CN100395339C - Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion - Google Patents
Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion Download PDFInfo
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Abstract
The present invention relates to the technical field of organism and medicine engineering. The world approximately has 1.7 hundred millions of HCV infected people, and more than 70% of HCV infection can be developed to chronic infection. The combination of E2 mediated HCV of HCV envelope protein and target cells relates to the most key protein of HCV infection, but E2 protein belongs to low expression protein. The protein expressed by mammalian cells obtained by a gene recombination method is quite difficult. The present invention aims to provide a method repressing hepatitis C virus envelope protein E2 by the high efficiency secretion of mammalian cells. The method constitutes novelmammalian cell expression plasmid which can express target gene E2 protein in high level, and express sieving marking glutamine synthetase in low level. The present invention can prepare recombination HCV coating E2 protein of HCV envelope protein natural biological function and antigenicity in a batch mode, establish foundation for a serologically detection reagent infected by HCV and HCV vaccine, and provide important material for the research of HCV molecular virology.
Description
Technical field:
The present invention relates to bio-pharmaceutical engineer technology domain, relate in particular to a kind of method of expressing hepatitis C virus (HCV) envelope protein E 2 with mammal cell with high efficient secretion.
Background technology:
Hepatitis C virus (HCV) is that present global HCV the infected has the HCV infection more than 1.7 hundred million, 60% can develop into chronic infection approximately through one of main virulence factor of the acute and chronic hepatitis of blood propagation.HCV chronic infection and liver cirrhosis, hepatocellular carcinoma have high correlation, and closely related with lymphoma, cryoglobulinemia etc.Though set up at present and commercial HCV Infect And Diagnose method, most widely used serological diagnostic method, promptly enzyme linked immunosorbent assay (ELISA) method also has certain loss, and needing to increase some has the virus antigen of diagnostic significance.In addition, also not succeeding in developing a kind of vaccine at present can effectively prevent HCV to infect.
The HCV envelope protein comprises two kinds of E1 and E2, and wherein combining of E2 mediation HCV and target cell relates to the infective most critical albumen of HCV.Report is arranged, and the synthetic Toplink of adding E2 improves the susceptibility that detects in the ELISA diagnostic reagent, reduces omission, therefore, is necessary this albumen is participated in the present ELISA detection based on HCV core protein and Nonstructural Protein.Because E2 albumen is in HCV and keying action during target cell combines, therefore, this albumen can be used as the important candidate antigens of HCV vaccine.But, have only the E2 albumen with high glycosylation and natural sterie configuration of mammalian cell expression just to have and target cell bonded biological function and natural antigenicity, and E2 albumen belongs to low expressing protein, and this albumen that obtains mammalian cell expression with the method for gene recombination has suitable technical difficulty.This also is not introduce the proteic major cause of E2 at present in the ELISA method, also is the major reason that hinders the HCV vaccine research.
Summary of the invention:
The object of the present invention is to provide a kind of method of expressing hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion.
The invention provides a kind of energy proteic technological method of mammalian cell high-level secretory expression HCV coating E2, at first made up a kind of novel mammalian cell expression plasmid vector, the expression of the eukaryotic promoter high level regulation and control HCV coating raq gene that this plasmid vector utilization is strong, and by means of the marker gene of the shearing function of intron control screening usefulness (glutaminase synthase gene, GS) low expression level only.With this expression plasmid transfection mammalian cell, cell clone with first sulfonyl sulfuric acid amine (MSX) screening stable transfection, because of MSX is the highly efficient depressor of GS, so have only the cell of high copy integrated plasmid encoding gene in the karyomit(e) to survive, and the cell of survival can the plasmid-encoded goal gene of high level expression--HCV coating raq gene.Using the Chinese hamster ovary cell (CHO) that is widely used in mammalian cell gene recombinant protein engineering at present most among the present invention is host cell, with this plasmid transfection CHO-K1 cell, successfully construct and stablize the proteic cell strain of high-level secretory expression HCV coating E2, and set up the serum-free suspension culture method of this cell strain, because of not containing protein ingredients such as bovine serum in the nutrient solution, thereby can obtain recombinant HCV coating E2 albumen with simple chromatography method purifying from nutrient solution.
Particular content of the present invention is as follows:
1.HCV the amplification of coating E2 albumen extracellular fragment gene: according to nucleotide sequence design, the synthetic primer of 1b HCV genotype coating E2 protein coding gene, with polymerase chain reaction (PCR) amplification HCV coating E2 albumen extracellular fragment gene, insert the T cloning vector, E2 extracellular fragment gene is carried out dna sequencing.
2. the clone of glutamine synthetase gene: according to the sequence of glutamine synthetase (GS) gene, the design synthetic primer, from Chinese hamster ovary cell (CHO) clone GS gene, insert the T cloning vector with reverse transcription (RT) PCR, the GS gene is carried out dna sequencing.
3. based on the structure of intron excision vector: the GS gene is inserted between shearing donor (SD) and shearing acceptor (SA) site of intron gene of eukaryon expression plasmid pCI-neo (U.S. Promega product), and HCV coating E2 protein gene is placed 3 of GS gene intron ' end.
4. expression plasmid transfection CHO cell: with the plasmid transfection Chinese hamster ovary celI, detect the proteic expression of HCV coating E2 with liposome.
5. the structure of the proteic Chinese hamster ovary celI strain of stably express HCV coating E2: with the GS inhibitor of lower concentration--the CHO-K1 cell clone of first sulfonyl sulfuric acid amine (MSX) screening stable transfection, the proteic CHO-K1 cell of high level expression HCV coating E2 is screened in the MSX pressurization that increases progressively with concentration again, substitute the nutrient solution that contains calf serum with serum-free medium gradually then, make cell be adapted to the serum-free culture growth fully.
6. the proteic antigenicity of recombinant HCV coating E2 is identified: with monoclonal antibody and many anti-proteic antigenicities of identifying from cells and supernatant of HCV coating E2.
The present invention can batch preparations have HCV envelope protein natural biological function and antigenic recombinant HCV coating E2 albumen, for serology detection reagent and HCV developing vaccines that HCV infects are laid a good foundation, and can provide important materials for the research of HCV Molecular Virology.
Description of drawings
Fig. 1: the structure of pCIDA-GS-E2-neo plasmid
Fig. 2: Western blot analyzes in the endochylema and secretes E2 albumen to the culture supernatant (with goat-anti E2 anti-detection the how)
Wherein 1, the CHO-K1 cell pyrolysis liquid of 2:pCIDA-GS-E2-neo transfection
The CHO-K1 cells and supernatant of 3:pCIDA-GS-E2-neo transfection
The CHO-K1 cell pyrolysis liquid of 4:pCIDA-GS-neo transfection
Fig. 3: the E2 albumen in the dot blot culture supernatant (detecting) with the E2 monoclonal antibody
Wherein 1, the CHO-K1 cells and supernatant of 2:pCIDA-GS-neo transfection
3, the CHO-K1 cells and supernatant of 4:pCIDA-GS-E2-neo transfection
The proteic SDS-PAGE of the HCV coating E2 of Fig. 4 secreting, expressing analyzes
Embodiment:
Below in conjunction with drawings and Examples the present invention is further described.
Embodiment 1:
1.HCV the amplification of coating E2 albumen extracellular fragment gene:
According to nucleotide sequence design, the synthetic primer of 1b HCV genotype coating E2 protein coding gene, with polymerase chain reaction (PCR) amplification HCV coating E2 albumen extracellular fragment gene.In order to make expression product be convenient to purifying, introduced the nucleotide sequence of 6 histidine residues of encoding at 3 ' end of E2 extracellular fragment gene.The template plasmid pGEM-HCJ4 of pcr amplification contains 1b type HCV gene complete sequence, for Yanagi doctor M of NIH makes up, provides (referring to Virology.1998; 244 (1): 161-72).DNA cloning reagent is U.S. Promega product.
Primer sequence: (downstream primer E2R contains the SalI restriction enzyme site)
E2F：5′-GAGACCCACACGACGGGGAG-3′
E2R：5′-GTCGACAACTCAGTGGTGGTGGTGGTGGTGTTCTGACCTATCCCTGTCC-3′
Set up following reaction system at 0.5 milliliter of centrifuge tube:
Plasmid pGEM-HCJ4 10ng
10 * Pfu polymeric enzyme reaction damping fluid, 5 μ l
Primer E2F (5mM) 2 μ l
Primer E2R (5mM) 2 μ l
dNTP(10mM) 1μl
Pfu polysaccharase (3U/ μ l) 0.5 μ l
Replenish two aqua sterilisas that steam to total reaction volume 50 μ l, carry out thermal cycle reaction: 94 ℃ of sex change 2 minutes, 94 ℃ of sex change 40 seconds with MJ PTC-100 type PCR instrument, annealed 40 seconds for 60 ℃, 72 ℃ were extended 1 minute 20 seconds, and carried out 30 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.Reaction product is carried out agarose gel electrophoresis, and the purpose band is reclaimed.
2.E2 the splicing of gene and human normal immunoglobulin signal peptide gene:
In order to make recombinant HCV coating E2 albumen energy secreting, expressing, introduce the human normal immunoglobulin signal peptide gene at 5 of this gene ' end.
Synthetic primer: (upstream primer ISF contains the NheI restriction enzyme site)
ISF：5′-GCTAGCGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCC-3′
ISR：5′-CTCCCCGTCGTGTGGGTCTCGGCGTCGCCGGTGCTGCCGGGCACCCACAGCAGCAGC-3′
Set up following reaction system at 0.5 milliliter of centrifuge tube:
10 * Taqase reaction buffer, 5 μ l
Primer I SF (5mM) 2 μ l
Primer I SR (5mM) 2 μ l
dNTP(10mM) 1μl
Taqase polysaccharase (5U/ μ l) 0.2 μ l
Replenish two aqua sterilisas that steam to total reaction volume 50 μ l, carry out thermal cycle reaction: 94 ℃ of sex change 2 minutes, 94 ℃ of sex change 20 seconds with MJ PTC-100 type PCR instrument, annealed 30 seconds for 60 ℃, 72 ℃ were extended 30 seconds, and carried out 30 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.
Carry out splicing reaction again:
Set up following reaction system at 0.5 milliliter of centrifuge tube:
E2 extracellular fragment gene reclaims product 2 μ l
Human normal immunoglobulin signal peptide gene reaction product 5 μ l
10 * Pfu polymeric enzyme reaction damping fluid, 5 μ l
Primer I SF (5mM) 2 μ l
Primer E2R (5mM) 2 μ l
dNTP(10mM) 1μl
Pfu polysaccharase (3U/ μ l) 0.5 μ l
Replenish two aqua sterilisas that steam to total reaction volume 50 μ l, carry out thermal cycle reaction: 94 ℃ of sex change 2 minutes, 94 ℃ of sex change 40 seconds with MJ PTC-100 type PCR instrument, annealed 40 seconds for 60 ℃, 72 ℃ were extended 1 minute 20 seconds, and carried out 30 circulations altogether, 72 ℃ were extended 5 minutes then, and temperature is reduced to 4 ℃ and finished reaction.Add Taqase 0.5U then in reaction tubes, 72 ℃ were reacted 20 minutes.Reaction product is carried out agarose gel electrophoresis, and the purpose band is reclaimed, and pMD18T is connected with the T carrier, and the ligation system is as follows:
Set up following reaction system at 0.5 milliliter of centrifuge tube:
The E2 fusion gene 6.5 μ l that reclaim
5 * T4DNA ligase enzyme damping fluid, 2 μ l
T carrier pMD18T 0.5 μ l
T4DNA ligase enzyme (2U/ μ l) 1 μ l
Mixing, place 16 ℃ of water-baths 16 hours, get 5 μ l transformed competence colibacillus bacillus coli DH 5 alphas, the amicillin resistance colony inoculation that obtains is in the LB nutrient solution that contains penbritin 100 μ g/ml, shaking culture is spent the night, extract recombinant plasmid again and carry out enzyme and cut evaluation, called after pMD-E2, the sequence of human normal immunoglobulin signal peptide wherein and HCV envelope protein E 2 fusion gene is shown in SEQID NO:1.
3.CHO the clone of cell GS gene
Go down to posterity with the DMEM nutrient solution that contains 5% new-born calf serum and to cultivate CHO-K1 cell (available from U.S. ATCC), cell reaches 80% when converging, and with Trizol Reagent (U.S. Gibco product) extracting cell total rna, operation is consulted and used explanation and carried out.The cell total rna that obtains is dissolved in the sterilization redistilled water that DEPC handles.
Reverse transcription reaction (RT): reverse transcription reaction reagent is U.S. Promega product.
Get RNA 2 μ l (about 5 μ g), place in one 50 μ l centrifuge tube, add following each component more successively:
5 * AMV reverse transcription reaction damping fluid, 5 μ l
dNTP(10mM) 1μl
oligo(dT)15(5pmol/μl) 1μl
In 70 ℃ of hot water baths 5 minutes, place ice-water bath more rapidly, add following component:
RNA enzyme inhibitors (30U/ μ l) 2 μ l
AMV reversed transcriptive enzyme (5U/ μ l) 1 μ l
The sterilization redistilled water that adds the DEPC processing is to final volume 25 μ l.
Mixing, in 42 ℃ of water-baths 40 minutes, 95 ℃ of 5 minutes deactivation reversed transcriptive enzymes carried out pcr amplification GS gene immediately again.
Sequences Design, synthetic primer (introducing ApaI and SacII restriction enzyme site among upstream and downstream primer GSF, the GSR respectively) according to the GS gene:
GSF：5′-GGGCCCATGGCCACCTCAGCAAGTTCC-3′
GSR：5′-CCGCGGTTAGTTTTTGTATTGGAAGGGCTCGTCGCC-3′
With the RT product is template amplification GS gene, and amplified production inserts T carrier pMD18T, obtains recombinant plasmid called after pMD-GS, again the GS gene is carried out dna sequencing, and working method is the same.The GS gene order of order-checking gained is consistent with Genbank accession X03495.
4. based on the structure of the expression vector of intron selection markers
With carrier for expression of eukaryon pCI-neo (U.S. Promega product) is basic framework, the GS gene is inserted in the intron gene, because of the intron gene is sheared from the mRNA chain after transcribing, encoding sequence wherein because 5 ' end lacks that cap-like structure and 3 ' end lacks poly adenosine sequence and only can low expression level, therefore will the selection markers gene insert in the intron and can improve the positive expression rate of screening the cell clone that obtains.
Sequence synthesized primer thing according to CMV cytomegalovirus promoter/enhanser in the pCI-neo carrier and chimeric intron:
InDF:5 '-GAGCTCGTTTAGTGAACCG-3 ' (containing the SacI restriction enzyme site)
InDR:5 '-GGGCCCTCTGTCTCGACAAGCCCAG-3 ' (containing the ApaI restriction enzyme site)
InAF:5 '-GGGCCCATACCGCGGAAGACTCTTGCGTTTCTG-3 ' (containing ApaI, SacII restriction enzyme site)
InAR:5 '-GTCGACCTATAGTGAGTCGT-3 ' (containing the NheI restriction enzyme site)
With plasmid pCI-neo is template, shears the donor site fragment with primer inDF, inDR amplification intron, shears the acceptor site fragment with primer inAF, inAR amplification intron.Respectively intron is sheared donor site fragment and acceptor site fragment and inserted T carrier pMD18T, obtain recombinant plasmid called after pMD-inD, pMD-inA, carry out dna sequencing again, working method is the same.
With SacI, ApaI digested plasmid pMD-inA, reclaim the intron that cuts out and shear donor site fragment inD; With ApaI, NheI digested plasmid pMD-inA, reclaim the intron that cuts out and shear donor site fragment inA.
With SacI, NheI digested plasmid pCI-neo, intron gene in the excision plasmid, reclaim the big fragment of carrier, the big fragment of carrier is connected with inD, inA fragment, the recombinant plasmid called after pCIDA-neo that obtains, the characteristics of this carrier are, have introduced two restriction enzyme site ApaI and SacII in the intron, can insert the selection markers gene.
Plasmid pMD-GS is cut with ApaI and SacII enzyme, and the GS gene that cuts out inserts among the pCIDA-neo, obtains recombinant plasmid pCIDA-GS-neo.Its characteristics are that the GS gene is inserted among the intron.
5.HCV the structure of coating raq gene expression vector:
Plasmid pMD-E2 cuts with NheI and SalI enzyme, the HCV coating raq gene that recovery cuts out, this gene is inserted the linearized vector pCIDA-GS-neo that cuts through NheI and SalI enzyme, promptly obtain E2 expression vector pCIDA-GS-E2-neo, its expression regulation structure is as shown in Figure 1: PCMV is a cytomegalovirus promoter, introD is the intron donor sequences, GS is the glutaminase synthase gene, introA is the intron receptor sequence, Sig is the human normal immunoglobulin signal peptide, E2 is a HCV coating raq gene, and polyA is a SV40 virus polyadenylic acid sequence.
6.HCV the expression of coating E2 albumen in Chinese hamster ovary celI
Go down to posterity with the DMEM nutrient solution that contains 5% new-born calf serum and to cultivate the CHO-K1 cell, when cell 80% merges, with 0.25% trypsin digestion and cell, passage is inoculated in the 35mm Tissue Culture Dish, next day with 2 μ g plasmid pCIDA-GS-E2-neo and control plasmid pCIDA-GS-neo respectively with 10 μ l lipofectamine Lipofectamine (American I nvitrogen product) mixings, transfection CHO-K1 cell, operation is carried out according to operation instruction.Tissue Culture Dish is placed 37 ℃, the cell culture incubator of 5% carbonic acid gas, saturated humidity, add the DMEM nutrient solution that 1ml contains 20% foetal calf serum after 12 hours, continue to cultivate 48 hours.Freezing-thawing and cracking cell then, cell pyrolysis liquid carries out sds polyacrylamide gel electrophoresis, carrying out western blot again detects, E2 antibody is the anti-HCV coating of goat E2 albumen how anti-(U.S. Biodesign product), and enzyme labelled antibody is the anti-goat IgG of donkey (magnificent biotechnology company limited product) of horseradish peroxidase (HRP) mark.Simultaneously culture supernatant is concentrated, concentrated solution carries out western blot and detects.Operation is all carried out with reference to " molecular cloning experiment guide second edition " (Science Press, 1995).There are the E2 albumen of various different molecular weight forms in result such as Fig. 2 in the visible cell lysate, the albumen of highest weight is complete glycosylation E2, and all the other are part glycosylation E2, wherein secrete the complete glycosylated E2 that is mainly to the culture supernatant.Cells and supernatant is carried out dot blot with anti-E2 monoclonal antibody (U.S. Biodesign product) and is detected, and as shown in Figure 3, also can detect and secrete E2 albumen to the culture supernatant.
7. the structure of the proteic CHO-K1 cell strain of stably express HCV coating E2:
With the CHO-K1 cell of plasmid pCIDA-GS-E2-neo transfection in inoculation 24 orifice plates, after 48 hours, with cell with 0.25% tryptic digestion, be inoculated in 5 100mm Tissue Culture Dishs, GMEM-S nutrient solution (JRH product) with the foetal calf serum that contains 10% dialysis is cultivated, add first sulfonyl sulfuric acid amine (MSX) (Sigma product) to final concentration 25 μ mol/L, cell begins death after 3-4 days, the colony (clone) that the back culture dish formation of two weeks is formed by individual cells propagation, select single clone, be inoculated in 24 orifice plates, continue to cultivate with the GMEM-S nutrient solution of 25 μ mol/L MSX, the back culture supernatant proteic expression of dot blot method detection E2 of one week, the highest clone of expression level with tryptic digestion, is inoculated 96 orifice plates with 105 cells/well, cultivate with MSX (the 100-1000 μ mol/L) pressurization that concentration increases progressively, after 2 weeks, to the expression of E2 in its culture supernatant of well-grown cell detection.The expression amount soprano carries out limiting dilution again, is inoculated in 96 orifice plates, and individual cells clone is carried out the expression level analysis, and the high-expression clone that obtains is as stand-by cell strain, called after CR9.
8. serum-free culture
Substitute the GMEM-S substratum gradually with serum free medium (JRH product), make the CR9 cell adapt to complete serum free medium gradually and from the adherent suspension growth that transfers to.Use suspension cell instead shake-flask culture, 150 milliliters of volume of culture.Since in the serum-free medium except E2 is expressed in emiocytosis, other protein contents (deriving from the cell of minority death) are extremely low, thereby greatly made things convenient for protein purification, culture supernatant is centrifugal, after the ultrafiltration, close the method for chromatography with the nickel ion metal-chelate and carry out preliminary purification (reagent adopts the Qiagen product), can obtain E2 protein 25 milligram in each hundred milliliters of nutrient solution.Fig. 4 is that the SDS-PAGE of different batches purifying protein analyzes.
SEQUENCE?LISTING
＜110〉Second Military Medical University, PLA
＜120〉a kind of method of expressing hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion
＜130〉specification sheets, claims
<160>1
<170>PatentIn?version?3.1
<210>1
<211>921
<212>DNA
＜213〉artificial sequence
<400>1
atggagaccg?acaccctgct?gctgtgggtg?ctgctgctgt?gggtgcccgg?cagcaccggc 60
gacgccgaga?cccacacgac?ggggagggtg?gccggccaca?ccacctccgg?gttcacgtcc 120
cttttctcat?ctggggcgtc?tcagaaaatc?cagcttgtga?ataccaacgg?cagctggcac 180
atcaacagga?ctgccctaaa?ttgcaatgac?tccctccaaa?ctgggttctt?tgccgcgctg 240
ttttacgcac?acaagttcaa?ctcgtccggg?tgcccggagc?gcatggccag?ctgccgcccc 300
attgactggt?tcgcccaggg?gtggggcccc?atcacctata?ctaagcctaa?cagctcggat 360
cagaggcctt?attgctggca?ttacgcgcct?cgaccgtgtg?gtgtcgtacc?cgcgtcgcag 420
gtgtgtggtc?cagtgtattg?tttcacccca?agccctgttg?tggtggggac?caccgatcgt 480
tccggtgtcc?ctacgtatag?ctggggggag?aatgagacag?acgtgatgct?cctcaacaac 540
acgcgtccgc?cacaaggcaa?ctggttcggc?tgtacatgga?tgaatagtac?tgggttcact 600
aagacgtgcg?gaggtccccc?gtgtaacatc?gggggggtcg?gtaaccgcac?cttgatctgc 660
cccacggact?gcttccggaa?gcaccccgag?gctacttaca?caaaatgtgg?ctcggggccc 720
tggttgacac?ctaggtgcct?agtagactac?ccatacaggc?tttggcacta?cccctgcact 780
ctcaattttt?ccatctttaa?ggttaggatg?tatgtggggg?gcgtggagca?caggctcaat 840
gccgcatgca?attggactcg?aggagagcgc?tgtaacttgg?aggacaggga?taggtcagaa 900
caccaccacc?accaccactg?a 921
Claims (4)
1. method of expressing hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion, it is characterized in that this method has made up a kind of mammalian cell expression plasmid, this expression plasmid will insert between shearing donor and the shearing acceptor site of intron gene of eukaryon expression plasmid pCI-neo as the glutaminase synthase gene of selection markers, and the hepatitis C virus envelope protein E 2 gene is placed 3 of glutaminase synthase gene intron ' end; And with hepatitis C virus envelope protein E 2 and human normal immunoglobulin signal peptide amalgamation and expression; Described mammalian cell is a Chinese hamster ovary cell.
2. according to claim 1ly a kind ofly express the method for hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion, the sequence of fusion gene that it is characterized in that hepatitis C virus envelope protein E 2 in the expression plasmid and human normal immunoglobulin signal peptide is shown in SEQ ID NO:1.
3. a kind of method of expressing hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion according to claim 1 and 2, it is characterized in that behind the expression plasmid transfection mammalian cell wherein, with the cell clone of first sulfonyl sulfuric acid amine screening stable transfection.
4. claim 1 or the 2 described a kind of application of method in the engineering cell strain that makes up the secreting, expressing hepatitis C virus envelope protein E 2 of expressing hepatitis C virus envelope protein E 2 with mammal cell with high efficient secretion.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000003021A2 (en) * | 1998-07-10 | 2000-01-20 | Jens Nielsen | Metabolically engineered microbial cell comprising a modified redox activity |
| CN1465700A (en) * | 2002-06-14 | 2004-01-07 | 中国科学院上海生命科学研究院生物化 | Hepatitis c virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use |
| CN1609213A (en) * | 2004-09-17 | 2005-04-27 | 中国人民解放军第二军医大学 | Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000003021A2 (en) * | 1998-07-10 | 2000-01-20 | Jens Nielsen | Metabolically engineered microbial cell comprising a modified redox activity |
| CN1465700A (en) * | 2002-06-14 | 2004-01-07 | 中国科学院上海生命科学研究院生物化 | Hepatitis c virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use |
| CN1609213A (en) * | 2004-09-17 | 2005-04-27 | 中国人民解放军第二军医大学 | Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence |
Non-Patent Citations (2)
| Title |
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| HCV核心-包膜E2抗原融合基因DNA疫苗的构建及对小鼠的免疫应答实验. 姜春鹏等.中华微生物学和免疫学杂志，第22卷第5期. 2002 |
| HCV核心-包膜E2抗原融合基因DNA疫苗的构建及对小鼠的免疫应答实验. 姜春鹏等.中华微生物学和免疫学杂志，第22卷第5期. 2002 * |
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