CN1185254C - First hypervariable region antigen of hepatitis C and fusion antigen - Google Patents

Abstract

From the reported different HVR1 sequences, 12 HVR1 sequences which are possibly provided with high cross reactions are screened for recombinant expression. After experiment verification, 4 HVR1 sequences with high cross reactions are selected for further combination and connection. Compared with single segment HVR1 antigen, the connected fusion antigen obviously enhances the cross reactions, the detection rate of hepatitis C patients reaches as high as 90/91, and the connected fusion antigen is an HVR1 antigen with the widest cross reactions at present. Because the HVR1 contains the unique neutral antigen epitope in hepatitis C, the present invention can be used for detecting the neutral antigen in the bodies of hepatitis C patients and preparing hepatitis C vaccines.

Description

Hepatitis c virus hypervariable region 1 antigen and fused antigen thereof

Technical field

The present invention relates to a series of fused antigens that have the hepatitis c virus hypervariable region 1 antigen of high cross reactivity and contain a plurality of different first hypervariable regions.

Background technology

Hepatitis C virus (HCV) gene is to clone first successfully in 1989, started upsurge (the Choo QL.et al.Isolation of a cDNA clone derived from blood-bornenon-A of research HCV since then, non-B viral hepatitis genome, Science, 1989; 244:359-362).HCV virus is single strand RNA virus, about 9500 bases of total length, and the polyprotein precursor that is about 3000 amino-acid residues of encoding under the effect of host and virogene proteins encoded enzyme, is processed into multiple maturation proteins such as C, E1, E2, NS2-5 then.The HCV whole genome has very high variability, wherein be positioned at 27 amino acid variation maximums of E2 district N end, be commonly referred to first hypervariable region (HVR1), because HVR1 is neutrality epitope (Farci P.etalPrevention of hepatitis C virus infection in chimpanzees after antibody-mediated invitro neutralization.Proc Natl Acad Sci USA, 1994 unique in the present known hepatitis C virus; 96:15394-15399), relevant (the Zibert.A etal Antibodies in Human Sera Specific toHypervariable Region 1 of Hepatitis C Virus Can Block Viral Attachment of the level of its antibody with the state of an illness, Virology, 1995; 203:653-661), so HVR1 antibody may be in close relations with prognosis of disease.Utilize to can be used for the HVR1 antibody horizontal behind the gene cloning and expression of method with HVR1 such as RT-PCR and detect, but this method program complexity, and can only detect the antibody of individual plant virus.Like this, still do not have simply and effectively to detect the means of HVR1 antibody so far, need HVR1 antigen badly, patient's HVR1 antibody is detected by ELISA with high cross reactivity.

Hepatitis C virus is a kind of hazardness virus greatly, the chronicity ratio is up to 70%, wherein there is 50% patient to develop into liver cirrhosis and hepatocellular carcinoma, mainly be Interferon, rabbit or Interferon, rabbit and virazole merging use to the therapy for hepatitis C method at present, cost an arm and a leg, side effect is big, weak curative effect (Fried MW andHoofnagle JH.Therapy of hepatitis C, Semin Liver Dis, 1995; 15:82-91).So the development of hcv vaccine receives much attention.But also because the neutrality epi-position of hepatitis C virus is positioned at the HVR1 of variability maximum, this brings very big difficulty for the development of HCV vaccine, and successful key is to obtain the hypervariable region antigen of high immunological cross-reaction.Have recently and studies have shown that much HVR1 still has certain cross-immunoreactivity, and preliminary screening is to more such HVR1 (Puntoriero G et al.Towards asolution for hepatitis C virus hypervariability:mimotopes of the hypervariableregion 1 can induce antibodies cross-reacting with a large number of viral variants, J.EMBO, 1998; 17:3521-3533).But we think that the intercrossing that causes by a HVR1 is limited after all, if we can link several HVR1 fragments with high cross-immunoreactivity together with the method for gene recombination imagination, may obtain and with most humans serum immunoreactive polypeptide to take place, can be vaccine research real effectively immunogen is provided.

The purpose of this invention is to provide a series of reorganization HVR1 antigens with single slice of high cross reactivity.

Another object of the present invention provides the fused antigen that contains a plurality of HVR1 of a high cross reactivity.

Summary of the invention

For achieving the above object, the present inventor filters out the HVR1 that may have high cross reactivity from the different HVR1 sequence of lot of documents report (domestic and external), synthetic gene is recombinant expressed, and its cross reactivity of experimental verification, their aminoacid sequence is:

STHVTGGVQGRTTRGLTSLFTPGPSQK

STHVTGGVQGHSLRGLTSLFTSGPAQK

ITRVTGGVQGHSLRSLTSLFTPGPAQK

STHVTGAVQGRSLQSFTSFLSPGPSQK

DTHVTGGAAARGASGLANLFTSGPAQK

GTYVTGGATAHTASGFASLFTTGSKQN

TTHVTAGTAAHATSSFTKLFAPGAKQN

ETHTSGGSVARAAFGLTSIFSPGKSQN

NTYVTGGSAAHTTSRFTSLFSPGPQQN

TTTTTGGVQGHTTRGLVRLFSLGSKQN

DTTVTGGQAARTTQSFTSLFPPGPSQK or

QTTTTGGSASHAVSSLTGLFSPGSKQN。

Method with gene recombination links together two or more sequences wherein, can obtain to have the reorganization HVR1 fused antigen of higher cross reaction, after preferably following four HVR1 genes being connected with connecting arm, and the reorganization fused antigen of expression:

STHVTGGVQGRTTRGLTSLFTPGPSQKSGGGSSGGGSTHVTGGVQ

GHSLRGLTSLFTSGPAQKGGGSSGGGSSTHVTGAVQGRSLQSFTSF

LSPGPSQKGGGSSGGGSETHTSGGSVARAAFGLTSIFSPGKSQN

The present invention can be achieved by the following technical programs.

(Military Medical Science Institute writes to utilize the Goldkey computer software, have commercially available) selected 12 representative HVR1 antigen sequences, behind the synthetic gene, recombinant expressed respectively, determine the cross reactivity of itself and serum then with ELASA, and therefrom select the high HVR1 sequence of cross reactivity to be used to be connected.

Because the HVR1 gene fragment has only 81 bases, not very long, so the synthetic employing chemical synthesis of antigen gene.The chemosynthesis gene can save the operation of extracting templet gene than the PCR method, and is fast convenient.Main also have only the gene that just can obtain corresponding sequence with chemosynthesis as required, also can be chosen in the synthetic HVR1 gene of colibacillary bias codon simultaneously, helps obtaining to express efficiently in E.coli, and this is that the PCR method is inaccessiable.Select chemical synthesis for use,, infer that nucleotide sequence is synthesized according to the bias codon of high expression level in the peptide sequence of epitope and the intestinal bacteria.The chemical method synthetic gene once can the synthetic limited length, generally is no more than 60 bases.Can synthesize two 3 ' ends earlier to the gene of HVR1 has one section complementary eclipsed gene fragment, and polymerase extension is used in the two annealing back, obtains positive and negative double-stranded DNA gene.

Interconnect for the ease of intergenic, and can obtain antigenic activity preferably after connection, between epitope one connecting arm need be arranged, we add G-G-G-S and S-G-G-G respectively in the upstream and downstream of epi-position, so should add corresponding gene when synthetic gene.Arrive expression vector for the ease of gene clone at last, two universal primers that match with connecting arm have also been synthesized, introduce two restriction enzyme site XhoI and XbaI, to be fit to expression vector pBVIL1 (referring to Chinese patent application: expression vector pBVIL1 and construction process thereof and purposes, application number 00100695.9).

The two ends of synthetic gene have been introduced restriction enzyme site XhoI and XbaI, so each gene fragment all can be inserted among the carrier pBVIL1 with the double digestion method easily, make up corresponding expression plasmid.The expression plasmid that makes up need be identified with the PCR method, inserts to prove that each gene fragment has obtained correctly.

Each single slice HVR1 antigen presentation plasmid, behind the Transformed E .coli HB101, cultivate by thermal induction, get full bacterium liquid and carry out the SDS-PAGE evaluation, prove that each expression plasmid has all obtained to express efficiently, through ion exchange column and gel-filtration purifying, all can obtain the pure product of HVR1 antigen of electrophoretically pure single slice again.With ELASA detection individual chip and HCV patients serum's cross reactivity, select the wherein HVR1 antigen sequence of high relatively cross reactivity, be used for next step connection.

Many HVR1 fused antigen expression plasmid is to make up to form on the basis of each HVR1 antigen presentation plasmid.One of them is contained the plasmid of HVR1-1# gene as carrier; Another plasmid that contains gene HVR1-2# is as template, with the universal primer that contains SpeI and contain former IL-1 gene 3 ' the end primer of BamHI, amplify the gene fragment that contains HVR1-2# and part IL-1, insert back, obtain to contain the plasmid of HVR1-1# and two genes of HVR1-2# with XhoI and the two HVR1-1# genes of cutting of XbaI.Because in the novel plasmid, the intergenic connection of HVR1-1# and HVR1-2# is by linking to each other between complementary enzyme XbaI and the SpeI, connects the back restriction enzyme site and has not existed, therefore, novel plasmid can be used as new carrier again, is connected with another amplification gene fragment that contains the HVR1-n# gene plasmid.Triplicate can obtain plasmid pBVIL1/HVR1-1#+2#+4#+8#.After transforming, inducing, express, obtain the HVR1 four fragment fused antigens of purifying.

Experimental result proves, different HVR1 antigen of the present invention all has certain intercrossing, the intercrossing of many HVR1 fused antigen more has the raising of essence, with HCV patients serum's at random cross reacting rate up to 90/91, be significantly higher than report (the Waranabe K et al.Thehypervariable Region 1 Protein of Hepatitis C Virus Broadly Reactive with Sera ofPatients with Chronic Hepatitis C Has a Similar Amino Acid Sequence with theConsensus Sequence of 60-75% crossing-over rate on the present document, Virology, 1999; 264:153-158), be the HVR1 fused antigen of the high cross reaction of real meaning.

Brief Description Of Drawings

Fig. 1 connects between HVR1 antigen and multi-epitope antigen expression plasmid structure synoptic diagram

Fig. 2 is the transformed bacteria SDS-PAGE synoptic diagram that obtains during HVR1 connects for four times

Fig. 3 is high cross reactivity HVR1 antigen sequence

Embodiment

Below in conjunction with accompanying drawing the present invention is done to describe in further detail, this embodiment is used for explaining and does not limit the present invention in any way.

1.12 selections of representing epitope of embodiment

We analyze domestic 123 HVR1 sequences by Goldkey software, obtained the common sequences 1# of HVR1 according to the height of the amino acid frequency of occurrences, and 6 HVR1 sequences have been selected as representative (sequence 2#～7#) by the difference of similarity between each sequence, sequence 8#, 9# be for delivering sequence abroad, and 10～12# is that bibliographical information is thought the HVR1 sequence that may have high cross reaction.

1# STHVTGGVQGRTTRGLTSLFTPGPSQK

2# STHVTGGVQGHSLRGLTSLFTSGPAQK

3# ITRVTGGVQGHSLRSLTSLFTPGPAQK

4# STHVTGAVQGRSLQSFTSFLSPGPSQK

5# DTHVTGGAAARGASGLANLFTSGPAQK

6# GTYVTGGATAHTASGFASLFTTGSKQN

7# TTHVTAGTAAHATSSFTKLFAPGAKQN

8# NTYVTGGSAAHTTSRFTSLFSPGPQQN

9# ETHTSGGSVARAAFGLTSIFSPGKSQN

10# TTTTTGGVQGHTTRGLVRLFSLGSKQN

11# DTTVTGGQAARTTQSFTSLFPPGPSQK

12# DTTVTGGQAARTTQSFTSLFTPGPSQK

Wherein, 1# is the common sequences of China, and 2#～7# is the HVR1 sequence of China, and by itself and the arrangement of the height of 1# homology, 8#, 9# are for delivering sequence abroad, and 10#～12# is external intercrossing higher H VR1 sequence, are mainly used to do the comparison with the intercrossing of 1～9#HVR1.

The antigenic cloning and expression of embodiment 2.HVR1#～12#

1, the structure that contains HVR1 single slice antigen presentation plasmid

1.1 the segmental design of synthetic gene is with synthetic

According to the needs of each synthetic gene, designed the following gene fragment of mentioning.All entrusting Shanghai to give birth to worker's biotechnology Services Co., Ltd after the gene fragment order design synthesizes.

1#

F：GGTGGTGGATCTTCCACCCACGTAACCGGCGGCGTACAGGGCCGAAC

CACCCGAGGCCTG

R：ACCTCCACCACTTTTCTGGGAAGGGCCAGGAGGGAACAGGGAGGTG

AAGGACTGGGTGGT

2#

F：GGTGGTGGATCTTCCACCCACGTAACCGGCGGCGTACAGGGCCACTC

CCTGCGAGGCCTG

R：ACCTCCACCACTGTTCTGTTTGGAGCCAGGGGAGAACAGGCCGGTCA

GGGAGGATACAGC

3#

F：GGTGGTGGATCTATCACCCGAGTAACCGGCGGCGTACAGGGCCACTCC

CTGCGATCCCTG

R：ACCTCCACCACTGTTCTGTTTGGAGCCCAGGGAGAACAGTCGTACCA

GGCCTCGGGTGGT

4#

F：GGTGGTGGATCTTCCACCCACGTAACCGGCGCTGTACAGGGCCGATCC

CTGCAGTCCTTC

R：ACCTCCACCACTGTTCTGGGATTTGCCAGGGGAGAAGATGGAGGTCA

GGCCGAAAGCAGC

5#

F：GGTGGTGGATCTGACACCCACGTAACCGGCGGCGCTGCTGCTCGAGG

CGCTTCCGGCCTG

R：ACCTCCACCACTGTTCTGCTGAGGGCCAGGGGAGAACAGGGAGGTG

AATCGGGAGGTGGT

6#

F：GGTGGTGGATCTGGCACCTACGTAACCGGCGGCGCTACCGCTCACACC

GCTTCCGGCTTC

R：ACCTCCACCACTGTTCTGTTTAGCGCCAGGAGCGAACAGTTTGGTGA

AGGAGGAGGTAGC

7#

F：GGTGGTGGATCTACCACCCACGTAACCGCTGGCACCGCTGCTCACGCT

ACCTCCTCCTTC

R：ACCTCCACCACTGTTCTGTTTGGAGCCGGTGGTGAACAGGGAAGCGA

AGCCGGAAGCGGT

8#

F：GGTGGTGGATCTGAAACCCACACCTCCGGCGGCTCCGTAGCTCGAGC

TGCTTTCGGCCTG

R：ACCTCCACCACTTTTCTGGGAAGGGCCAGGGGACAGGAAGGAGGTG

AAGGACTGCAGGGA

9#

F：GGTGGTGGATCTAACACCTACGTAACCGGCGGCTCCGCTGCTCACACC

ACCTCCCGATTC

R：ACCTCCACCACTTTTCTGAGCAGGGCCGGAGGTGAACAGGTTAGCCA

GGCCGGAAGCGCC

10#

F：GGTGGTGGATCTACCACCACCACCACCGGCGGCGTACAGGGCCACAC

CACCCGAGGCCTG

R：ACCTCCACCACTTTTCTGAGCAGGGCCAGGGGTGAACAGGGAGGTCA

GGGATCGCAGGGA

11#

F：GGTGGTGGATCTCAGACCACCACCACCGGCGGCTCCGCTTCCCACGC

TGTATCCTCCCTG

R：ACCTCCACCACTTTTCTGAGCAGGGCCGGAGGTGAACAGGGAGGTCA

GGCCTCGCAGGGA

12#

F：GGTGGTGGATCTGACACCACCGTAACCGGCGGCCAGGCTGCTCGAAC

CACCCAGTCCTTC

R：ACCTCCACCACTTTTCTGGGAAGGGCCAGGGGTGAACAGGGAGGTCA

GGCCTCGGGTGGT

Universal primer F1:GCCTCGAGGGTGGTGGATCT

Universal primer R1:GCTCTAGAACCTCCACCACT

Universal primer F2:GCACTAGTGGTGGTGGATCT

Universal primer R2:GCGGATCCTTAGGAAGACACAAA

1.2PCR method obtains HVR1#～12# gene

The method for synthesizing gene of HVR1#～12#, except that used synthetic gene fragment difference, all use the two-step pcr synthesis method:

The first step: in the pcr amplification pipe, add successively: H ₂O 41 μ l, 10 * PCR Buffer, 5 μ l, add each 1 μ l of a pair of gene fragment of synthetic of gene (20pmol/ μ l) R, F separately respectively, 10mmol/L 4 dNTP 1ul, the Taq archaeal dna polymerase 1ul of 2U/ μ l, mixing, centrifugal 30 seconds of 1000rpm puts into pcr amplification instrument (GeneAmp PCR System 2400) amplification then.The PCR reaction conditions is 94 ℃ after 1 minute, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, 5 circulations.

Second step: the PCR product with the first step is that template increases.Promptly in the pcr amplification pipe, add successively: H ₂O 40 μ l, 10 * PCR Buffer, 5 μ l, each 1 μ l of 20pmol/ μ l universal primer R1, F1,1/10 the first step PCR product 1 μ l, 10mmol/L 4 dNTP 1 μ l, the TaqDNA polysaccharase 1 μ l of 2U/ μ l, mixing, centrifugal 30 seconds of 1000rpm puts into the pcr amplification instrument then and increases.The PCR reaction conditions is 94 ℃ after 3 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, after 30 circulations again 72 ℃ extended 5 minutes.

The PCR product purification

" PCR product purification in a small amount " test kit of producing with Shanghai China Shun biotechnology company limited carries out purifying, promptly adds PB 0.25ml in the PCR product, and full dose moves into adsorption column, and the by specification method is cleaned and wash-out then, and electrophoresis is identified then.

1.3 the structure of expression plasmid

1.3.1PCR product and expression vector pBVIL1 double digestion

PCR product and each 30ul of pBVIL1 expression vector of getting above purifying are put in respectively in the Eeppendorf centrifuge tube, each adds 10 * buffer (H) 4ul, XbaI (10u/ul) and each 1ul of XhoI (12u/ul), add sterile purified water to 40ul, put 37 ℃ of water-bath enzymes and cut and spend the night.

Enzyme is cut the agarose gel electrophoresis purifying and the recovery of product: PCR product and carrier pBVIL1 carry out purifying with 1.2% sepharose behind double digestion, and concrete grammar is undertaken by the method for " molecular cloning " (Science Press, second edition).The a small amount of glue that purified genes is produced with Shanghai China Shun biotechnology company limited again reclaims test kit and reclaims: promptly downcut the agarose that contains plasmid and goal gene under ultraviolet lamp respectively; be put in the Eeppendorf centrifuge tube; each adds S1 liquid; putting 55 ℃ of water-baths separated peptization in 10 minutes; add the equivalent Virahol, mixing, 55 ℃ of temperature were bathed 1 minute; after moving into adsorption column respectively then, carry out purifying by the test kit specification sheets.

1.3.2 connect: the carrier after the above-mentioned enzyme of adding is cut in sterilization Eeppendorf centrifuge tube and each 1ul of goal gene, 10 * T4 DNA Ligase buffer 1ul, T4 DNA Ligase (12u/ul) 1ul, add sterile purified water to 10ul, put 16 ℃ and spend the night.

1.3.3 transform: in Bechtop, get 100 μ l competent cells (competent cell is undertaken by the method for " molecular cloning " (Science Press, second edition)) suspension with aseptic suction nozzle and in Eppendorf, add above-mentioned connector 5 μ l, rotate mixing gently, ice bath 30 minutes.Transfer to immediately in 42 ℃ of water-baths and placed 2 minutes, every pipe adds 0.5ml LB substratum (not added with antibiotic), and 30 ℃ of shaking baths were cultivated after 60 minutes, respectively gets 0.2ml and is applied to respectively on the LB agar culture plate and (contains microbiotic), after room temperature is dried, put 37 ℃ of thermostat containers and be inverted overnight incubation.Select several bacterium colonies, be inoculated in respectively (5ml/ pipe) among the LB, overnight incubation is respectively got 0.1ml next day and is transferred to (2ml/ pipe) among the LB, cultivated 3 hours for 32 ℃, inducing culture 4h in 42 ℃ of shaking baths receives bacterium, identify with SDS-PAGE, select goal gene to obtain the high expression level bacterial strain, do further to identify.

1.3.4 plasmid extracts and identifies

Get the bacterial strain that above-mentioned SDS-PAGE assay certificate goal gene obtains high expression level, extract plasmid by " molecular cloning " (Science Press, second edition) method.With the plasmid is template, carries out pcr amplification (method is with above-mentioned second step) with the F1 primer of each gene and the general R2 of drawing, and identifies with agarose gel electrophoresis then.

2. antigenic expression and purification

2.1 the cultivation of expression strain: the expression strain 20ul that gets-70 ℃ of preservations is inoculated in (100ml LB/500ml triangular flask) in the LB substratum, 30 ℃ of air shaking table overnight incubation, next day, in LB substratum (the same), 30 ℃ of air shaking tables were cultivated about 3 hours, worked as OD in 5% ratio transferred species ₆₀₀Value reaches at 0.7 o'clock, immediately culturing bottle is forwarded in 42 ℃ of shaking baths to inducing culture 4 hours.Bacterium liquid is merged, and centrifugal 20 minutes of 6000rpm abandons supernatant, the collecting precipitation part.

2.2 extraction inclusion body: will precipitate and claim weight in wet base, will precipitate with the 20mmol/L pH8.0 TE damping fluid of 10 times of volumes and hang, adding N,O-Diacetylmuramidase (1mg/ml suspension), at room temperature magnetic agitation is 10 minutes.Ultrasonic disruption bacterium in ice bath surpassed for 30 seconds at every turn, at interval 30 seconds, surpassed altogether 10 times.8 ℃, 12000rpm, centrifugal 20 minutes, abandon supernatant, precipitation is washed once with 1mol/L NaCl (preparing with TE), washes 2 times collecting precipitation again with TE.Precipitation adds 1% beta-mercaptoethanol with 8M urea (with PH8.0 TE preparation) dissolving.In 20 ℃, 12000rpm centrifugal 10 minutes, goes precipitation to get supernatant again.

2.3 purifying: above-mentioned dissolved inclusion body solution is crossed Q-Sepharose FF anion-exchange column, with balance liquid (pH8.0,20mmol/L TE contains the 6mol/L urea, 0.1% beta-mercaptoethanol) after the cleaning, NaCl (preparing) wash-out with different concns with balance liquid, collect each elution peak, identify, collect 0.05mol/L NaCl elution peak through SDS-PAGE.After Sephardex G-50 gel-filtration column, collect first elution peak.Various purification of Recombinant epitope antigens are all identified with SDS-PAGE.

3. single slice HVRl antigenic activity is identified

With the single slice antigen of purifying and, be diluted to 2.0ug/ml with the phosphate buffered saline buffer of pH7.2, bag, to 15 parts of HCV positive serums that blood station, Red Cross Society of China Beijing provides, is measured after sealing by the ELISA assay plate.The result is as shown in table 1.

Embodiment 3. selects with China HCV and infects the HVR1 that strain has high cross reactivity

According to table 1 experimental result, we select 4 HVR1 peptide sequence 1#, 2#, 4#, 8# with high cross reactivity.The comprehensive intersection performance of these 4 HVR1 polypeptide covers other HVR1 polypeptide.

The method of embodiment 4. usefulness molecular recombination is carried out the connection of 4 HVR1

The plasmid that contains the HVR1# gene is as carrier; Another plasmid that contains gene HVR2# is as template, with the universal primer F2 that contains SpeI and contain former IL-1 gene 3 ' the end universal primer R2 of BamHI, amplify the gene fragment that contains HVR2# and part IL-1, insert back, obtain to contain the plasmid (Fig. 1) of HVR1# and two genes of HVR2# with XhoI and the two HVR1# genes of cutting of XbaI.Because in the novel plasmid, the intergenic connection of HVR1# and HVR2# is by linking to each other between complementary enzyme XbaI and the SpeI, connecting the back restriction enzyme site has not existed, therefore, novel plasmid can be used as new carrier again, with and another plasmid that contains the HVR1 gene increases and is connected, repeat said process, take turns the plasmid that circulation can obtain to contain four fragment HVR1 through 3 again.Fig. 2 can find out obviously that the HVR1 sequence connects one by one, and obtains well to express.

The acquisition of embodiment 5 four fragment HVR1 fused antigens and the mensuration of high intercrossing

By 2 antigenic purifying and expression among the embodiment 2, can obtain electrophoretically pure four fragment HVR1 fused antigens (L4HVR1) to the operation that contains four fragment expression bacterial classifications.Antigenic cross property after ELISA connects has had essential raising, can with 15 parts of all HCV positive serum generation cross reactions.In order further to identify the recall rate of four fragment HVR1 fused antigens in the HCV positive serum, we detect (the results are shown in Table 2) recall rate up to 90/91 part with 92 parts of HCV positive serums of 302 hospitals (determining through magnificent company test kit), prove absolutely that HVR1 four fragment fused antigen polypeptide are high cross reactions, this antigenic sequence as shown in Figure 3.

Table 1:12 bar is represented HVR1 polypeptide antigen and 15 parts of positive serum intercrossing (black matrix is represented positive)

	1#	2#	3#	4#	5#	6#	7#	8#	9#	10#	11#	12#	L4HVR1
														1564	0.232	0.147	0.082	0.350	0.087	0.156	0.103	0.090	0.435	0.139	0.026	0.213	1.048
P6	0.070	0.204	0.622	0.395	0.057	0.082	0.006	0.351	0.479	0.224	0.557	0.069	1.660
														P7	0.414	0.031	0.026	0.441	0.010	0.024	0.026	0.175	0.421	0.128	0.677	0.005	0.781
P9	0.157	0.013	0.077	0.011	0.013	0.012	0.011	0.010	0.039	0.013	0.055	0.009	0.367
														P13	0.030	0.011	0.046	0.050	0.024	0.024	0051	0.116	0.031	0.059	0.038	0.017	0.376
30	0.058	0.041	0.060	0.133	0.042	0.054	0.027	0.050	0.022	0.035	0.025	0.050	0.244
														41	0.117	0.380	0.468	0.272	0.115	0.058	0.010	0.589	0.197	0.028	0.265	0.483	1.235
44	0.038	0.037	0.097	0.024	0.030	0.058	0.006	0.028	0.069	0.050	0.044	0.017	0.254
														50	0.056	0.132	0.050	0.014	0.010	0.016	0.003	0.127	0.110	0.027	0.019	0.019	0.518
51	0.501	0.951	0.516	0.006	0.007	0.009	0.013	1.031	0.852	0.339	0.357	0.020	1.052
														52	1.114	1.066	0.246	1.154	0.314	0.020	0.000	0.645	0.083	0.018	0.098	0.191	1.519
55	0.066	0.003	0.029	0.014	0.015	0.014	0.008	0.025	0.025	0.751	0.018	0.010	0.612
														166	0.120	0.117	0.105	0.109	0.062	0.108	0.059	0.076	0.025	0.123	0.138	0.417	1.696
156	0.117	0.138	1.059	2.071	0.078	0.019	0.702	1.255	0.068	0.031	0.604	0.024	0.770
														1693	0.130	0.089	0.085	0.088	0.077	0.106	0.111	0.095	0.017	0.056	0.050	0.089	0.525
Total	9/15	8/15	7/15	7/15	2/15	3/15	3/15	8/15	5/15	6/15	6/15	4/15	15/15

Table 2.HVR1 four fragment fused antigen polypeptide (L4HVR1) activity identification

OD detects umber % accumulative total %

＞2.0 56 61.5 61.5

1.5～2.0 14 15.4 76.9

1.0～1.5 13 14.3 91.2

0.5～1.0 6 6.6 97.8

0.371 1 1.1 98

0.065 1 1.1

Claims (2)

1. the fused antigen of hepatitis c virus hypervariable region 1 is characterized in that said hepatitis c virus hypervariable region 1 is following four seed amino acid sequences:

STHVTGGVQGRTTRGLTSLFTPGPSQK，

STHVTGGVQGHSLRGLTSLFTSGPAQK

STHVTGAVQGRSLQSFTSFLSPGPSQK and

ETHTSGGSVARAAFGLTSIFSPGKSQN。

2. the described fused antigen of claim 1 is characterized in that the sequence after said hepatitis c virus hypervariable region 1 sequence merges is:

STHVTGGVQGRTTRGLTSLFTPGPSQKSGGGSSGGGSTHVTGGVQ

GHSLRGLTSLFTSGPAQKSGGGSSGGGSTHVTGAVQGRSLQSFTSF

LSPGPSQKSGGGSSGGGSETHTSGGSVARAAFGLTSIFSPGKSQN。

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