 100) of anti-different strain HCV Envelope 2 protein in rabbit anteserum.
Embodiment
Below in conjunction with embodiments of the invention and accompanying drawing, enforcement of the present invention is elaborated; following examples are to implement under take technical solution of the present invention as prerequisite; provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1: the amplification containing HVR1 with the 1-6 HCV genotype Envelope 2 protein gene of deletion HVR1:
1a, 1b, 2a type HCV envelope protein E1 raq gene are presented by professor C.M.Rice of Rockefeller university of the U.S.; 3a, 4,5,6 type HCV envelope protein E1 raq genes are presented by professor J.K.Ball of Nottingham university of Britain.Gene order is in 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 secretion signal peptide of E2 albumen).The sequence of amplimer (upstream primer FP1, downstream primer RP) is in table 2.Pcr amplification reagent is Promega company of U.S. product.
Set up following reaction system at 0.5 milliliter of centrifuge tube:
Supplement two aqua sterilisas that steam to total reaction volume 50 μ l, carry out thermal cycle reaction with MJ PTC-100 type PCR instrument: 94 ℃ of sex change 2 minutes, 94 ℃ of sex change 40 seconds, anneal 40 seconds for 60 ℃, 72 ℃ are extended 1 minute 20 seconds, carry out altogether 30 circulations, then 72 ℃ are extended 5 minutes, and temperature is down to 4 ℃ and is finished reaction.Reaction product is carried out agarose gel electrophoresis, object band is reclaimed, 1a, 1b, 2a, 3a, 4,6 type PCR products are cut with NheI/XbaI enzyme, 5 type PCR products are cut with XhoI/XbaI enzyme, and enzyme is cut product and inserted mammalian cell expression plasmid vector pCI-neo (Promega company of U.S. product).The gene inserting is carried out to 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 with two-step PCR preparation.The sequence of the amplimer (upstream primer FP1, upstream primer FP2, downstream primer RP) of each pnca gene is in table 2.FP2 and the RP for primer of PCR reaction, other components are the same, and thermal cycle reaction condition is the same, reaction product is carried out agarose gel electrophoresis, object band is reclaimed, then carry out PCR reaction, FP1 and RP for primer, other components are the same, thermal cycle reaction condition is the same, and reaction product is carried out agarose gel electrophoresis, and object band is reclaimed, cut with NheI/NotI enzyme, enzyme is cut product and is inserted mammalian cell expression plasmid vector pCI-neo (Promega company of U.S. product).The gene inserting is carried out to DNA sequencing.The raq gene called after E2t Δ of amplification, the E2 expression plasmid called after pCI-E2t Δ building, its expression regulation structure is as shown in Figure 2: PCMV is cytomegalovirus promoter, and E2 is HCV coating raq gene E2t or E2t Δ, and polyA is SV40 virus polyadenylic acid sequence.
Table 1:HCV envelope protein E1 raq gene sequence
| 1a type H77 strain | Genbank Accession：AF009606.1GI：2316097 | As shown in SEQ ID NO:1 |
| 1b type Con1 strain | Genbank Accession：AJ238799.1GI：5420376 | As shown in SEQ ID NO:2 |
| 2a type J6 strain | Genbank Accession：D00944.1GI：221650 | As shown in SEQ ID NO:3 |
| 3a type UKN3A1.28C strain | As shown in SEQ ID NO:4 | |
| 4 type UKN4.21.16 strains | As shown in SEQ ID NO:5 | |
| 5 type UKN5.15.7 strains | As shown in SEQ ID NO:6 |
| 6 type UKN6.5.8 strains | As 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 Envelope 2 protein expression 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% merges, with 0.25% trypsin digestion and cell, go down to posterity and be inoculated in 35mm Tissue Culture Dish, overnight incubation.By the 1-6 genotype of the above-mentioned structure of 4 μ g plasmid, totally 7 strain HCV Envelope 2 protein expression plasmid pCI-E2t (containing HVR1), pCI-E2t Δ (having deleted HVR1) and empty carrier pCI-neo mix with 10 μ l transfection reagent liposome Lipofectamine (American I nvitrogen company product) respectively, transfection HEK 293T cell, operation is carried out according to operation instruction.After plasmid-lipidosome mixture is added to Tissue Culture Dish, Tissue Culture Dish is placed in to 37 ℃, the cell culture incubator of 5% carbonic acid gas, saturated humidity, after 8 hours, adds the complete DMEM nutrient solution of 1ml containing 10% foetal calf serum, continue to cultivate 48 hours.Then sucking-off culture supernatant, the cell with 400 μ l containing phosphate buffered saline buffer (PBS) the cracking adherent growth of 2%SDS and proteinase inhibitor (Roche company of U.S. product).Cell culture supernatant and cell pyrolysis liquid are carried out respectively to sds polyacrylamide gel electrophoresis, carry out again western blot analysis, detecting antibody is goat-anti HCV Envelope 2 protein polyclonal antibody (BioDesign company of U.S. product), enzyme mark second antibody is the anti-sheep IgG of rabbit (Sigma company of U.S. product) of horseradish peroxidase (HRP) mark, with chemoluminescence method detection reaction band.Operation is all carried out with reference to " the molecular cloning experiment guide second edition " (Science Press, 1995).Result shows: 1-6 genotype totally 7 strain HCV Envelope 2 proteins 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, contain HVR1 similar with the expression and secretion level of the E2 albumen of deletion HVR1.
Further analyze the impact of the expression and secretion of HVR1 on E2 albumen with enzyme-linked immunosorbent assay (ELISA).HCV envelope protein is high glycosylation albumen, lectin can be special N-glucosides on absorption E2 albumen, thereby can be used for the detection of E2 albumen.Lectin GNA (Sigma company of U.S. product) is dissolved into concentration 20 μ g/ml with PBS, add enzyme mark microwell plate (Corning company of U.S. product), every hole 0.1ml, be placed in 4 ℃ of refrigerator overnight, absorb GNA next day, every hole is washed once with 0.2ml PBS damping fluid, then every hole adds PBS damping fluid (confining liquid) 0.1ml containing 3% bovine serum albumin bletilla 0.05%Tween 20, room temperature is placed 2 hours, then absorb confining liquid, every hole adds 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 is placed 2 hours, then absorb cell pyrolysis liquid or culture supernatant, every hole adds the goat-anti HCV Envelope 2 protein polyclonal antibody 0.1ml with 1000 times of dilutions of confining liquid, room temperature is placed 40 minutes, then absorb serum, with PBS damping fluid (washing lotion) hole flushing containing 0.05%Tween 20 5 times, then every hole adds goat anti-mouse igg (Sigma company of the U.S. product) 0.1ml with the HRP mark of confining liquid dilution, room temperature is placed 40 minutes, then absorb antibody, with washing lotion hole flushing 5 times, every hole adds the substrate solution 0.1ml containing TMB, room temperature lucifuge was placed after 10 minutes, add 2M sulfuric acid and the each 0.05ml of 30% superoxol, mix, detect the absorbance value of 450nm wavelength by microplate reader, reference wavelength 630nm.Result, in shown in Fig. 3, all can detect HCV Envelope 2 protein in cell pyrolysis liquid and cells and supernatant, and whether HVR1 exists the expression and secretion that does not affect E2 albumen.
Embodiment 3: mouse genetic immunization and antibody test:
With a large amount of above-mentioned 1-6 genotype of preparation and purification totally 7 strain HCV Envelope 2 protein expression plasmid pCI-E2t, pCI-E2t Δ and the empty carrier pCI-neo of plasmid extraction kit (German Qiagen company product), 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 three times altogether, two weeks interval times.Each immune the day before yesterday, (time was respectively 0,2,4 week, blood sampling time is decided to be 0 week first) and last immunity after every two weeks (totally 7 times, time is respectively 6,8,10,12,14,16,18 weeks) get blood from mouse orbit, with E2t (containing HVR1), E2t Δ (having deleted HVR1) and HVR1 antibody in enzyme-linked immunosorbent assay (ELISA) detection mice serum.E2t, the antibody test of E2t Δ: by 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 (Sigma company of U.S. product) is dissolved into concentration 20 μ g/ml with PBS, add enzyme mark microwell plate (Corning company of U.S. product), every hole 0.1ml, be placed in 4 ℃ of refrigerator overnight, absorb GNA next day, every hole is washed once with 0.2ml PBS damping fluid, then every hole adds PBS damping fluid (confining liquid) 0.1ml containing 3% bovine serum albumin bletilla 0.05%Tween 20, room temperature is placed 2 hours, then absorb confining liquid, every hole adds the HEK 293T cells and supernatant 0.1ml of plasmid pCI-E2t or the transfection of pCI-E2t Δ, room temperature is placed 2 hours, then absorb cells and supernatant, every hole adds the mouse immune serum 0.1ml with confining liquid dilution, room temperature is placed 40 minutes, then absorb serum, with PBS damping fluid (washing lotion) hole flushing containing 0.05%Tween 20 5 times, then every hole adds goat anti-mouse igg (Sigma company of the U.S. product) 0.1ml with the HRP mark of confining liquid dilution, room temperature is placed 40 minutes, then absorb antibody, with washing lotion hole flushing 5 times, every hole adds the substrate solution 0.1ml containing TMB, room temperature lucifuge was placed after 10 minutes, add 2M sulfuric acid and the each 0.05ml of 30% superoxol, mix, detect the absorbance value of 450nm wavelength by microplate reader, reference wavelength 630nm.Under identical extent of dilution, pCI-E2t and pCI-E2t Δ immune serum absorbance value exceed 2.1 times of empty carrier pCI-neo immune serum average light absorption value for antibody positive.
HVR1 antibody test: 1-6 genotype totally 7 strain HCV Envelope 2 protein HVR1 synthetic peptides (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, be placed in 4 ℃ of refrigerator overnight, absorb HVR1 synthetic peptide solution next day, every hole is washed once with 0.2ml PBS damping fluid, then every hole adds confining liquid 0.1ml, room temperature was placed after 2 hours, and with washing lotion hole flushing 5 times, then every hole adds the mice serum of dilution, detect HVR1 antibody, follow-up working method is the same.
Result: 1-6 genotype is (totally 14 kinds of plasmids in the mouse of totally 7 strain HCV raq gene immunity, 10 mouse of every kind of plasmid immunity), after first immunisation, in the mice serum of pCI-E2t plasmid immunity, exceed 80% and homophyletic HVR1 antibody and homophyletic E2t (containing HVR1) antibody can be detected, but only have 1a, 2a, 3 and 5 genotype homophyletic E2t Δ antibody can be detected, positive rate is respectively 10%, 30%, 20% and 20% (table 3).For the second time after immunity, homophyletic HVR1 in mice serum and homophyletic E2t antibody horizontal obviously raise (table 4), positive rate all reaches 100% (table 3), and homophyletic E2t Δ antibody horizontal also has rising, but positive rate is all lower than 50% (table 3).After immunity, the E2t Δ antibody horizontal in mice serum further raises by (table 4) for the third time, but positive rate is all no more than 60% (table 3).
After first immunisation, in the mice serum of plasmid pCI-E2t Δ immunity, homophyletic E2t Δ antibody positive rate meets or exceeds 90% (table 5), for the second time after immunity, the anti-homophyletic E2t of each group mice serum Δ antibody horizontal 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, not consistent containing HVR1 in the E2 albumen that this and plasmid pCI-E2t Δ are encoded.
With the corresponding antibodies in different strain HCV Envelope 2 protein detection mice serum, the antibody horizontal for homophyletic E2t Δ in its trend and mice serum is basically identical.Corresponding antibodies in the mice serum of other 6 strain coatings E2t Protein Detection 1a, 1b and 2a type pCI-E2t and the immunity of pCI-E2t Δ plasmid for table 7,8,9 shows, the antibody horizontal for different strain E2 albumen in pCI-E2t plasmid immune serum is significantly lower than the mice serum of pCI-E2t Δ plasmid immunity.3, the also remarkable mice serum lower than the immunity of pCI-E2t Δ plasmid of the antibody horizontal for different strain E2 albumen in 4,5 and 6 type pCI-E2t plasmid immune serums.
Result shows: HVR1 can efficiently induce antibody response, in the time there is HVR1 in E2, effect for the epitope induction antibody response beyond HVR1 significantly reduces, and 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 detecting for other genotype E2 protein antibodies in table 7:1a type pCI-E2t and pCI-E2t Δ plasmid immune serum
The absorbance value detecting for other genotype E2 protein antibodies in table 8:1b type pCI-E2t and pCI-E2t Δ plasmid immune serum
The absorbance value detecting for other genotype E2 protein antibodies in table 9:2a type pCI-E2t and pCI-E2t Δ plasmid immune serum
Embodiment 4: in the virus of mouse immune serum and activation analysis:
With HCVpp and HCVcc be 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 double-stranded RNA, coding has the reporter gene (green fluorescence protein gene or luciferase gene) of being convenient to detection, surface is peplos, is inlaid with HCV coating E1, E2 albumen in coating.The infection characterization of HCVpp is similar to HCV, and conventional Bel7402 Huh7 or its derivative clone, if Huh7.5 is as the target cell of its infection, are the important models of evaluating antibody neutralization.HCV envelope protein expression plasmid and retrovirus packaging plasmid cotransfection HEK 293T cell can be obtained to HCVpp.
Analyze the neutralization activity of antibody with HCVpp: infectivity is mixed with certain dilution mice serum (comprising the control group mice serum of empty plasmid immunity and the mice serum of raq gene immunity) respectively at a certain amount of HCVpp of linearity range, place 45 minutes for 37 ℃, then add inoculation to have 96 orifice plates interior (first absorbing former substratum before adding virus) of Huh7.5 target cell, 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 cell pyrolysis liquid, uciferase activity is the quantitative target that HCVpp infects, with relative fluorescence element, unit of enzyme activity represents.Cell pyrolysis liquid and luciferase detection reagent are Promega company of U.S. product.In and percentage=(control group serum relative fluorescence element unit of enzyme activity-raq gene immune serum of processing hole is processed the relative fluorescence element unit of enzyme activity in hole) ÷ control group serum process relative fluorescence element unit of enzyme activity × 100% in hole.
HCV (the cell culture produced HCV that cell cultures produces, HCVcc) be real HCV, the 2a genotype JFH-1 strain HCV full length genomic rna transfected with human hepatoma cell line Huh7 obtaining by in-vitro transcription or its derivative clone, as Huh7.5, can obtain HCVcc.Only have at present JFH-1 strain can prepare HCVcc, and other strains HCV can not prepare HCVcc.The structure gene of other strains HCV (comprising envelope protein gene in structure gene) and the nonstructural gene of JFH-1 strain are recombinated, may obtain chimeric HCVcc, this is also the truer model for evaluating antibody neutralization.
Analyze the neutralization activity of antibody with HCVcc: infectivity is mixed with certain dilution mice serum (comprising the control group mice serum of empty plasmid immunity and the mice serum of raq gene immunity) respectively at 1000FFU (foci forming unit)/mlHCVcc 0.4ml, final volume 0.8ml, place 45 minutes for 37 ℃, then add inoculation to have 96 orifice plates interior (first absorbing former substratum before adding virus) of Huh7.5 target cell, cultivate after 5 hours for 37 ℃ and change complete DMEM substratum, continue to cultivate 48 hours, then absorb nutrient solution, use formaldehyde fixed cell, detect the Huh7.5 cell of expression of HCV albumen with immunofluorescence method, detecting antibody is the monoclonal antibody for HCV Nonstructural Protein NS5A, tracer antibody is fluorescein-labeled anti-mouse IgG, after EO, under fluorescent microscope, count the cell of the fluorescence positive.In and percentage=(control group serum is processed fluorescencepositive cell number-raq gene immune serum in hole and processed the fluorescencepositive cell number in hole) ÷ control group serum process fluorescencepositive cell number × 100% in hole.
Result: table 10 shows that immunity for the third time rear 1a, 1b and 2a type raq gene immune serum are to homophyletic and different strain HCVpp neutralizing effect, visible 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 is significantly better than 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, are deleted the be significantly increased neutralization activity of mouse immune serum of HVR1.
With JFH1, J6-JFH1 and the chimeric HCVcc of H77-JFH1 be the neutralization activity of model evaluation mouse immune serum, Fig. 4 shows 1a, 1b and the neutralizing effect of 2a genotype E2 immune serum to this three strain HCVcc, and visible pCI-E2t Δ immune serum is all significantly better than the neutralizing effect of pCI-E2t immune serum to the neutralizing effect of three strain HCVcc.
The mice serum of table 10:1a, 1b and the immunity of 2a type raq gene is in homophyletic and different strain pseudovirus (pp) and percentage (%)
Embodiment 5: the expressing cho cell plasmid that builds 1b type HCV Envelope 2 protein (containing HVR1 with not containing HVR1):
Respectively take plasmid pCI-E2t, pCI-E2t Δ as template, 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 is in table 2.3 ends of downstream primer RP-C are containing the encoding sequence of 6 Histidines, and 6 histidine residues are as the label of protein purification.PCR product is cut with NheI and Sa1I enzyme, then inserts the expressing cho cell plasmid pCIDA-GS-neo with independent intellectual property right that this laboratory builds.PCR reaction system and thermal cycle conditions are with reference to described in embodiment 1.Operation is all carried out with reference to " the molecular cloning experiment guide second edition " (Science Press, 1995).The plasmid structure obtaining is as shown in Figure 5: PCMV is cytomegalovirus CMV promotor, introD is intron donor sequences, GS is glutaminase synthase gene, introA is intron receptor sequence, E2 is HCV coating raq gene E2t (containing HVR1) or E2t Δ (not containing HVR1), and polyA is SV40 virus polyadenylic acid sequence.
Embodiment 6: the structure of suspension growth CHO-K1 cell strain and expression and the purifying of E2 albumen of stably express HCV Envelope 2 protein (containing HVR1 with not containing HVR1):
With going down to posterity and cultivate CHO-K1 cell containing the DMEM nutrient solution of 10% foetal calf serum, when cell 80% merges, with 0.25% trypsin digestion and cell, be inoculated in 35mm Tissue Culture Dish, mix the above-mentioned E2 expression plasmid of 4 μ g and 10 μ l lipofectamine Lipofectamine (American I nvitrogen product) next day, transfection CHO-K1 cell, operation is carried out according to operation instruction.After plasmid-lipidosome mixture is added to Tissue Culture Dish, Tissue Culture Dish is placed in to 37 ℃, the cell culture incubator of 5% carbonic acid gas, saturated humidity, after 8 hours, adds the DMEM nutrient solution of 1ml containing 10% foetal calf serum, continue to cultivate 48 hours.Then by cell with 0.25% tryptic digestion, be inoculated in 5 100mm Tissue Culture Dishs, with GMEM-S nutrient solution (Sigma company of the U.S. product) cultivation of the foetal calf serum that contains 10% dialysis, add first sulfonyl sulfuric acid amine (MSX) (Sigma company of U.S. product) to final concentration 25 μ mol/L, after 3-4 days, cell starts death, culture dish forms by individual cells and breeds and the clone that forms after two weeks, choose single clone and be inoculated in 96 orifice plates, continue to cultivate with the GMEM-S nutrient solution containing 25 μ mol/L MSX, after one week, culture supernatant detects the expression of E2 albumen by westernblot method, 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 little square vase later, use subsequently serum free medium (JRH product) to substitute gradually GMEM-S substratum, make cell adapt to gradually complete serum free medium, 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, with centrifugal removal cell, then concentrates with ultrafiltration, carries out purifying (German Qiagen company product) subsequently by the method that nickel ion metal-chelate is closed chromatography, in each hundred milliliters of nutrient solution, can obtain 30 milligrams, E2 albumen.Fig. 6 is that the SDS-PAGE of purified product E2t and E2t Δ albumen analyzes.
Embodiment 7: use E2t and E2t Δ protein immunization rabbit:
Respectively recombinant expressed 1b type E2t and E2t Δ albumen are adjusted to concentration to 0.25mg/ml with PBS damping fluid, fully mix with equal-volume oil/water emulsion-type adjuvant ISA720 (French Seppic company product), room temperature is placed 30 minutes, then in 2 subcutaneous injection immunizing rabbits of back part, every some injection 0.2ml, 0.4ml altogether, containing E2t or E2t Δ albumen 50 μ g, two weeks once, altogether immunity three times.With same dosage bovine serum albumin (Sigma company of U.S. product) associating ISA720 adjuvant immunity rabbit in contrast.Every group of 10 rabbit.
Embodiment 8: in the antibody test in rabbit anteserum and virus and activation analysis
Regularly detect E2t antibody, E2t Δ antibody, HVR1 antibody and the neutralization activity of serum to HCV in rabbit anteserum.In antibody test, the goat anti-rabbit igg (Sigma company of U.S. product) of HRP mark for enzyme labelled antibody, the consumptive material of other operations and use, reagent are all with described in embodiment 3.
Result: the rabbit of E2t protein immunization after first immunisation in serum the positive rate of rotation of homophyletic E2t and HVR1 antibody be 100%, homophyletic E2t Δ antibody male rotary rate is 30%.After immunity, homophyletic E2t Δ antibody male rotary rate is 50% for the second time.After 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 serum the positive rate of rotation of homophyletic E2t and E2t Δ antibody be 90%, after immunity, homophyletic E2t and E2t Δ antibody male rotary rate are 100% for the second time.HVR1 antibody do not detected always, this with in E2t Δ albumen, do not conform to containing HVR1.
With the corresponding antibodies in different strain HCV Envelope 2 protein detection mice serum, the antibody horizontal for homophyletic E2t Δ in its trend and mice serum is basically identical.The E2 antibody horizontal (table 11) that anti-other 6 strains HCV Envelope 2 protein antibody positive rate of E2t protein immunization induction is significantly induced lower than E2t Δ protein immunization.The E2 antibody horizontal that anti-other 6 strains HCV Envelope 2 protein antibody horizontal of E2t protein immunization induction is also significantly induced lower than E2t Δ protein immunization.
Result shows: the feature similarity of the antibody response of recombinant protein immunity and genetic immunization induction, it is the antibody response that HVR1 significantly suppresses conserved epitope induction in E2 albumen, deletion HVR1 can significantly strengthen the immunogenicity of conserved epitope in E2 albumen, improves the protein induced cross-reacting antibody level of E2.
By the neutralization activity of HCVpp model evaluation rabbit anteserum, method is with described in embodiment 4.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 is significantly 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 be the neutralization activity of model evaluation mouse immune serum, method is with described in embodiment 4.Result shows: the rabbit anteserum of E2t Δ protein immunization is all significantly 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 antibody positive rate (%) of the anti-different strain HCV Envelope 2 protein of different time points
Embodiment 9: with the E2 antibody in E2t and E2t Δ Protein Detection HCV infected person anteserum
E2t and E2t Δ albumen are diluted to concentration 20 μ g/ml with PBS damping fluid, add enzyme mark microwell plate (Corning company of U.S. product), every hole 0.1ml, be placed in 4 ℃ of refrigerator overnight, absorb E2 albumen next day, every hole is washed once with 0.2ml PBS damping fluid, then every hole adds PBS damping fluid (confining liquid) 0.1ml containing 3% bovine serum albumin bletilla 0.05%Tween20, room temperature is placed 2 hours, every hole adds HCV infected person anteserum or the HCV with confining liquid dilution in 1: 100, all negative serum 0.1ml of health adult of HBV antibody and detection of nucleic acids, room temperature is placed 40 minutes, then absorb serum, with PBS damping fluid (washing lotion) hole flushing containing 0.05%Tween 20 5 times, then every hole adds goat anti-human igg's (Sigma company of U.S. 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 containing TMB, room temperature lucifuge is placed 10 minutes, then add 2M sulfuric acid and the each 0.05ml of 30% superoxol, mix, detect the absorbance value of 450nm wavelength by microplate reader, reference wavelength 630nm.
Detected all negative health adult's serum of 30 parts of HCV infected person anteserums and 10 parts of HCV, HBV antibody and detection of nucleic acids, as shown in figure 11, the antibody horizontal of E2t Δ Protein Detection is apparently higher than the antibody of E2t Protein Detection result.
Claims (5)
1. modified 1b type the third liver Hepatitis virus Envelope 2 protein, is characterized in that described modified hepatitis C virus Envelope 2 protein refers to the peptide section of having deleted the hypervariable region 1 in hepatitis C virus Envelope 2 protein.
2. the application of modified 1b type hepatitis C virus Envelope 2 protein according to claim 1 in preparation prevention and therapeutic hcv vaccine.
3. the application of modified 1b type hepatitis C virus Envelope 2 protein according to claim 1 in the immunological diagnostic reagent of preparing infection with hepatitis C virus.
4. the application of the encoding gene of a modified 1b type hepatitis C virus Envelope 2 protein as claimed in claim 1 in preparation prevention and therapeutic hcv vaccine.
5. the application of the encoding gene of a modified 1b type hepatitis C virus Envelope 2 protein as claimed in claim 1 in the immunological diagnostic reagent of preparing infection with hepatitis C virus.
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