CN105330730A - Preparation and application of hepatitis C virus recombinant protein - Google Patents
Preparation and application of hepatitis C virus recombinant protein Download PDFInfo
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- CN105330730A CN105330730A CN201410367643.XA CN201410367643A CN105330730A CN 105330730 A CN105330730 A CN 105330730A CN 201410367643 A CN201410367643 A CN 201410367643A CN 105330730 A CN105330730 A CN 105330730A
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Abstract
The present invention discloses preparation and application of a hepatitis C virus (HCV) recombinant protein, specifically discloses a separated HCV antigen peptide, which is derived from an E2 protein of HCV virus; the antigenic peptide can bind to an anti-HCV antibody; and the HCV is a Con1 strain of HCV 1b genotype. The invention provides a composition containing the above antigen peptide and a preparation method thereof. The HCV antigen peptide of the invention can effectively and specifically combine with anti-E2 antibody in serum of an infected patient in a broad-spectrum way, and can be used to develop a diagnostic kit for the detection of HCV infection.
Description
Technical field
The invention belongs to biomedicine field, specifically, the present invention relates to a kind of preparation and application thereof of hepatitis C virus recombinant protein.
Background technology
It is one of main inducing causing mankind's liver cirrhosis, liver cancer that hepatitis C virus (HepatitisCvirus, HCV) infects, and 170,000,000 people that worldwide has an appointment infects, and infection rate is up to 3%, and great threat the health of the mankind.At present for hepatitis C virus, there is no preventative or therapeutic vaccine can use.
Along with going deep into of clinical study, increasing evidence shows that neutralizing antibody plays an important role in the control, scavenging process of HCV infection.In patient body, the rapid induction of neutralizing antibody, titre, the range of neutralizing antibody are directly related with the removing of HCV infection.No matter chimpanzee experiment or patients clinical sample analysis all shows, in HCV acute infection process, the neutralizing antibody that rapid induction goes out can effectively control and remove HCV infection; And in chronic infection patient body, if do not induce high titre neutralizing antibody in early days in infection, even if late period, Immune inducing in vivo went out neutralizing antibody, the also persistent infection of uncontrollable virus.Therefore, the neutralizing antibody reaction of induced high titers is an important indicator of early stage HCV vaccine research and development.But, because the parcel of lipoprotein component in the variability of HCV itself, serum, glycosylation are to reasons such as the effects of the shielding of neutralizing site and interference antibody, the antibody that early stage HCV candidate vaccine produces often only has neutralization to identical subtype virus, does not have broad spectrum.
Therefore, the wide spectrum neutralizing antibody for all or most of subtype virus can be induced in the urgent need to exploitation in this area, for use in exploitation HCV preventative vaccine.
Summary of the invention
The object of the present invention is to provide a kind of preparation and application thereof of hepatitis C virus recombinant protein.
A first aspect of the present invention, provide a kind of HCV antigen peptide of separation, described antigen peptide is derived from the E2 albumen (especially the extracellular region of E2 albumen) of HCV virus, and the length of described antigen peptide is 200-350 amino acid, and described antigen peptide can be incorporated into the antibody of HCV-Ab IgG.
In another preference, described HCV virus is HCV1b C-type virus C; Preferably, be Con1 strain.
In another preference, described antigen peptide has the ability of induction wide spectrum neutralizing antibody, and described wide spectrum neutralizing antibody can be incorporated at least 6 kinds or the whole HCV strains being selected from lower group: Con1, PR52, PR79L9, JFH1, S52, HK6a, H77/JFH1, Con1/JFH1, J6/JFH1, PR26, PR79L9, J8/JFH1, S52/JFH1, ED43/JFH1, PR26C3mt, SA13/JFH1, HK6a/JFH1 and QC69/JFH1.
In another preference, ratio A1/B1≤0.7 of the B1 of described antigen peptide and the binding activities A1 of acceptor SR-BI and the binding activities of corresponding wild-type E2 albumen and acceptor SR-BI, preferably≤0.5, more preferably≤0.2.
In another preference, described antigen peptide is selected from lower group:
(A) there is the polypeptide of aminoacid sequence shown in SEQIDNO:5, SEQIDNO.:3, SEQIDNO.:4 or SEQIDNO.:2;
(B) there is arbitrary shown aminoacid sequence >=80% homology (homology of preferably, >=90% in (A); Deng the homology of preferably >=95%; Most preferably, the homology of >=97%) polypeptide, and described polypeptide has the binding ability with the antibody of HCV-Ab IgG;
(C) aminoacid sequence shown in arbitrary in SEQIDNO:2-5 is formed through the replacement of 1-5 amino-acid residue, disappearance or interpolation, and retain and the derivative polypeptide of HCV antigen/antibody combination binding ability.
In another preference, described HCV antigen/antibody combination comprises HCV-Ab IgG wild-type E2 protein antibodies, comprising the monoclonal antibody of the corresponding zone of antiserum(antisera) or HCV-Ab IgG wild-type E2 albumen.
In another preference, described antigen peptide is selected from lower group: SEQIDNO:5, SEQIDNO.:3, SEQIDNO.:4, SEQIDNO.:2 or its combination.
In another preference, described antigen peptide adopts chemosynthesis or biological engineering method to obtain, and comprises recombinant protein, fusion rotein.
In another preference, described antibody comprises monoclonal antibody, polyclonal antibody or antiserum(antisera).
In another preference, described E2 albumen comprises the E2 sequence of wild-type and saltant type.
A second aspect of the present invention, provides a kind of fusion rotein, and described fusion rotein contains antigen peptide described in first aspect present invention and optional sequence label and/or catenation sequence.
In another preference, described sequence label is 6His sequence.
A third aspect of the present invention, provides a kind of polynucleotide of separation, the described antigen peptide described in polynucleotide encoding first aspect present invention or the fusion rotein described in second aspect present invention.
In another preference, described polynucleotide are selected from lower group:
The polynucleotide of (a) coding polypeptide as shown in SEQIDNO.:2-5;
The polynucleotide of (b) sequence as shown in SEQIDNO.:9, SEQIDNO.:8, SEQIDNO.:7 or SEQIDNO.:6;
The polynucleotide of homology >=95% (preferably >=98%) of sequence shown in (c) nucleotide sequence and (b);
D () be the 5' end of polynucleotide and/or the polynucleotide of 3' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as Suo Shi (b);
The polynucleotide of the polynucleotide complementation e () is arbitrary with (a)-(d) described in.
A fourth aspect of the present invention, provides a kind of expression vector, and described expression vector contains the polynucleotide described in third aspect present invention.
A fifth aspect of the present invention, provides a kind of host cell, and described host cell contains the expression vector described in fourth aspect present invention, or in genome, be integrated with the polynucleotide described in third aspect present invention.
In another preference, described host cell comprises prokaryotic cell prokaryocyte and eukaryotic cell.
In another preference, described host cell comprises Drosophila S 2 cells, intestinal bacteria, yeast, Chinese hamster ovary celI, DC cell etc.
A sixth aspect of the present invention, provide a kind of pharmaceutical composition, described pharmaceutical composition contains the antigen peptide described in first aspect present invention, the fusion rotein described in second aspect present invention, the polynucleotide described in third aspect present invention, the expression vector described in fourth aspect present invention or the host cell described in fifth aspect present invention, and pharmaceutically acceptable carrier and/or auxiliary material.
In another preference, described pharmaceutical composition is vaccine.
A seventh aspect of the present invention, provide a kind of vaccine composition, described vaccine composition contains the antigen peptide described in first aspect present invention, the fusion rotein described in second aspect present invention, the polynucleotide described in third aspect present invention, the expression vector described in fourth aspect present invention or the host cell described in fifth aspect present invention, and acceptable carrier and/or auxiliary material in immunology.
In another preference, described vaccine composition is also containing adjuvant.
In another preference, described adjuvant comprises aluminum oxide, saponin(e, quilA, Muramyl dipeptide, mineral oil or vegetables oil, adjuvant, non-ionic block copolymer or deae dextran based on vesica, cytokine (comprising IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12 and CpG).
In another preference, described adjuvant is selected from: aluminium adjuvant, aluminium adjuvant mixed C pG and freund's adjuvant.
A eighth aspect of the present invention, provide the purposes of the antigen peptide described in first aspect present invention, the fusion rotein described in second aspect present invention, the polynucleotide described in third aspect present invention, the expression vector described in fourth aspect present invention or the host cell described in fifth aspect present invention
A () is for the preparation of the antibody for described antigen peptide; And/or
B () is for the preparation for the treatment of or the medicine preventing HCV infection relative disease; And/or
C () is for the preparation of the reagent or the test kit that detect HCV; And/or
D () is for detecting the antibody of HCV-Ab IgG.
In another preference, described " detecting reagent or the test kit of HCV " comprises detection lug, check-out console, protein chip, liquid base chip, magnetic bead.
In another preference, the HCV antigen/antibody combination in described HCV antigen/antibody combination behaviour humoral sample.Preferably, described body fluid is blood or saliva.
A ninth aspect of the present invention, provides a kind of immunity detection reagent, and described test kit comprises the antigen peptide for detecting HCV antigen/antibody combination described in first aspect present invention.
In another preference, the antigen peptide for detecting HCV antigen/antibody combination described in one or more first aspect present invention in described test kit, can be comprised.
In another preference, described test kit also comprises carrier, and described antigen peptide is coated on described carrier.
In another preference, described immunity detection reagent also comprises two of the HCV antigen/antibody combination of mark and resists.Two anti-being used for detect the IgG antibody in human serum, IgM, or IgA. marker used can be horseradish peroxidase, alkaline phosphatase, fluorescence molecule FITC (or other fluorescent mark), chemiluminescence detection.Detection method can be development process, fluorescent method, chemoluminescence, and Electrochemiluminescince carries out qualitative and quantitative analysis.
In another preference, described two anti-be that two of horseradish peroxidase-labeled resists.
A tenth aspect of the present invention, provides the preparation method of the antigen peptide described in a kind of first aspect present invention, comprises the following steps:
A () cultivates the host cell described in fourth aspect present invention, thus give expression to the antigen peptide described in first aspect present invention;
Antigen peptide described in (b) separation.
In another preference, described host cell is Drosophila S 2 cells.
In another preference, before step (a), described method is further comprising the steps of:
(1) provide the polynucleotide sequence of a coding HCV E2 albumen, described polynucleotide sequence is as shown in SEQIDNO.:6-9;
(2) preparation contains the carrier of polynucleotide sequence described in (1) in steps;
(3) by the vector introduction host cell in step (2), thus obtained host cell according to claim 4.
In another preference, described carrier is inducible expression vector.
In another preference, described carrier is pcDNA3.1 (+) plasmid.
In another preference, in described step (a), when the carrier imported in described host cell is inducible expression vector, in cell density>=1 × 10
7inductor is added again after/ml.
A eleventh aspect of the present invention, provide a kind of methods for the treatment of, give the object needed use described in the antigen peptide described in first aspect present invention, the fusion rotein described in second aspect present invention, the polynucleotide described in third aspect present invention, the expression vector described in fourth aspect present invention or the host cell described in fifth aspect present invention, sixth aspect present invention pharmaceutical composition or vaccine composition described in seventh aspect present invention.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the expression of sE2 albumen in S2 cell conditioned medium, qualification and purifying.(A) S2 cell sE2 expression plasmid pMT-Bip/sE2/His schematic diagram.(B) sE2 original coding gene (WT) with codon optimized after encoding gene (OPTI) be cloned into after on above-mentioned carrier, with calcium phosphate method transient transfection S2 cell.Optimised sequence can express sE2 more efficiently.(C) chromium chloride induction after respectively at the the 0th, 3,5, within 7,9 days, collect cell conditioned medium, Westernblot detects the expression of sE2 albumen, primary antibodie is the mouse-anti His monoclonal antibody (abmart) of 1:1000 dilution, the two mountain sheep anti-mouse iggs resisting the HRP for 1:4000 dilution to mark.(D) the sE2 albumen after SDS-PAGE glue coomassie brilliant blue staining analysis ni-sepharose purification, size is about 46kDa.
Fig. 2 shows the specificity analysis of sE2 albumen.(A) detect without PNGase process with through sE2 albumen Westernblot after SDS-PAGE gel electrophoresis that PNGase enzyme is cut, respectively with the mouse His monoclonal antibody (abmart) of 1:1000 dilution and the mouse E2 monoclonal antibody (AP33) of 1:1000 dilution as primary antibodie, the mountain sheep anti-mouse igg of the AP mark of 1:4000 dilution is anti-as two.(B) respectively with identifying the monoclonal antibody E2C1 of conformational epitope and identifying that the monoclonal antibody AP33 of linear epitope carries out ELISA detection to sE2, to check its antigenicity.α-Core monoclonal antibody is negative control.(C) sE2 and wild-type CHO cells in conjunction with situation.(D) sE2 and stably express HCV acceptor clone CHO-CD81 in conjunction with situation, the cell of about 76.2% can be combined with sE2.(E) sE2 and stably express HCV acceptor clone CHO-SRB1 in conjunction with situation, the cell of about 64.5% can be combined with sE2.(F) sE2 contestable suppresses HCV infection Huh7.5.1 cell.The sE2 of different concns or 37 DEG C, BSA and Huh7.5.1 cell are hatched 1 hour, then add HCVCon1/JFH-1 virus to infect, carry out the immunofluorescence dyeing of HCVNS3 albumen after 72h, counting to the fluorescent spot in every hole under fluorescent microscope (Leica) counts.Count as blank group with the fluorescent spot of 0 μ g/mL, infect inhibition (%)=(fluorescent spot of blank group counts-respective aperture in fluorescent spot count) fluorescent spot of/blank group counts * 100%.
Fig. 3 shows sE2 can specific detection HCV infection patient blood plasma.The elisa plate of sE2 bag quilt, hatches with the human normal plasma of 1:2000 or 1:20000 dilution and the third hepatopathy human plasma respectively, hatches two anti-, colour developings subsequently, read OD450 after stopping colour developing.Patients serum when 1:20000 dilutes also can show nearly 1.0 read value.
Fig. 4 shows sE2 albumen can induce good humoral immune reaction in BALB/c mouse.(A) immunization experiment grouping.SE2 respectively with aluminium adjuvant (Alum), aluminium adjuvant+CpG or freund's adjuvant (FA) for adjuvant, and establish PBS control group.(B) respectively at the the 0th, 2,4 week abdominal injection immunity BALB/c mouse, got blood respectively at the the the the the 0th, 4,6,13,17,22,25 week, detect the terminal titre for sE2 specific antibody in serum by the method for ELISA.For detecting immunological memory, once adding in the 25th week and exempting from, and got at the 27th week the terminal titre that specific antibody in serum is surveyed in blood examination.Antibody terminal titre after three immune group mouse booster immunizations reaches 10
5-10
6.
The titre changing conditions in time of the specificity height antibody gone out at rabbit Immune inducing in vivo after Fig. 5 shows sE2 immunity.With freund's adjuvant and sE2 mixed immunity new zealand rabbit, immunization interval was two weeks, every blood sampling in 2 weeks.Three exempt to put to death for latter three weeks, collect serum.Antibody terminal titre after immunity reaches 10
6-10
7.
Fig. 6 show sE2 immune serum can in and 1-7 type HCV virus.(A) sE2 respectively with aluminium adjuvant, aluminium adjuvant+CpG or freund's adjuvant for adjuvant, abdominal injection immune mouse.The mouse antiserum(antisera) 1:40 of the 27th week carries out external Neutralizing test after diluting, and determines the neutralization to the different HCV virus of 13 strains of all 7 hypotypes.(B) after often organizing the antiserum(antisera) equal-volume mixing of 10 mouse, in every strain virus and the most high dilution of 50%.Neutralization (%)=(fluorescent spot of preimmune serum group counts-respective aperture in fluorescent spot count) fluorescent spot of/preimmune serum group counts * 100%.Three immune group mice serum major parts efficiently can neutralize the virus (>50%) of all 7 hypotypes, and comparatively speaking, sE2+ aluminium adjuvant+CpG demonstrates the effect of best induction neutralizing antibody.
Fig. 7 shows sE2 protein immunization rabbit serum to the Neutralization effect of different HCV virus.(A) three exempt from the antiserum(antisera) of latter two weeks after carrying out 1:20 dilution, measure the neutralization that 13 strain different virus are infected.(B) two rabbits three exempt from after the antiserum(antisera) of two weeks in every strain virus and the most high dilution of 50%.Neutralization (%)=(fluorescent spot of preimmune serum group counts-respective aperture in fluorescent spot count) fluorescent spot of/preimmune serum group counts * 100%.Rabbit serum all can efficiently neutralize all virus.
Fig. 8 shows the expression of sE2 Δ HVR1, purifying and qualification.(A) sE2 and the sE2 Δ HVR1 albumen after SDS-PAGE glue coomassie brilliant blue staining analysis ni-sepharose purification, size is about 40kDa.(B) with monoclonal antibody AR3A, ELISA detection is carried out to sE2 and sE2 Δ HVR1, to check its antigenicity and epi-position exposure.Neutralizing antibody AR3A to the identification of sE2 and sE2 Δ HVR1 without remarkable difference.(C) sE2 Δ HVR1 and wild-type CHO cells, clone CHO-CD81, clone CHO-SRB1 in conjunction with situation.SE2 Δ HVR1 and sE2 compares, and the combination of CHO-CD81 is without significantly difference (75.9% and 71.0%), and the combination of CHO-SRB1 then significantly weakens (92.2% and 27.1%).(D) sE2 Δ HVR1 is in competitive inhibition HCV infection, and comparatively sE2 weakens to some extent.(E) the same with sE2, sE2 Δ HVR1 also can specific detection HCV infection patient blood plasma.
What Fig. 9 showed sE2 Δ HVR1 immune serum has wide spectrum Neutralization effect.(A) sE2 Δ HVR1, sE2 and PBS are respectively as antigen immune BALB/c mouse, and sE2 Δ HVR1 albumen can induce good antibody response in BALB/c mouse, and after exempting from three, the specific antibody terminal titre of two weeks is a little more than sE2 group.(B) the three antiserum(antisera) 1:40 exempted from latter two weeks carry out external Neutralizing test after diluting, and determine the neutralization to the different HCV viruses of 13 strains of all 7 hypotypes.The neutralization of the neutralizing antibody that sE2 Δ HVR1 induces to this strain Con1 virus is more weak, all can induce the neutralizing antibody suitable with sE2 immune group to all the other virus strain.
Neutralization to HCV virus when the lower Specific antibody titre of Figure 10 .sE2 immune mouse induction.(A) sE2 albumen and freund's adjuvant carry out emulsification, subcutaneous injection immunity BALB/c mouse (n=3).ELISA detection was carried out to specificity E2 antibody in mice serum in after three immunity two weeks.(B) antiserum(antisera) 1:40 carries out external Neutralizing test for Con1 and JFH1 two strain virus after diluting.When antibody titers is lower, antiserum(antisera) weakens the neutralization of HCV virus is also corresponding.
Embodiment
The present inventor is by extensive and deep research, obtain a kind of HCV truncation type E2 albumen (sE2 Δ HVR1) through optimizing, be surprised to find that through experiment, the E2 albumen of described truncation type, significantly weaken with the binding activities of acceptor SR-BI on the one hand, retain the ability of induction wide spectrum neutralizing antibody on the other hand.Described sE2 Δ HVR1 all can induce the neutralizing antibody suitable with sE2 immune group to 1-7 C-type virus C, and therefore when being used as vaccine, sE2 Δ HVR1 produces the HCV antigen/antibody combination of broad spectrum while significantly can reducing vaccine side effect and occurring in inductor.Complete the present invention on this basis.
In addition, the present inventor has also carried out a series of optimization to the codon of expressing E2 albumen, through a large amount of screening, the polynucleotide sequence obtained is particularly suitable for expressing in Drosophila S 2 cells, expression output significantly improves, at least improve compared with wild-type 5 times (compared with before optimizations), 50 times of E2 protein yield of the HEK293T cell expressing of report before being.
HCV virus
HCV is strand underlying stock RNA viruses, belongs to flaviviridae hepatitis virus and belongs to.HCV genome comprises 5' non-coding region, coding region and 3' non-coding region, wherein about 9000 alkali yl codings 10 viral proteins of coding region: structural protein Core, E1, E2, Nonstructural Protein NS2, NS3, NS4A, NS4B, NS5A, NS5B (1). wherein virus envelope protein E1 (amino-acid residue 192 – 383), E2 (amino-acid residue 384 – 746) is two transmembrane glycoproteins of composition HCV peplos, in endoplasmic reticulum, be wrapped in the surface of core protein Core in an assembling process and discharge thus, therefore E1, E2 is exposed to surfaces of viral particles, it is the major target class (2) of neutralizing antibody.Simultaneously, HCV passes through E1, receptors bind on E2 and cytolemma thus mediate retroviral enters host cell, four kinds of acceptors are had to take part in the process entered: CD81, ScavengerreceptorclassBtypeI (SR-BI), Claudin-1 (CLDN-1) and Occludin (OCLN).Research has before identified many neutralizing epitopes and has all been positioned at E2, comprise the hypervariableregion1 (HVR1 of highly divergent isolate, 384aa-410aa), and the comparatively conservative epi-position be positioned on CD81 binding site, and conserved epitope to a great extent hide by HVR1 epi-position, the neutralizing antibody broad spectrum causing virus induction to go out is poor.
In an embodiment of the invention, the aminoacid sequence of described HCVE2 albumen is:
GTYVTGGTMAKNTLGITSLFSPGSSQKIQLVNTNGSWHINRTALNCNDSLNTGFLAALFYVHKFNSSGCPERMASCSPIDAFAQGWGPITYNESHSSDQRPYCWHYAPRPCGIVPAAQVCGPVYCFTPSPVVVGTTDRFGVPTYSWGENETDVLLLNNTRPPQGNWFGCTWMNSTGFTKTCGGPPCNIGGIGNKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRLWHYPCTVNFTIFKVRMYVGGVEHRLEAACNWTRGERCNLEDRDRSELSPLLLSTTEWQVLPCSFTTLPALSTGLIHLHQNVVDVQYLYGIGSAVVSFAIKWEYVLLLFLLLADARVCACLWMMLLIAQAEA(SEQIDNO.:1)
The existing preventative vaccine for HCV is studied, and no matter is DNA vaccination, recombinant protein vaccine, virus sample particle vaccines, and Bacterial vector vaccines or vector-viral vaccine, design mostly based on envelope protein E1 and E2.Wherein, the HCVE1E2/MF59 for envelope protein E1 E2 (NCT00500747) of expressing cho cell enters clinical trial.The above vaccine immunity animal induce the antiserum(antisera) of generation to the protection of same strain virus better, but limited to other hypotype neutralization.And above-mentioned most of vaccine is all design based on the H77 strain of genotype 1a, limited for the most popular 1b C-type virus C neutralization of China and East Asia Region.Therefore, how to select a kind of immunogenicity better, wide spectrum neutralizing antibody can be induced, especially for China and the East Asia Region good antigen of epidemic strain effect as vaccine, become the problem of the present inventor's concern.
In this study, the present inventor finds to utilize Drosophila S 2 cells to prepare two kinds of clipped forms of the E2 albumen (SolubleE2, sE2) of solubility: sE2 and sE2 Δ HVR1; These two kinds of albumen can be combined with HCV acceptor CD81, SR-BI thus suppress HCV infection sensitive cells in vitro; ELISA specific detection HCV infection patients serum can be passed through; The antiserum(antisera) produced with these two kinds of protein immunization mouse and rabbit not only has good neutralization to the 1b C-type virus C strain Con1 of homotype, and the four strain 1b C-type virus Cs that in energy and 1-7 hypotype laboratory adapts to virus strain and obtains from clinical sample.These results of study prompting sE2 and sE2 Δ HVR1 can be used for developing HCV diagnostic kit and HCV preventative vaccine further.
Active polypeptide
As used herein, term " HCV antigen ", " HCV antigen peptide ", " C hepatitis virus antigen peptide ", " C hepatitis virus antigen ", " antigen of antibody of HCV ", " antigenic polypeptide " are used interchangeably.Refer to that peptide sequence comes from the polypeptide of hepatitis C virus E 2 albumen.
As used herein, term " HCV antigen of the present invention ", " HCV antigen peptide of the present invention ", " C hepatitis virus antigen peptide of the present invention ", " C hepatitis virus antigen peptide section of the present invention " are used interchangeably, and refer to the antigen peptide (polypeptide as shown in SEQIDNO:1-5) that can be incorporated into anti-HCV.In addition, described term also comprise have in conjunction with anti-hepatitis c virus antibody function, the variant form of SEQIDNO:1-5 sequence.These variant forms comprise (but being not limited to): 1-3 (is generally 1-2, more preferably 1) amino acid whose disappearance, insertion and/or replacement, and to add or disappearance one or several (is generally within 3 at C-terminal and/or N-terminal, within being preferably 2, within being more preferably 1) amino acid.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, to add or disappearance one or several amino acid also can not change the structure and function of protein usually at C-terminal and/or N-terminal.In addition, described term also comprises the polypeptide of the present invention of monomer and multimeric forms.This term also comprises linear and nonlinear polypeptide (as cyclic peptide).
Of the present invention one preferred embodiment in, the aminoacid sequence of described HCV antigen peptide sE2 is:
gTYVTGGTMAKNTLGITSLFSPGSSQKIQLVNTNGSWHINRTALNCNDSLNTGFLA ALF yVHKFNSSGCPERMASCSPIDAFAQGWGPITYNESHSSDQRPYCWHYAPRPCGIVP AA qVCGPVYCFTPSPVVVGTTDRFGVPTYSWGENETDVLLLNNTRPPQGNWFGCTWMN S tGFTKTCGGPPCNIGGIGNKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPY RL wHYPCTVNFTIFKVRMYVGGVEHRLEAACNWTRGERCNLEDRDRSE
sE2(SEQIDNO.:2)。
Of the present invention one preferred embodiment in, the aminoacid sequence of described HCV antigen peptide sE2 is:
GTYVTGGTMAKNTLGITSLFSPGSSQKIQLVNTNGSWHINRTALNCNDSLNTGFLAALF YVHKFNSSGCPERMASCSPIDAFAQGWGPITYNESHSSDQRPYCWHYAPRPCGIVPAA QVCGPVYCFTPSPVVVGTTDRFGVPTYSWGENETDVLLLNNTRPPQGNWFGCTWMNS TGFTKTCGGPPCNIGGIGNKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRL WHYPCTVNFTIFKVRMYVGGVEHRLEAACNWTRGERCNLEDRDRSESRGPFEGKPIPNPLLGLDSTRTGHHHHHH(SEQIDNO.:3)；
Wherein, underscore part is sE2, and rest part is label protein and link amino acid.
Of the present invention one preferred embodiment in, described sE2 coding nucleotide sequence (after codon optimized) is:
ggcacatacgtgacaggcggcacaatggccaagaacaccctgggcatcaccagcctgttcagccccggcagcagcca gaaaatccagctggtgaacaccaacggcagctggcacatcaaccggaccgccctgaactgcaacgactccctgaataccggctt cctggccgccctgttctacgtgcacaagttcaacagcagcggctgccccgagcggatggccagctgtagccctatcgatgccttc gcccagggctggggccctatcacctacaacgagagccacagcagcgaccagcggccctactgctggcactacgcccccagac cttgcggcattgtgcctgccgctcaggtctgcggccctgtgtactgcttcacccccagccccgtggtggtgggaaccaccgatag attcggcgtgccaacctacagctggggcgagaacgagacagacgtgctgctgctgaacaacaccagacccccccagggcaatt ggttcggctgcacctggatgaacagcaccggcttcaccaagacctgcggcggacccccctgcaacatcggcggcatcggcaac aagaccctgacatgccccaccgattgcttcagaaagcaccccgaggccacctacaccaagtgcggctctggcccctggctgacc cccagatgcctggtgcactacccctaccggctgtggcactacccttgcaccgtgaacttcaccatcttcaaagtgcggatgtatgtg ggcggagtggaacaccggctggaagccgcctgcaactggaccagaggcgagcggtgcaacctggaagatcgggacagaag cgag(SEQIDNO.:6)。
Of the present invention one preferred embodiment in, described sE2 coding nucleotide sequence (after codon optimized) is:
ggcacatacgtgacaggcggcacaatggccaagaacaccctgggcatcaccagcctgttcagccccggcagcagcca gaaaatccagctggtgaacaccaacggcagctggcacatcaaccggaccgccctgaactgcaacgactccctgaataccggctt cctggccgccctgttctacgtgcacaagttcaacagcagcggctgccccgagcggatggccagctgtagccctatcgatgccttc gcccagggctggggccctatcacctacaacgagagccacagcagcgaccagcggccctactgctggcactacgcccccagac cttgcggcattgtgcctgccgctcaggtctgcggccctgtgtactgcttcacccccagccccgtggtggtgggaaccaccgatag attcggcgtgccaacctacagctggggcgagaacgagacagacgtgctgctgctgaacaacaccagacccccccagggcaatt ggttcggctgcacctggatgaacagcaccggcttcaccaagacctgcggcggacccccctgcaacatcggcggcatcggcaac aagaccctgacatgccccaccgattgcttcagaaagcaccccgaggccacctacaccaagtgcggctctggcccctggctgacc cccagatgcctggtgcactacccctaccggctgtggcactacccttgcaccgtgaacttcaccatcttcaaagtgcggatgtatgtg ggcggagtggaacaccggctggaagccgcctgcaactggaccagaggcgagcggtgcaacctggaagatcgggacagaag cgagtctagagggcccttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattga(SEQIDNO.:7)
Underscore part is the gene of sE2, and rest part is label protein and link amino acid whose gene.
Of the present invention another preferred embodiment in, the aminoacid sequence of described HCV antigen peptide sE2 Δ HVR1 is:
QLVNTNGSWHINRTALNCNDSLNTGFLAALFYVHKFNSSGCPERMASCSPIDA
FAQGWGPITYNESHSSDQRPYCWHYAPRPCGIVPAAQVCGPVYCFTPSPVVVGTTD
RFGVPTYSWGENETDVLLLNNTRPPQGNWFGCTWMNSTGFTKTCGGPPCNIGGIG
NKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRLWHYPCTVNFTIFKVRM
YVGGVEHRLEAACNWTRGERCNLEDRDRSE
sE2ΔHVR1(SEQIDNO.:4)。
Of the present invention another preferred embodiment in, the aminoacid sequence of described HCV antigen peptide sE2 Δ HVR1 is:
qLVNTNGSWHINRTALNCNDSLNTGFLAALFYVHKFNSSGCPERMASCSPIDA fAQGWGPITYNESHSSDQRPYCWHYAPRPCGIVPAAQVCGPVYCFTPSPVVVGTTD rFGVPTYSWGENETDVLLLNNTRPPQGNWFGCTWMNSTGFTKTCGGPPCNIGGIG nKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRLWHYPCTVNFTIFKVRM yVGGVEHRLEAACNWTRGERCNLEDRDRSEsRGPFEGKPIPNPLLGLDSTRTGHHHHHHsE2 Δ HVR1 (SEQIDNO.:5); Underscore part is sE2 Δ HVR1, and rest part is label protein and link amino acid.
Of the present invention another preferred embodiment in, described sE2 Δ HVR1 coding nucleotide sequence (after codon optimized) is:
cagctggtgaacaccaacggcagctggcacatcaaccggaccgccctgaactgcaacgactccctgaataccggcttc ctggccgccctgttctacgtgcacaagttcaacagcagcggctgccccgagcggatggccagctgtagccctatcgatgccttcg cccagggctggggccctatcacctacaacgagagccacagcagcgaccagcggccctactgctggcactacgcccccagacc ttgcggcattgtgcctgccgctcaggtctgcggccctgtgtactgcttcacccccagccccgtggtggtgggaaccaccgatagat tcggcgtgccaacctacagctggggcgagaacgagacagacgtgctgctgctgaacaacaccagacccccccagggcaattg gttcggctgcacctggatgaacagcaccggcttcaccaagacctgcggcggacccccctgcaacatcggcggcatcggcaaca agaccctgacatgccccaccgattgcttcagaaagcaccccgaggccacctacaccaagtgcggctctggcccctggctgaccc ccagatgcctggtgcactacccctaccggctgtggcactacccttgcaccgtgaacttcaccatcttcaaagtgcggatgtatgtgg gcggagtggaacaccggctggaagccgcctgcaactggaccagaggcgagcggtgcaacctggaagatcgggacagaagc gag(SEQIDNO.:8)。
Of the present invention another preferred embodiment in, described sE2 Δ HVR1 coding nucleotide sequence (after codon optimized) is:
cagctggtgaacaccaacggcagctggcacatcaaccggaccgccctgaactgcaacgactccctgaataccggcttc ctggccgccctgttctacgtgcacaagttcaacagcagcggctgccccgagcggatggccagctgtagccctatcgatgccttcg cccagggctggggccctatcacctacaacgagagccacagcagcgaccagcggccctactgctggcactacgcccccagacc ttgcggcattgtgcctgccgctcaggtctgcggccctgtgtactgcttcacccccagccccgtggtggtgggaaccaccgatagat tcggcgtgccaacctacagctggggcgagaacgagacagacgtgctgctgctgaacaacaccagacccccccagggcaattg gttcggctgcacctggatgaacagcaccggcttcaccaagacctgcggcggacccccctgcaacatcggcggcatcggcaaca agaccctgacatgccccaccgattgcttcagaaagcaccccgaggccacctacaccaagtgcggctctggcccctggctgaccc ccagatgcctggtgcactacccctaccggctgtggcactacccttgcaccgtgaacttcaccatcttcaaagtgcggatgtatgtgg gcggagtggaacaccggctggaagccgcctgcaactggaccagaggcgagcggtgcaacctggaagatcgggacagaagc gagtctagagggcccttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattga(SEQIDNO.:9)
Underscore part is the gene of sE2 Δ HVR1, and rest part is label protein and link amino acid whose gene.
The present invention also comprises the active fragments of C hepatitis virus antigen peptide, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " refer to the polypeptide of the function that maintenance substantially combines with the antibody of anti-hepatitis c virus or activity.Polypeptide fragment of the present invention, derivative or analogue can be the polypeptide that (i) has that one or several is guarded or non-conservative amino acid residue (preferred conservative amino acid) is substituted, or (ii) has the polypeptide of substituted radical in one or more amino-acid residue, or (iii) antigen peptide and another compound (such as extend the compound of polypeptide transformation period, such as polyoxyethylene glycol) merge the polypeptide formed, or (iv) additional aminoacid sequence be blended in this peptide sequence and the polypeptide formed (with leader sequence, the fusion rotein that the sequence label such as secretion sequence or 6His merges and formed).According to instruction herein, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
The preferred reactive derivative of one class refers to compared with the aminoacid sequence of formula I or formula II, has 3 at the most, preferably at the most 2, more preferably at the most 1 amino acid replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out amino acid replacement according to Table A and produce.
Table A
Invention also provides the analogue of C hepatitis virus antigen peptide.The difference of the polypeptide shown in these analogue and SEQIDNO:1-5 can be the difference on aminoacid sequence, can be also the difference do not affected on the modified forms of sequence, or have both at the same time.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide exemplified.
(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as carried out glycosylation modified and polypeptide that is that produce in those in the synthesis of polypeptide and processing or further procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) by being exposed to by polypeptide and completing.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.
Polypeptide of the present invention can also with by pharmaceutically or the derivative salt form of the acceptable acid of physiology or alkali use.These salt include, but is not limited to the salt formed with following acid: spirit of salt, Hydrogen bromide, sulfuric acid, citric acid, tartrate, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succsinic acid, oxalic acid, fumaric acid, toxilic acid, oxaloacetic acid, methylsulfonic acid, ethyl sulfonic acid, Phenylsulfonic acid or isethionic acid.Other salt comprise: the salt formed with basic metal or alkaline-earth metal (as sodium, potassium, calcium or magnesium), and with the form of " prodrug " of ester, carbamate or other routines.
Encoding sequence
The invention still further relates to the polynucleotide of coding C hepatitis virus antigen peptide.
Polynucleotide of the present invention can be DNA form or rna form.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can the varient of or degeneracy identical with the sequence of the polypeptide shown in coding SEQIDNO:1-5.As used herein, " varient of degeneracy " refers to that coding has the polypeptide shown in SEQIDNO:1-5 in the present invention, but the differentiated nucleotide sequence of corresponding encoded region sequence.
In the present invention preferably embodiment, described polynucleotide sequence as shown in SEQIDNO.:6-9.
In an embodiment of the invention, the polynucleotide sequence of the coding hepatitis C virus of wild-type is as shown in SEQIDNO.:10.
At one preferably in embodiment of the present invention, the polynucleotide of the coding hepatitis C virus described in after codon optimized are as shown in SEQIDNO.:11.
Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.At present, the DNA sequence dna of code book invention polypeptide (or its fragment, or derivatives thereof) can be obtained completely by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA molecular (or as carrier) as known in the art and cell.
The present invention also relates to the carrier comprising polynucleotide of the present invention, and with the host cell that carrier of the present invention or polypeptid coding sequence produce through genetically engineered.
As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.
Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding and the present invention have protein fragments, the sum analogous to general Dedekind sum of identical aminoacid sequence.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function changing in fact its code book invention enzyme.
As used herein, term " primer " refers to and is matching with template, can with it for starting point carries out synthesizing the general name with the oligonucleotide acid of the DNA chain of template complementation under the effect of archaeal dna polymerase.Primer can be natural RNA, DNA, also can be any type of natural nucleotide.Primer can be even that non-natural Nucleotide is as LNA or ZNA etc.The complementary that primer " haply " (or " substantially ") is special with on a chain in template.Primer could must start to extend with the abundant complementation of the chain of in template, but the sequence of primer need not with the sequence complete complementary of template.Such as, hold at 3' end and the 5' of the primer of template complementation and adds the preceding paragraph and the not complementary sequence of template, such primer still haply with template complementation.As long as there is sufficiently long primer can be combined fully with template, the primer of non-fully complementation also can form primer-template complex with template, thus increases.
The Nucleotide full length sequence of polypeptide of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.
Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.
In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.
The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR suitably can be selected according to sequence information of the present invention disclosed herein, and using conventional procedures synthesis.Using conventional procedures is as the DNA/RNA fragment increased by gel electrophoresis abstraction and purification.
The present invention also relates to the carrier comprising polynucleotide of the present invention, and with the host cell that carrier of the present invention or albumen coded sequence produce through genetically engineered, and the method for enzyme of the present invention is produced through recombinant technology.
By the recombinant DNA technology of routine, polynucleotide sequence of the present invention can be utilized express or produce HCV antigen peptide.In general following steps are had:
(1). with the polynucleotide (or varient) of code book invention polypeptide of the present invention, or transform or suitable host cell of transduceing with the recombinant expression vector containing these polynucleotide;
(2). in suitable substratum, cultivate host cell;
(3). separation, purifying HCV antigen peptide from substratum or cell.
Method well-known to those having ordinary skill in the art can be used for building the DNA sequences encoding containing enzyme of the present invention and the suitable expression vector of transcribing/translating control signal.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, synthesizes to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as Tetrahydrofolate dehydrogenase, neomycin resistance and green fluorescent protein (GFP) that eukaryotic cell is cultivated, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, Bacillus subtillis, the bacterial cell of streptomyces; Fungal cell is as pichia spp, brewing yeast cell; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast etc. of CHO, NS0, COS7 or 293 cells.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, and with the process of CaCl2 method, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.
The transformant obtained can be cultivated by ordinary method, expresses the protein of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.
Protein in the above methods can be expressed or be secreted into extracellular in cell or on cytolemma.If need, can utilize its physics, chemistry with other characteristic by various separation method abstraction and purification albumen.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Preparation method
Polypeptide of the present invention can be recombinant polypeptide or improvement on synthesis.Polypeptide of the present invention can be chemosynthesis, or restructuring.Correspondingly, polypeptide using conventional procedures synthetic of the present invention, also can produce with recombination method.
A kind of preferred method uses liquid phase synthesis techniques or solid phase synthesis technique, as Boc solid phase method, Fmoc solid phase method or two kinds of method conbined usage.Solid phase synthesis can obtain sample fast, can select suitable resin carrier and synthesis system according to the sequence signature of object peptide.Such as, in Fmoc system, preferred solid phase carrier is as being connected with the Wang resin of C terminal amino acid in peptide, and Wang resin structure is polystyrene, and the arm between amino acid is 4-alkoxyl group benzylalcohol; By 25% hexahydropyridine/dimethyl formamide room temperature treatment 20 minutes, to remove Fmoc blocking group, and held one by one to the extension of N end by C according to given aminoacid sequence.After having synthesized, the proinsulin related peptides of synthesis cut down from resin with the trifluoroacetic acid containing 4% p-methyl phenol and remove protecting group, after can crossing filtering resin, ether sedimentation is separated and obtains thick peptide.After the solution freeze-drying of products therefrom, the peptide needed for purifying by gel-filtration and reverse phase HPLC method.When using Boc system to carry out solid phase synthesis, preferred resin is the PAM resin being connected with C terminal amino acid in peptide, and PAM resin structure is polystyrene, and the arm between amino acid is 4-methylol phenylacetamide; In Boc synthesis system, going to protect, in and, in the circulation of coupling, remove blocking group Boc with TFA/ methylene dichloride (DCM) and (DIEA/ methylene dichloride neutralizes with diisopropylethylamine.After peptide chain condensation completes, with the hydrogen fluoride (HF) containing p-cresol (5-10%), at 0 DEG C, process 1 hour, peptide chain is cut from resin, removes blocking group simultaneously.With 50-80% acetic acid (containing a small amount of mercaptoethanol) extracting peptide, use molecular sieve SephadexG10 or Tsk-40f separation and purification after solution freeze-drying further, and then obtain required peptide through high-pressure liquid phase purifying.Various coupling agent known in chemistry of peptides field and each amino-acid residue of coupling method coupling can be used, such as can use dicyclohexylcarbodiimide (DCC), hydroxyl benzotriazole (HOBt) or 1,1,3,3-tetra-urea phosphofluoric acid ester (HBTU) carries out direct coupling.For synthesizing the small peptide obtained, its purity and structure can be confirmed with RP-HPLC and mass spectroscopy.
In one embodiment, C hepatitis virus antigen peptide of the present invention, by its sequence, adopts the method preparation of solid phase synthesis, row high-efficient liquid phase chromatogram purification, obtains high purity object peptide freeze-dried powder ,-20 DEG C of storages.
Another kind method produces polypeptide of the present invention with recombinant technology.By the recombinant DNA technology of routine, polynucleotide of the present invention can be utilized express or produce C hepatitis virus antigen peptide of the present invention.In general following steps are had:
(1). with the polynucleotide (or varient) of coding C hepatitis virus antigen peptide of the present invention, or transform or suitable host cell of transduceing with the recombinant expression vector containing these polynucleotide;
(2). the host cell cultivated in suitable substratum;
(3). separation, protein purification from substratum or cell.
Recombinant polypeptide can be expressed or be secreted into extracellular in cell or on cytolemma.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Because polypeptide of the present invention is shorter, multiple polypeptide therefore can be considered to be cascaded, the expression product of recombinant expressed rear acquisition multimeric forms, then forms required little peptide by the enzyme method such as to cut.
Pharmaceutical composition and application process
Antigen peptide of the present invention or fusion rotein can be used as immunogen and induce the antibody and T lymphocytes activity that produce HCV-Ab IgG or HCV positive cell in vivo, thus reach anticancer therapy effect.In addition, antigen peptide of the present invention has excellent specificity and immunocompetence, therefore can be used for the vaccine preparing immunotherapy or prevention.
On the other hand, present invention also offers a kind of medicine (comprising vaccine) composition, it contains polypeptide of the present invention or its pharmacy acceptable salt (or its encoding sequence) of (a) safe and effective amount; And (b) pharmaceutically acceptable carrier or vehicle.The quantity of polypeptide of the present invention is generally 10 microgram-100 milligrams/agent, is preferably 100-1000 microgram/agent.
For the purposes of the present invention, effective dosage for giving individuality about 0.01 mg/kg to 50 mg/kg, the preferably polypeptide of the present invention of 0.05 mg/kg to 10 mg/kg body weight.In addition, polypeptide of the present invention can be alone, also can use together with other treatment agent (as being formulated in same pharmaceutical composition).
Pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration.This term refers to some medicament carriers like this: they itself are not induced and produce the antibody harmful to the individuality accepting said composition, and do not have undue toxicity after administration.These carriers are well known to those of ordinary skill in the art.Discussing fully about pharmaceutically acceptable vehicle can be found in Remington'sPharmaceuticalSciences (MackPub.Co., N.J.1991).This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol, adjuvant and combination thereof.
In therapeutic composition Chinese materia medica, acceptable carrier can contain liquid, as water, salt solution, glycerine and ethanol.In addition, in these carriers, also may there is complementary material, as wetting agent or emulsifying agent, pH buffer substance etc.
Usually, therapeutic composition can be made injectable agent, such as liquor or suspension; Also can be made into be applicable to allocating in solution or suspension before the injection, the solid form of liquid vehicle.
Once be made into composition of the present invention, it can be carried out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously, subcutaneous, intracutaneous or topical.Wait that the object preventing or treat can be animal; Especially people.
When pharmaceutical composition of the present invention is used to actual therapeutic, the pharmaceutical composition of various different dosage form can be adopted according to service condition.It is preferably injection.
These pharmaceutical compositions or can dissolve and prepare according to conventional methods by mixing, dilution, and add suitable medicated premix once in a while, as vehicle, disintegrating agent, tackiness agent, lubricant, thinner, buffer reagent, isotonic agent (isotonicities), sanitas, wetting agent, emulsifying agent, dispersion agent, stablizer and solubility promoter, and this process for preparation can carry out according to formulation usual way.
Pharmaceutical composition of the present invention can also sustained release formulation administration.Such as, it is in the pill of carrier or micro-capsule that C hepatitis virus antigen peptide or its salt can be impregnated in release polymer, then by this pill or the micro-capsule tissue by Operation people.As the example of release polymer, what can exemplify has ethylene-vinylacetate multipolymer, poly-hydroxyl-metacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose gum, lactic acid polymer, lactic acid-ethanol copolymer etc., and what preferably can exemplify is that biodegradable polymkeric substance is as lactic acid polymer and lactic acid-ethanol copolymer.
When pharmaceutical composition of the present invention is used to prevention or treatment, as the C hepatitis virus antigen peptide of activeconstituents or the dosage of its pharmacy acceptable salt, can according to the body weight of each object (patient) waiting to prevent or treat, age, sex, symptom degree and reasonably being determined.
Vaccine of the present invention (composition) can be preventative (i.e. preventing disease) or curative (namely at ill rear disease therapy).
These vaccines comprise immunising antigen (comprising recombinant protein of the present invention), and usually combine with " pharmaceutically acceptable carrier ", and these carriers comprise any carrier of itself not inducing and producing the antibody that the individuality accepting said composition is harmful to.Suitable carrier normally large, metabolism macromole slowly, as protein, polysaccharide, poly(lactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer, lipid aggregates (as oil droplet or liposome) etc.These carriers are well known to those of ordinary skill in the art.In addition, these carriers can play immunostimulant (" adjuvant ") effect.In addition, antigen also can with bacterial toxoid (toxoid as pathogenic agent such as diphtheria, tetanus, cholera, helicobacter pyloris) coupling.
The preferred adjuvant strengthening immune composition effect includes but not limited to: (1) aluminium salt (alum), as aluminium hydroxide, aluminum phosphate, Tai-Ace S 150 etc.; (2) oil-in-water emulsion formula, such as, (a) MF59 (see WO90/14837), (b) SAF, and (c) Ribi
tMadjuvant system (RAS) (RibiImmunochem, Hamilton, MT), (3) saponin adjuvant; (4) Freund Freund's complete adjuvant (CFA) and Freund Freund's incomplete adjuvant (IFA); (5) cytokine, as interleukin (as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), Interferon, rabbit (as IFN-γ), macrophage colony stimulating factor (M-CFS), tumour necrosis factor (TNF) etc.; (6) the detoxification varient of bacterial ADPribosylating toxin (as E.coli LT LT); And (7) carry out other material of enhancing composition effect as immunostimulant.
Comprise the vaccine composition (such as, antigen, pharmaceutically acceptable carrier and adjuvant can be comprised) of immunogenic composition, usually containing thinner, as water, salt solution, glycerine, ethanol etc.In addition, auxiliary substances, as wetting agent or emulsifying agent, pH buffer substance etc. can be present in this kind of vehicle.
More specifically, comprise the vaccine of immunogenic composition, comprise the immunogenic polypeptide of immunological effective amount, and above-mentioned component needed for other." immunological effective amount " refers to that it is effective for giving individual amount to treatment or prevention with single dose or a continuous agent part.This consumption can according to treat that the classification (as people) of individuality is treated by individual healthy state and physiological situation, institute, the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician determine the assessment of medical conditions and other correlative factor.Estimate that this consumption is by relatively wide scope, determines by normal experiment.
Usually, vaccine composition or immunogenic composition can be made injectable agent, such as liquor or suspension; Also can be made into the solid form being applicable to allocating into solution or suspension, liquid excipient before the injection.Said preparation also can emulsification or be encapsulated in liposome, to strengthen adjuvant effect.
In addition, vaccine composition of the present invention can be unit price or polyvalent vaccine.
Major advantage of the present invention is:
(1) HCV antigen peptide of the present invention is derived from the E2 albumen (preferred 1b C-type virus C) of HCV, can efficient, special, wide spectrum in conjunction with the anti-E2 antibody in infected patient serum, can be used for developing the diagnostic kit detecting HCV infection;
(2) after using antigen peptide immunising mammals of the present invention, the antibody of generation has the neutralising capacity of wide spectrum, all shows neutralization to each hypotype of HCV, can in and the strain of HCV1-7 hypotype;
(3) using the codon through optimizing of the present invention in Drosophila S 2 cells, express antigen peptide of the present invention, expressing output and significantly improving, at least improve 5 times compared with wild-type, 50 times of E2 protein yield of the HEK293T cell expressing of report before being.
(4) when using antigen peptide immune animal of the present invention, the titre that CpG can significantly improve neutralizing antibody in immunological adjuvant, is added.
(5) binding activities of further truncation type antigen peptide sE2 Δ HVR1 of the present invention to acceptor SR-BI significantly declines, and can reduce the interference to body normal physiological activity, reduce immunogenic toxic side effect.
(6) antigen peptide sE2 of the present invention has the conformation different from natural HCVE2 virus, but still can bring out the immune response of animal, experiment proves that it can expose more epitope, bring out the immune response of higher titre, 6His label wherein, makes protein purification more simple.
Below in conjunction with specific embodiment, state the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted detailed conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Materials and methods
1 cell
Fruit bat Schneider2 (S2) cell is purchased from Invitrogen company, S2 cell cultures is in interpolation 10% foetal calf serum (Gibco), 1% dual anti-(Gibco), in the Schneider'sDrosophilaMedia (Gibco) of 1%L-glutamine (Gibco) or add 1% dual anti-(Gibco), the Express of 1%L-glutamine (Gibco)
in SFM substratum (Gibco), 28 DEG C of incubators are cultivated.Huh7.5.1 cell cultures is in interpolation 10% foetal calf serum (Gibco), 1% dual anti-(Gibco), 1%L-Glutamine (Gibco), 10mMHEPESbuffer (Gibco), in the DMEM (Hyclone) of 1%NonessentialAminoAcids (Gibco), 37 DEG C of CO2gas incubator are cultivated.Chinese hamster ovary celI is incubated at interpolation 10% foetal calf serum (Gibco), and in the Ham'sF-12K substratum (Gibco) of 1% dual anti-(Gibco), 37 DEG C of CO2gas incubator are cultivated.Cell strain CHO-CD81 and CHO-SRB1 of stably express HCV acceptor CD81 and SR-B1 can reference (3).
2 viruses
All HCV viruses used by the present invention are as shown in table 1.HCV2a C-type virus C JFH-1 is prepared according to document (4) method.In brief, 10 μ gpUC-vJFH recombinant plasmids are by XbaI enzyme cutting linearizing, get 1 μ g after reclaiming linear DNA product to illustrate by AmbionT7transcriptionkit as masterplate and carry out in-vitro transcription, be dissolved in 30 μ L nuclease free ultrapure waters after purifying, be stored in-80 DEG C of refrigerators for subsequent use.Resuspended to cell count 1 × 10 with Opti-MEM after the enzymic digestion of Huh7.5.1 cell tryptase
7/ mL, gets 400 μ L and adds electric revolving cup, and adds the above-mentioned RNA of 10 μ g.Electric shock condition is adjusted to 0.27kV, and 100Ohms and 950 μ F, shocks by electricity, then cell sucking-off is added complete DMEM, and 37 DEG C of CO2gas incubator are cultivated.Be passaged to when there is cytopathic effect and start to collect cell conditioned medium, after the viral supernatants collected measures titre, to be stored in-80 DEG C of refrigerators for subsequent use in packing.Chimeric HCV1b C-type virus C Con1/JFH-1,1a C-type virus C H77/JFH-1,2a C-type virus C Jc1, by document (5,6) method preparation, Core to NS2 first cross-film district of embedded virus is made up of the aminoacid sequence of corresponding virus strain, and genome rest part derives from JFH-1.(see reference the recombinant plasmid of structure 2-7 type embedded virus document DevelopmentandcharacterizationofhepatitisCvirusgenotype1-7cellculturesystems:roleofCD81andscavengerreceptorclassB typeIandeffectofantiviraldrugsHepatology (Baltimore, Md.) .2009.10.1002/hep.22673), electricity is gathered in the crops after turning amplification as stated above, comprise HCV2b C-type virus C J8/JFH1, 3a C-type virus C S52/JFH1 (I793S, K1404Q), 4a C-type virus C ED43/JFH1 (T827A, T977S), 5a C-type virus C SA13/JFH1 (A1022G, K1119R), 6a C-type virus C HK6a/JFH1 (F350S, and 7a C-type virus C QC69/JFH1 N417T).
Clinical separation strain PR26, PR52, PR79 derive from three accept IFN-α+Ribavirin treatment chronic HCV infection patient (document that sees reference (7)) at Medical College, Shanghai Communication Univ.'s Ruijin Hospital.This experiment obtains the approval of Ruijin Hospital Ethics Committee, and has obtained sufferers themselves's license.From 140 μ l patient blood samples, extract total serum IgE (QIAampViralRNAminikit), be dissolved in 40 μ L nuclease free ultrapure waters, be stored in-80 DEG C of refrigerators for subsequent use.TaKaRaAMVXLreversetranscriptase test kit and HCV Auele Specific Primer is utilized to be cDNA by HCVRNA reverse transcription.1b hypotype is defined as by order-checking and gene type (HCVgenotypinggenechipkit).Afterwards, through RT-PCR, the DNA sequence dna in coding Core to NS2 first cross-film district is increased with primer, and carry out over-lap PCR with the 5'UTR of JFH-1 and remaining NS2 to NS3 region.The pUC-vJFH carrier that PCR primer after purifying cuts through with enzyme after EcoRI and SpeI double digestion carries out ligation.The present inventor's Stochastic choice 20-30 correct clone carries out in-vitro transcription as above and turns with electricity.Last the present inventor selects four strain clone PR26C3M, PR26C6M, PR52B6M and PR79.
1-7 type HCVcc virus used in table 1. embodiment
3 antibody
Mouse-anti HCVNS3 monoclonal antibody purchased from Abmart company, people's HCV-Ab IgG E2 monoclonal antibody AR3A (document that sees reference (8)).Mouse-anti His label monoclonal antibody is purchased from Abmart (for Westernblot) and Sigma (detecting for FACS).HRP marks mountain sheep anti-mouse igg, goat anti-rabbit igg two is anti-is purchased from Sigma, HRP marks the anti-Abcam.AP that is purchased from of Goat anti human IgG bis-and marks the anti-Promega.HRP that is purchased from of mountain sheep anti-mouse igg two and mark goat against murine IgG1 and HRP and mark the anti-Southernbiotech.AlexaFluor-488 that is purchased from of goat against murine IgG2a bis-and mark mountain sheep anti-mouse igg two and resist, and AlexaFluor-555 marks that mountain sheep anti-mouse igg two is anti-is purchased from Invitrogen.
4 plasmid constructions
Insect cell expression vector is that pMT/BiP/V5-HisA and screening plasmid pCoBlast is purchased from Invitrogen company.Recombinant plasmid pcDNA3.1-CE1E2wt and pcDNA3.1-CE1E2opti to be connected on pcDNA3.1 (+) plasmid (purchased from Invitrogen company) by EcoRI and XbaI by the HCVCon1 strain structural protein Core-E1-E2 encoding gene of wild-type and the Con1 strain Core-E1-E2 encoding gene after codon optimized (Geneart company) formed.
The E2 albumen (amino-acid residue 384-661) of brachymemma is implemented between Bip signal peptide sequence and V5 and His label by carrier pMT/BiP/V5-HisA.According to sequences Design upstream and downstream primer:
5'CTGCCATGGGGAACCTATGTGACAGGGGGGACG3'(SEQIDNO.:12)，
5'GCCTCTAGACTCTGATCTGTCCCTGTCCTCCAG3'(SEQIDNO.:13)，
With plasmid pcDNA3.1-CE1E2wt for masterplate, pcr amplification sE2wt fragment; Design primer:
5'CTGCCATGGCCGGCACATACGTGACAG3'(SEQIDNO.:14)，
5'GCACTCTAGACTCGCTTCTGTCCCGAT3'(SEQIDNO.:15)，
With plasmid pcDNA3.1-CE1E2opti for masterplate, pcr amplification sE2opti fragment.Fragment NcoI after purifying and XbaI double digestion, cut and to be connected to after glue reclaims on pMT/BiP/V5-HisA carrier that NcoI and XbaI double digestion crosses, obtain recombinant plasmid pMT-BiP/sE2wt/His and pMT-BiP/sE2opti/His with this.
For expressing the sE2 cutting out HVR1, the present inventor utilizes primer:
5'CCCCATGGCAGCTGGTGAACACCAACGGCAGC3'(SEQIDNO.:16)；
5'GCCTCTAGACTCTGATCTGTCCCTGTCCTCCAG3'(SEQIDNO.:17)，
Recombinant clone pMT-BiP/sE2opti Δ HVR1/His is obtained by above-mentioned same procedure.
The structure of the S2 clone of 5 expression sE2 and sE2 Δ HVR1 albumen and screening
For transient expression, by wild-type S2 cell dilution to 3 × 10
6individual/hole is laid in 6 orifice plates, after about 12h, calcium robin carries out transfection, by 19 μ gpMT-BiP/sE2wt/His or pMT-BiP/sE2opti/His transfection in S2 cell, hatch 16-24h for 27 DEG C, repeatedly rinse cell twice with fresh Schneider'sDrosophilaMedia afterwards, finally cell is resuspended in 6 orifice plates.After 27 DEG C of cultivation 1-3d, get 2 × 10
6individual/hole is laid in 6 orifice plates, and add the chromium chloride that final concentration is 5 μMs, inducing cell expressing protein, collects cell conditioned medium after 3-5d, adopts Westernblot to detect protein expression.
For stably express, by wild-type S2 cell dilution to 3 × 10
6individual/hole is laid in 6 orifice plates, after about 12h, calcium robin carries out transfection, 1 μ g is screened plasmid pCoBlast and 19 μ gpMT-BiP/sE2opti/His cotransfections in S2 cell, hatch 16-24h for 27 DEG C, cell is repeatedly rinsed twice afterwards with fresh Schneider'sDrosophilaMedia, finally cell is resuspended in 6 orifice plates, liquid is changed after 27 DEG C of cultivation 1-3d, to be that cell is resuspended in 6 orifice plates by the complete Schneider'sDrosophilaMedia of 25 μ g/mL blasticidins containing concentration, cultivate 1-2weeks for 27 DEG C.When cell density reaches 6 ~ 20 × 10
6individual/mL time, with containing concentration be the complete Schneider'sDrosophilaMedia of 10 μ g/mL blasticidins by passage, after this with the S2 cell after this substratum enlarged culturing transfection.From this passage cell, get 3mL in 6 orifice plates, add the chromium chloride that final concentration is 5 μMs, inducing cell expressing protein, collects cell conditioned medium after 3-5d, adopts Westernblot to detect protein expression.For screening the cell monoclonal of stably express, surely turn cell dilution by above-mentioned and be laid on 96 orifice plates with the density of 1.5 cells/well, to be that the complete Schneider'sDrosophilaMedia of 10 μ g/mL blasticidins cultivates about 2 weeks containing concentration, to select mono-clonal enlarged culturing and detect expression.The S2 clone same procedure expressing sE2 Δ HVR1 builds and screening.
The expression and purification of 6sE2 and sE2 Δ HVR1 albumen
When the monoclonal cell enlarged culturing of stably express sE2 and sE2 Δ HVR1 is used for great expression, substratum is replaced with the dual anti-(Pen .-Strep (Penicillin-Streptomycin) of interpolation 1%, Gibco), 1%L-glutamine (Gibco), the serum-free Express of 10 μ g/mL blasticidins
sFM substratum (Gibco).Cell is inoculated in magnetic agitation shaking flask, is cultured to cell density 4 × 10
6individual/mL time add the chromium chloride abduction delivering that final concentration is 5 μMs.Supernatant is collected after 5d, uf processing is carried out with the ultra-filtration centrifuge tube (Millipore) of 3kDa after 0.45 μm of membrane filtration, concentrated about 20-30 times volume be bindingbuffer (0.5MNaCl by solution replacement, 20mMTris, 10mM imidazoles, pH7.9), and after through with nickel post (Novagen) purifying, be combined in albumen washingbuffer (0.5MNaCl, 20mMTris, the 50mM imidazoles of nickel post, pH7.9) elutingbuffer (0.5MNaCl is used again after washing foreign protein off, 20mMTris, 500mM imidazoles, pH7.9) wash-out.Gained albumen carries out SDS-PAGE (12%acrylamide) and Westernblot and analyzes.
The qualification of 7sE2 albumen and analysis
SDS-PAGE and Westernblot detects after protein sample to be checked mixes with SDS-PAGE load solution, and 100 DEG C are boiled 5min, 12%SDS-PAGE electrophoretic analysis, coomassie brilliant blue staining; Sample 100V voltage 1h after 12%SDS-PAGE electrophoresis forwards pvdf membrane (PALL) to, 5% skimming milk room temperature closes 2h, the mouse-anti His monoclonal antibody of 1:1000 dilution or the AP33 monoclonal antibody of 1:1000 dilution are primary antibodie room temperature effect 2h, the mountain sheep anti-mouse igg of the HRP mark of 1:4000 dilution is two anti-room temperature effect 1h, appropriate ECL chemical illuminating reagent is substrate, and LAS4000 scanning analysis instrument (Fujifilm) obtains Westernblot result.During AP colour developing, be two anti-room temperature effect 1h with the mountain sheep anti-mouse igg of the AP mark of 1:3000 dilution, film drip BCIP/NBTColorDevelopmentSubstrate (Promega) and carries out color reaction.
The de-glycosylation of sE2 is got 10 μ gsE2 albumen and is added 1XGlycoproteinDenaturingBuffer (NEB), 100 DEG C of heating 10min.Add 10 × NP-40 subsequently, G7ReactionBuffer and PNGaseF (NEB), 37 DEG C of reaction 1h carry out de-glycosylation reaction.The deglycosylated sE2 protein site of gained, after 12%SDS-PAGE glue carries out electrophoresis, carries out Westernblot detection as stated above.
SE2 detection of antigenicity utilizes the method for ELISA to detect the antigenicity of sE2.SE2 protein 10 0ng/ hole bag is by elisa plate (Costar), 4 DEG C are spent the night, the closed 1h of 5% skimming milk 37 DEG C, every hole 200 μ l, after rinsing, every hole adds the HCVE2 monoclonal antibody E2-C1 of 2 times of gradient dilutions, AP33, and contrast monoclonal antibody (anti-HCVCore), hatch 2h for 37 DEG C, wash 3 times afterwards every hole add the mountain sheep anti-mouse igg of the HRP mark of 1:6000 dilution, hatch 1h for 37 DEG C, TMB colour developing after washing 5 times, room temperature lucifuge effect 10min, after 1M phosphoric acid termination reaction, the multi-functional readout instrument working sample of ThermoScientificVarioskanFlash is at the light absorption value of A450.
Complete DMEM doubling dilution sE2 albumen to the 100 μ g/mL of receptor binding assays, by adherent Chinese hamster ovary celI, CHO-CD81 cell and CHO-SRB1 cell 0.02%EDTA process make cell take off wall, by cell piping and druming evenly, the centrifugal rear above-mentioned 100 μ g/mLsE2 solution of 150 μ L of using respectively are resuspended, hatch 1h for 37 DEG C.After washing 2 times with PBS, add mouse-anti His label monoclonal antibody (Sigma) of 1:100 dilution, hatch 1h for 4 DEG C.After washing 2 times with PBS again, the AlexaFluor-555 adding 1:200 dilution marks mountain sheep anti-mouse igg two and resists, and hatches 30min for 4 DEG C.After PBS washes three times, resuspended with 500 μ LPBS, flow cytometer LSRII (BDBiosciences, SanDiego, CA) detects, acquired results FlowJo software analysis.
HCV infection blocking test is by Huh7.5.1 cell dilution to 1 × 10
4individual/hole is laid on 96 orifice plates, after about 12h, with complete DMEM doubling dilution sE2 albumen to 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.5625 μ g/mL, 0.78125 μ g/mL, and 0 μ g/mL and cell hatch, 37 DEG C of 1h, use HCVCon1/JFH-1 cells infected afterwards.After infecting, 4h changes liquid, after 37 DEG C of cultivation 72h, carries out the immunofluorescence dyeing experiment of HCVNS3 albumen.Add Fixationbuffer (4%paraformaldehydeinPBS) room temperature and fix 30min, suck stationary liquid immediately, PBS washes 2 times, add Blockingbuffer (3%BSA, 0.3%TritonX-100inPBS+10%FBS) room temperature closes 1h, PBS washes 2 times, add Bindingbuffer (3%BSA, HCV-Ab IgG NS3 monoclonal antibody (1:1000) room temperature effect 1h 0.3%TritonX-100inPBS) diluted, PBS washes 3 times, then two anti-AlexaFluor-488 are marked mountain sheep anti-mouse igg (1:1000) and Hoechst dyestuff (1:5000, MolecularProbes) be jointly diluted in Bindingbuffer, room temperature effect 1h, PBS washes 4 times, last counting to the fluorescent spot in every hole under fluorescent microscope (Leica) counts.Count as blank group with the fluorescent spot of 0 μ g/mL, infect inhibition (%)=(fluorescent spot of blank group counts-respective aperture in fluorescent spot count) fluorescent spot of/blank group counts * 100%.
8sE2 Protein Detection HCV infection patients serum
Determine that can sE2 be used for the HCV specific antibody detected in infected patient by ELISA.First sE2 albumen is diluted to 100ng/ μ L with PBS, 4 DEG C of bags that spend the night are by elisa plate, the closed 1h of 5% skim-milk 37 DEG C, wash off after milk with 6 Healthy Human Serums of 1% skim-milk 10 doubling dilution and 54 the third hepatopathy human plasmas (36 1b type infected patients, 18 2a type infected patients; Available from medical college of Jilin University) as primary antibodie, hatch 2h for 37 DEG C, resist with the Goat anti human IgG bis-of 1% skim-milk 1:5000 dilution HRP mark after cleaning, hatch 1h for 37 DEG C.TMB colour developing after washing 5 times, room temperature lucifuge effect 10min, after 1M phosphoric acid termination reaction, the multi-functional readout instrument working sample of ThermoScientificVarioskanFlash is at the light absorption value of A450.
9 animal immunes
By sE2 respectively with aluminium adjuvant (Pierce), aluminium adjuvant+CpG2006 (TCGTCGTTTTGTCGTTTTGTCGTT (SEQIDNO.:18)) or complete Freund's adjuvant (Sigma) emulsification mixing, abdominal injection immunity 10 female Balb/c mouse (6 week age), every mouse immune 50 μ gsE2, uses PBS as negative control group (Fig. 4 A) simultaneously.Within 2 weeks and 4 weeks, carry out second and three immunity respectively after just exempting from, and within 25 weeks, carry out the memory of the 4th immune again after just exempting from.The freund's adjuvant group full freund's adjuvant (Sigma) that cannots be used up when booster immunization carries out emulsification.Respectively at just exempting from rear 0th, 4,6,13,17,22,25, eye socket being carried out to mouse in 27 weeks and getting blood, separation of serum.
For comparing the immunogenicity of sE2 and sE2 Δ HVR1 and inducing the ability of neutralizing antibody, the present inventor carries out emulsification to two kinds of albumen respectively with aluminium adjuvant (Pierce), abdominal injection immunity 6 female Balb/c mouse (6 week age), every mouse immune 50 μ gsE2, uses aluminium adjuvant as negative control group simultaneously.Just exempt within 2 weeks and 4 weeks, to carry out second and three immunity respectively afterwards, respectively at just exempting from afterwards, within the 0th, 4,6 weeks, carrying out eye socket to mouse gets blood, separation of serum.
By sE2 and complete Freund's adjuvant emulsification mixing, subcutaneous multi-point injection immunity two new zealand rabbits, every rabbit immunity 200 μ gsE2.Within 2 weeks and 4 weeks, carry out second and three immunity respectively after just exempting from, freund's adjuvant group carries out emulsification at two full freund's adjuvants of exempting to cannot be used up when exempting from three.Blood sampling before each immunity, carries out carotid artery blood sampling on the 7th week and collects serum and put to death.
10sE2 its specific antibody determination
ELISA is utilized to measure the terminal titre of mouse resisting anteserum and rabbit anti-serum.With sE2100ng/ hole bag by elisa plate (Costar), 4 DEG C are spent the night, the closed 1h of 5% skimming milk 37 DEG C, every hole 200 μ l, after rinsing, every hole adds the serum 100 μ l of 2 times of gradient dilutions, hatch 2h for 37 DEG C, wash 3 times afterwards every hole add the mountain sheep anti-mouse igg of the HRP mark of 1:6000 dilution, hatch 1h for 37 DEG C, TMB colour developing after washing 5 times, room temperature lucifuge effect 10min, after 1M phosphoric acid termination reaction, the multi-functional readout instrument working sample of ThermoScientificVarioskanFlash is at the light absorption value of A450.With the corresponding extent of dilution of the mice serum of PBS control group for benchmark, OD450 is greater than benchmark more than 0.1 and is then judged to be the positive, to present the terminal titre that positive most high dilution is this serum.
11 serum Neutralizing test
Huh7.5.1 cell measures and obtains serum to the neutralization of different subtype HCV virus.By Huh7.5.1 cell dilution to 1 × 10
4individual/hole is laid on 96 orifice plates, carries out neutralization test after about 12h.Dilute mouse resisting anteserum with complete DMEM according to 1:40, dilute rabbit anti-serum according to 1:20.HCV viral dilution is to 4 × 10
3fFU/mL, HCV virus (about 200FFU) mixing that the serum sample that 50 μ L dilute and 50 μ L dilute, set preimmune serum group and positive neutralizing antibody control group (the HCV virus that E2-C1 monoclonal antibody+50 μ L that 50 μ L dilute dilutes), 37 DEG C at 5%CO simultaneously
2incubator hatches 2h, is added to by serum-virus mixture in the hole being covered with Huh7.5.1 cell afterwards, changes liquid after 4-6h, carries out the immunofluorescence dyeing experiment of HCVNS3 albumen after 37 DEG C of cultivation 72h according to abovementioned steps.Last counting to the fluorescent spot in every hole under fluorescent microscope (Leica) counts.Count as benchmark with the fluorescent spot of serologic group before immunity, neutralization (%)=(fluorescent spot of preimmune serum group counts-respective aperture in fluorescent spot count) fluorescent spot of/preimmune serum group counts * 100%.
For measuring half-inhibition concentration (Halfmaximalinhibitoryconcentration, IC
50), by the serum sample equal-volume of each group of 10 mouse mixing after press 1:20 dilute, then 2 doubling dilutions, by above-mentioned Neutralizing test method measure often group to the half-inhibition concentration of different virus.The serum sample of every rabbit also measures after method doubling dilution like this.
The expression of embodiment 1sE2 in Drosophila S 2 cells
For expression of HCV sE2 albumen in Drosophila S 2 cells recombinant plasmid as shown in Figure 1A, carry sE2 wild-type encoding gene or codon optimized gene, coded target protein is the extracellular region (comprising 384-661 amino acids) of E2 albumen.To express the carrier transient transfection Drosophila S 2 cells respectively of wild-type sE2 encoding gene or codon optimized rear gene, after transfection, 48h takes out 2 × 10
6cell adds the chromium chloride abduction delivering that final concentration is 5 μMs, and the sE2 collected after 72h after the visible optimization of supernatant detection expresses significantly enhancing (Figure 1B).Therefore the present inventor selects codon optimized expression vector to carry out subsequent experimental.By pMT-Bip/sE2opti/His and pCoBlast corotation Drosophila S 2 cells, through blasticidin screening, obtain monoclonal cell strain sE2A.By sE2A cell enlarged culturing in 3L Spinner flask, when cell density reaches 4 × 10
6individual/mL time add the chromium chloride abduction delivering that final concentration is 5 μMs, get supernatant respectively at different time points and detect sE2 expressing quantity.
Within after Fig. 1 C shows induction the 3rd day, can go out a large amount of albumen to supernatant by expression-secretion, increase progressively from Tot Prot accumulation in 3-9 days, this albumen is comparatively stable as seen, not easily degrades.9th day harvested by centrifugation supernatant, after ultrafiltration and concentration, ni-sepharose purification, by 12%SDS-PAGE glue coomassie brilliant blue staining, find that the sE2 protein band of purifying is single, purity is higher, and molecular weight is about 46kDa (Fig. 1 D).
The specificity analysis of embodiment 2sE2 albumen
Use His monoclonal antibody and E2 monoclonal antibody (AP33) to detect purifying sE2 albumen by Westernblot respectively, all produce about 46kDa positive band, show that this albumen is target sE2 albumen (Fig. 2 A) really.Use same antibody test with after the de-glycosylation of PNGaseF process sE2 albumen, find that positive band position is about 34kDa (Fig. 2 A), the sE2 albumen of prompting S2 insect cell expression exists glycosylation modified.
Detected by ELISA, sE2 can combine the monoclonal antibody AR3A identifying discontinuous conformational epitope very well, and its ELISA reads value and monoclonal antibody concentration is proportionate; Necessarily combine though sE2 and the monoclonal antibody AP33 of identification linear epitope have but react strong (Fig. 2 B).This result prompting restructuring sE2 albumen correctly folds, and obtains the conformation with virus surface E2 protein similar.
E2 albumen has with receptors bind thus mediates the function that HCV virion enters cell.The present inventor utilizes the Chinese hamster ovary celI of stably express HCV acceptor CD81 or SR-B1 to analyze the binding ability of sE2 and acceptor, and the method can reference (3,11), is the system of the comparatively maturation of qualification E2 and receptors bind.
Visible by FACS detected result, compare the Chinese hamster ovary celI (Fig. 2 C) of wild-type, the CHO-CD81 cell (Fig. 2 D) of expressing CD81 and the CHO-SRB1 cell (Fig. 2 E) of expressing SR-B1 all can be combined with sE2.Prove that sE2 albumen is similar to the envelope protein under native state, can directly in conjunction with CD81 and SR-BI.
Whether can by carrying out competitive inhibition HCV virus infection in conjunction with cell surface receptor in order to probe into sE2, after the sE2 of the present inventor's different concns or BSA and Huh7.5.1 cell incubation, add HCVCon1/JFH-1 virus infected cell.Significantly can suppress HCV infection (Fig. 2 F) relative to reference protein BSA, sE2, and present dose-dependence.
Embodiment 3sE2 can specific detection HCV infection patients serum
For determining that sE2 whether can HCV specific antibody in specific binding HCV infection person body, the present inventor with sE2 albumen bag by after elisa plate, hatch with the Healthy Human Serum diluted and two kinds of hypotype (1b and 2a) third hepatopathy human serums respectively, then detect the specific IgG antibodies in conjunction with sE2.
Result shows, compare Healthy Human Serum, patients serum produces very high binding activities, the mean OD value when 1:20000 dilutes still up to 0.5-1.0 (Fig. 3), show sE2 can efficient, special, wide spectrum in conjunction with the anti-E2 antibody in infected patient serum.Therefore, sE2 can be used for developing simple and quick HCV infection diagnostic kit.
The immunogenicity of embodiment 4sE2
With the sE2 protein immunization mouse of purifying, be adjuvant with aluminium adjuvant, aluminium adjuvant+CpG or freund's adjuvant respectively, PBS group in contrast.
Can find out from Fig. 4 B, compare control group, no matter sE2 all can induce the sE2 specific antibody of high titre in BALB/c mouse using aluminium adjuvant, aluminium adjuvant+CpG or freund's adjuvant as adjuvant, and can maintain higher level (4-22 week) for 18 weeks.Be down to lower level to antibody titers when the 25th week, but after a booster immunization, antibody titers rises to very high level again, illustrate that the immunity of sE2 has induced good immunological memory in Mice Body.Between different adjuvant group, immunogenic peci-order is sE2+ freund's adjuvant >sE2+ aluminium adjuvant+CpG>sE2+ aluminium adjuvant generally.
With the specific antibody detected after sE2 immune rabbit in serum, find that sE2 immunize rabbit serum also exists the specific antibody in conjunction with sE2 (Fig. 5) of high titre.
Embodiment 5sE2 immune serum there is wide spectrum Neutralization effect
By external Neutralizing test, detect sE2 immune serum to the neutralization of HCV virus (HCVcc).Fig. 6 A shows, under the Dilution ratio of 1:40, three experimental group immune serums all can significantly in and HCV virus, not only for the Con1 virus with sE2 homology, and viral for the H77 of 1a type, the JFH-1 virus of 2a type, Jc1 virus, the J8 virus of 2b type, the S52 virus of 3a type, the ED43 virus of 4a type, the SA13 virus of 5a type, the HK6a virus of 6a type, and the QC69 virus of 7a type all has good neutralizing effect.In addition, these three groups of immune serums are to the clinical separation strain PR26C3M of four strain 1b hypotypes, PR52B6M and PR79 has neutralization in various degree.Wherein remarkable with sE2+ aluminium adjuvant+CpG neutralization, except in QC69 and efficiency be about except 50%, in other each strain virus and efficiency all more than 70%.Its half to 13 strain virus suppression extent of dilution is measured after often organizing the mixing of mice serum sample equal-volume.
Result as shown in Figure 6B, visible three immune group mice serums in all 13 strain virus and titre all more than 40, wherein most senior middle school and titre are up to 1280 (sE2+ aluminium adjuvant and sE2+ aluminium adjuvant+CpG group for JFH1, sE2+FA group for PR79L9).For most of virus, sE2+ aluminium adjuvant group neutralization and sE2+ freund's adjuvant group are suitable, and the application of CpG can significantly improve the titre that sE2 induces neutralizing antibody, demonstrate the highest in and titre.
Rabbit immune serum is carried out 1:20 dilution, then has mixed with virus and carry out Neutralizing test.As can be seen from Fig. 7 A, under 1:20 extent of dilution, except the immune serum of #2 rabbit is to H77, S52, beyond the neutralization of SA13 is slightly weak, to the Neutralization effect of other viruses all more than 70%, and the immune serum of #1 rabbit effectively can neutralize 13 strain virus of all 7 hypotypes.The serum of two rabbits suppresses extent of dilution as shown in Figure 7 B to the half of 7 strain virus, #1 and #2 rabbit serum is in S52 and titre lower (20 and 10), #2 is in SA13 and HK6a and beyond titre lower (10 and 20), can reach more than 40 with titre in all the other, the highest titre can reach 1280.
These results show, the neutralizing antibody of the comparatively wide spectrum that can induce in animal body with sE2 protein immunization, and therefore this albumen can be used for developing HCV vaccine further.
In addition, the present inventor is by subcutaneous injection sE2, and the lower titre (Figure 10 A) obtained in BALB/c mouse body, titre is about 10
4.The antiserum(antisera) lower by this titre carries out Neutralizing test, and the neutralization of acquisition obviously weakens (Figure 10 B and Fig. 6 A) compared with the result obtained before, reduces 10% and 20% approximately respectively for JFH1 and Con1 two strain virus.Therefore can conclude, neutralization is closely-related with the titre of E2 antibody, and sE2 albumen of the present invention can induce higher antibody titers, therefore also can be significantly increased in neutralization.
The expression and purification of embodiment 6sE2 Δ HVR1 and qualification
Because HVR1 region has variability, and be considered to hide wide spectrum neutralizing epitope on viral native conformation, therefore, the present inventor constructs sE2 Δ HVR1 recombinant protein (411aa-661aa) of further brachymemma.Can find out from SDS-PAGE, the albumen after brachymemma has less molecular weight compared with sE2, is about 40kDa (Fig. 8 A).SE2 and sE2 Δ HVR1 is wrapped by elisa plate, with identifying that the AR3A antibody of CD81 binding site detects.
The identification activity of Fig. 8 B visible AR3A antibody to sE2 with sE2 Δ HVR1 is similar, all presents dosage positive correlation.This result prompting recombinant protein sE2 Δ HVR1 correctly folds and forms similar conformation, but with virus unlike, in the system of the present inventor, the existence of HVR1 does not affect the exposure of CD81 binding site and conservative neutralizing epitope, the exposure of more multi-epitope is there is in prompting sE2 compared with viral E2, thus in vaccine application aspect, compared with existing HCVE2 albumen, there is significant advantage.
In receptor activity modifying proteins, due to the disappearance of HVR1, the combination of sE2 Δ HVR1 and SR-BI significantly weakens; And the combination of itself and CD81 is not affected (Fig. 8 C).Because receptor activity modifying proteins is affected, in the ability that sE2 Δ HVR1 infects at blocking virus, comparatively sE2 also obviously weakens (Fig. 8 D).But be unexpectedly, sE2 Δ HVR1 can be used for the specific antibody IgG in detection third hepatopathy human serum equally, very high binding activities (Fig. 8 E) be all show to 1b and 2a type third hepatopathy human serum, mean OD value when 1:20000 dilutes is equally up to 0.5-1.0, similar with sE2 (Fig. 3), shows that sE2 Δ HVR1 can equally for developing simple and quick HCV infection diagnostic kit.
Embodiment 7sE2 Δ HVR1 immune serum has wide spectrum Neutralization effect
For comparing the Neutralization effect of sE2 and sE2 Δ HVR1, the sE2 Δ HVR1 protein immunization mouse of the present inventor's purifying, with PBS as negative control, sE2 is as positive control, and three groups are all carried out emulsification with aluminium adjuvant before immunity.
Can find out from Fig. 9 A, sE2 Δ HVR1 has induced the sE2 specific antibody of high titre in BALB/c mouse, and antibody titers significantly improves than sE2 group.In induction neutralizing antibody, sE2 Δ HVR1 all can induce the neutralizing antibody (Fig. 9 B) suitable with sE2 immune group to 1-7 C-type virus C.
Because the combination of sE2 Δ HVR1 and SR-BI acceptor significantly weakens, but but remain the ability of good induction of immunity reaction, when being used as vaccine, because itself and receptor binding capacity reduce the impact that can significantly reduce body normal physiological function, reduce the generation of side effect, and the ability of good induction of immunity reaction can the generation of HCV antigen/antibody combination in inductor.
Discuss
Inventor developed a kind of high yield, easy purifying, there is the recombinant HCV E 2 albumen being similar to viral native conformation, for the exploitation of HCV diagnostic kit and preventative vaccine provides a kind of New Policy.
In the experimentation of the present inventor, the present inventor has carried out series of optimum for the working condition of sE2.Be optimized rear discovery to each experiment condition, on expressing viral impact significantly, in rotating and culturing bottle, cell density is lower than 1 × 10 for cell density
6/ mL can affect the growth of cell and the expression of albumen, and therefore the present inventor is greater than 1 × 10 in cell density
7start induction during/mL, during to results viral supernatants, final cell density can reach 4 × 10
7/ more than mL.Because sE2 of the present invention is with 6 × His label, therefore purifying is simple.Output after purifying can reach 50-100mg/L cells and supernatant, 50 times (see document (9)) of the sE2 output that the HEK293T reported before being expresses.But the deficiency of this system is that the glycosylation in S2 cell is comparatively simple relative to mammalian cell.Because sE2 is with 11 N-link glycosylation sites, wherein first 10 is all complicated glycosylation site, only have C to hold last simple high mannose type glycosylation, the sE2 therefore with mammalian cell source compares, and its molecular size range about decreases about 15kDa.However, experimental result of the present invention shows, this species diversity does not cause remarkably influenced to its receptor activity modifying proteins and immunogenicity.On the contrary, can neutralizing epitope be covered due to glycosylation thus help the identification of HCV escape neutralizing antibody, the virus of glycosylation site mutation is more responsive to neutralizing antibody, therefore S2 insect cell source sE2 due to glycosylation comparatively simple, exposing more neutralizing epitope, inducing neutralizing antibody as being more conducive to during immunogen.
Good preventative vaccine should induce high titre, for the wide spectrum neutralizing antibody of multiple subtype virus, to remove the virus of different subtype.Antibody titers aspect, the present inventor all can obtain terminal titre up to 10 by three immunity in mouse and rabbit body
6specific antibody.By external Neutralizing test, the neutralizing antibody level that sE2 induces is assessed.The antiserum(antisera) that sE2 immune mouse and rabbit produce all illustrates the neutralization of wide spectrum, all presents good neutralization for all 7 subtype virus of HCV, its broad spectrum and in and intensity be better than before relevant report.The present inventor constructs 4 strain clinical separation strain PR26C3M by Molecular Virology method from clinical sample, and PR26C6M, PR52B6M, PR79 wish by the Neutralizing test to clinical virus, evaluates this vaccine and is applied to clinical potential effect.The immune serum that sE2 goes out mouse or rabbit Immune inducing in vivo is all ideal to the clinical virus effectiveness of this four strain, especially under low extension rate in PR79 virus and can close to 100%.
Be considered to mask CD81 receptor binding site due to HVR1 and E2 guards neutralizing epitope, the HVR1 amputation on antigen be conducive to the induction of wide spectrum neutralizing antibody.But in the present invention, the existence of HVR1 does not affect the combination (Fig. 8 C) that E2 guards neutralizing epitope (Fig. 8 B) and CD81, has pointed out sE2 of the present invention and natural E2 albumen structurally to there are differences, there is more antibody and expose.On the contrary, sE2 Δ HVR1, therefore cannot at the neutralizing antibody of mouse Immune inducing in vivo for HVR1 owing to lacking hypervariable region HVR1, therefore in and vaccine strain Con1 virus time, its neutralising capacity comparatively sE2 has and necessarily weakens, although in and other virus strain time, present the effect equal with sE2.Therefore, the amputation of HVR1 does not affect the generation of wide spectrum neutralizing antibody.SE2 Δ HVR1 can induce wide spectrum neutralizing antibody and efficiently in conjunction with the albumen of specific antibody in patients serum equally as a kind of, and molecular weight, reduces receptor binding capacity, is very suitable for the exploitation of vaccine and diagnostic tool.
In sum, the present inventor utilizes Drosophila S 2 cells to develop, and a kind of output is high, cost is low, be easy to purifying, different from HCV virus E2 albumen native conformation but still there is sE2 albumen and the clipped form sE2 Δ HVR1 thereof of immune interpretative function, by mouse and rabbit immunization experiment, specific antibody reaction and cell immune response are induced in vivo, the intensity of the neutralizing antibody induced and broad spectrum comparatively before report compare and all increase, and also ideal to the neutralization of clinical strains, be very suitable for the exploitation of HCV preventative vaccine.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Reference:
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3.D.Lavillette,Y.Morice,G.Germanidis,P.Donot,A.Soulier,E.Pagkalos,G.Sakellariou,L.Intrator,B.Bartosch,J.M.PawlotskyandF.L.Cosset,HumanserumfacilitateshepatitisCvirusinfection,andneutralizingresponsesinverselycorrelatewithviralreplicationkineticsattheacutephaseofhepatitisCvirusinfection.JVirol.79,6023-34(2005).
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Claims (10)
1. the HCV antigen peptide be separated, it is characterized in that, described antigen peptide is derived from the E2 albumen of HCV virus, and the length of described antigen peptide is 200-350 amino acid, and described antigen peptide can be incorporated into the antibody of HCV-Ab IgG; Preferably, described HCV virus is HCV1b C-type virus C; More preferably, be Con1 strain.
2. antigen peptide as claimed in claim 1, wherein, described antigen peptide is selected from lower group:
(A) there is the polypeptide of aminoacid sequence shown in SEQIDNO:5, SEQIDNO.:3, SEQIDNO.:4 or SEQIDNO.:2;
(B) there is arbitrary shown aminoacid sequence >=80% homology (homology of preferably, >=90% in (A); Deng the homology of preferably >=95%; Most preferably, the homology of >=97%) polypeptide, and described polypeptide has the binding ability with the antibody of HCV-Ab IgG;
(C) aminoacid sequence shown in arbitrary in SEQIDNO:2-5 is formed through the replacement of 1-5 amino-acid residue, disappearance or interpolation, and retain and the derivative polypeptide of HCV antigen/antibody combination binding ability.
3. a fusion rotein, is characterized in that, described fusion rotein contains antigen peptide according to claim 1 and optional sequence label.
4. the polynucleotide be separated, is characterized in that, described polynucleotide encoding antigen peptide according to claim 1 or fusion rotein according to claim 3;
More preferably, described polynucleotide are selected from lower group:
The polynucleotide of (a) coding polypeptide as shown in SEQIDNO.:2-5;
The polynucleotide of (b) sequence as shown in SEQIDNO.:9, SEQIDNO.:8, SEQIDNO.:7 or SEQIDNO.:6;
The polynucleotide of homology >=95% (preferably >=98%) of sequence shown in (c) nucleotide sequence and (b);
D () be the 5' end of polynucleotide and/or the polynucleotide of 3' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as Suo Shi (b);
The polynucleotide of the polynucleotide complementation e () is arbitrary with (a)-(d) described in.
5. an expression vector, is characterized in that, described expression vector contains polynucleotide according to claim 4.
6. a host cell, is characterized in that, described host cell contains expression vector according to claim 5, or in genome, be integrated with polynucleotide according to claim 4.
7. a pharmaceutical composition, it is characterized in that, described pharmaceutical composition contains claim 1-2 arbitrary described antigen peptide, fusion rotein according to claim 3, polynucleotide according to claim 4, expression vector according to claim 5 or host cell according to claim 6, and pharmaceutically acceptable carrier and/or auxiliary material.
8. a vaccine composition, it is characterized in that, described vaccine composition contains claim 1-2 arbitrary described antigen peptide, fusion rotein according to claim 3, polynucleotide according to claim 4, expression vector according to claim 5 or host cell according to claim 6, and acceptable carrier and/or auxiliary material in immunology.
9. the purposes of claim 1-2 arbitrary described antigen peptide, fusion rotein according to claim 2, polynucleotide according to claim 3, expression vector according to claim 4 or host cell according to claim 5,
A () is for the preparation of the antibody for described antigen peptide; And/or
B () is for the preparation for the treatment of or the medicine preventing HCV infection relative disease; And/or
C () is for the preparation of the reagent or the test kit that detect HCV; And/or
D () is for detecting the antibody of HCV-Ab IgG.
10. a preparation method for antigen peptide according to claim 1, is characterized in that, comprises the following steps:
A () cultivates host cell according to claim 6, thus give expression to antigen peptide according to claim 1;
Antigen peptide described in (b) separation.
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