CN1465700A - Hepatitis c virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use - Google Patents
Hepatitis c virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use Download PDFInfo
- Publication number
- CN1465700A CN1465700A CNA021120897A CN02112089A CN1465700A CN 1465700 A CN1465700 A CN 1465700A CN A021120897 A CNA021120897 A CN A021120897A CN 02112089 A CN02112089 A CN 02112089A CN 1465700 A CN1465700 A CN 1465700A
- Authority
- CN
- China
- Prior art keywords
- expression
- hcv
- hepatitis
- protein
- pqe8
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 36
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 14
- 241000711549 Hepacivirus C Species 0.000 title claims abstract description 12
- 101710132593 Protein E2 Proteins 0.000 title abstract 3
- 241000588724 Escherichia coli Species 0.000 title description 4
- MEYZYGMYMLNUHJ-UHFFFAOYSA-N tunicamycin Natural products CC(C)CCCCCCCCCC=CC(=O)NC1C(O)C(O)C(CC(O)C2OC(C(O)C2O)N3C=CC(=O)NC3=O)OC1OC4OC(CO)C(O)C(O)C4NC(=O)C MEYZYGMYMLNUHJ-UHFFFAOYSA-N 0.000 title 1
- 208000005176 Hepatitis C Diseases 0.000 claims abstract description 11
- 230000002265 prevention Effects 0.000 claims abstract description 5
- 238000003745 diagnosis Methods 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 24
- 101710091045 Envelope protein Proteins 0.000 claims description 23
- 101710188315 Protein X Proteins 0.000 claims description 23
- 230000000968 intestinal effect Effects 0.000 claims description 21
- 235000018102 proteins Nutrition 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- 108010003533 Viral Envelope Proteins Proteins 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 102100021696 Syncytin-1 Human genes 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 63
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 101710125507 Integrase/recombinase Proteins 0.000 abstract description 2
- 241001465754 Metazoa Species 0.000 abstract description 2
- 239000002775 capsule Substances 0.000 abstract 2
- 230000003053 immunization Effects 0.000 abstract 1
- 238000002560 therapeutic procedure Methods 0.000 abstract 1
- 238000010367 cloning Methods 0.000 description 25
- 230000035772 mutation Effects 0.000 description 25
- 230000010363 phase shift Effects 0.000 description 25
- 102100034349 Integrase Human genes 0.000 description 22
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 20
- 150000001413 amino acids Chemical class 0.000 description 19
- 210000002966 serum Anatomy 0.000 description 19
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 229940024606 amino acid Drugs 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 12
- 229910021529 ammonia Inorganic materials 0.000 description 10
- 230000008521 reorganization Effects 0.000 description 8
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000003119 immunoblot Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 208000006454 hepatitis Diseases 0.000 description 5
- 230000002209 hydrophobic effect Effects 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 241000700618 Vaccinia virus Species 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 231100000283 hepatitis Toxicity 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 206010046865 Vaccinia virus infection Diseases 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 208000002672 hepatitis B Diseases 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 102220369447 c.1352G>A Human genes 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000013522 chelant Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N ferric oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- 102000008482 12E7 Antigen Human genes 0.000 description 1
- 108010020567 12E7 Antigen Proteins 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- 101710153593 Albumin A Proteins 0.000 description 1
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- ODKSFYDXXFIFQN-SCSAIBSYSA-N D-arginine Chemical compound OC(=O)[C@H](N)CCCNC(N)=N ODKSFYDXXFIFQN-SCSAIBSYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 1
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 1
- 101000666856 Homo sapiens Vasoactive intestinal polypeptide receptor 1 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 1
- PTRKPHUGYULXPU-KKUMJFAQSA-N Leu-Phe-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O PTRKPHUGYULXPU-KKUMJFAQSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010076039 Polyproteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- VMVNCJDKFOQOHM-GUBZILKMSA-N Ser-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CO)N VMVNCJDKFOQOHM-GUBZILKMSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- ABSXSJZNRAQDDI-KJEVXHAQSA-N Tyr-Val-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ABSXSJZNRAQDDI-KJEVXHAQSA-N 0.000 description 1
- PVPAOIGJYHVWBT-KKHAAJSZSA-N Val-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N)O PVPAOIGJYHVWBT-KKHAAJSZSA-N 0.000 description 1
- 102100038388 Vasoactive intestinal polypeptide receptor 1 Human genes 0.000 description 1
- 241000726445 Viroids Species 0.000 description 1
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 102220369445 c.668T>C Human genes 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 102220023257 rs387907546 Human genes 0.000 description 1
- 102220023258 rs387907548 Human genes 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Abstract
The present invention discloses a hepatitis C virus capsule protein E2 gene capable of implementing total-length expression in colibacillus and its coding protein. Said hepatitis C virus capsule protein E2 with total length expression has antigenicity of E2 and its immunogenicity, can be used for immunizing animal and can produce antibody for resisting E2, so that it can create the condition for researching the antigenicity and immunogenicity of complete HCV E2, the purified and complete total-length E2 protein and prepared E2 antibody can be used for diagnosis, prevention and therapy or hepatitis C.
Description
Technical field
The present invention relates to the genetically engineered field, particularly a kind of can be in intestinal bacteria total length expressed hepatitis C virus envelope protein raq gene, and proteins encoded and its application in the medicine of preparation diagnosis, prevention and treatment hepatitis C.
Background technology
Hepatitis C virus (HCV, Hepatitis C Virus) is hepatitis C pathogenic former of present serious harm human health, by the blood propagation hepatitis C.1-3% is arranged according to statistics by this virus infection in the whole world population.Wherein 85% chronic hepatitis be will develop into, and wherein 20% liver cirrhosis and liver cancer caused the most at last.The vaccine of development prevention of hepatitis c and the effective medicine of development, and the development of effective diagnostic techniques is to control the top priority that hepatitis C is propagated at present.
Have now and studies show that HCV is a positive chain RNA virus, about 10,000 Nucleotide of genome total length only contain single reading frame, 3,000 the amino acid whose polyproteins of encoding.By with other generic virus relatively, two glycosylated envelope protein E1 and E2 are considered to be positioned at the surface of virion.Some immune response that envelope protein evoked of planting viroid can produce neutrality antibody and can remove virus effectively.
Because HCV titre in blood samples of patients is very low, the method that also lacks at present effective in-vitro multiplication, can not directly study virus, thereby can only pass through external translating system, range gene recombinant expression system, as the albumen of system expression HCV such as intestinal bacteria, insect viruses, mammalian cell and vaccinia virus.
HCV envelope protein (E1, E2) is considered to contain main immunologic determinants, particularly E2 wherein, thereby research envelope protein E2 has great importance in researchs such as HCV Molecular Virology, development vaccine undoubtedly.
The HCV E2 384-746 amino acids of encoding, and contain two hypervariable region HVR1 (the 386-411 position of encoding) and HVR2 (the 470-480 position of encoding) (people Biochem.Biophys.Res.Commun.1991 175:220-228 such as Hijikata M; People Virology 1991 180:842-848 such as Weiner AJ).The carboxylic end regions tool strong-hydrophobicity of E2 (coding 662-746 position), and contain and stride film district (TMD, transmembranedomain) the delay signal of E2 in the endoplasmic reticulum system (people J.Biol.Chem.1998 273:32088-32095 such as Duret S is contained in coding 718-746 position; People J.Virol.199872:2183-2193 such as Cocquerel L).E2 is at yeast expression system (people Res.Microbiol.1999150:179-187 such as Mustlli AC), at insect viruses expression system (people Virus Research 1996 45:45-57 such as H ü ssy R), at mammalian cell expression system (people Proc.Natl.Acad.Sci.U.S.A.199693:1759-1763 such as Rosa D), (people Proc.Natl.Acad.Sci.U.S.A.1994 91:1294-1298 such as Choo QL) etc. all realized expression in the recombinant vaccinia expression system.
If remove behind the carboxylic end hydrophobic region or add secretion signal, in above-mentioned eukaryotic expression system, can realize secreting, expressing.Though its expression product has saccharification processing, may more approaching natural virus structure, factors such as, purification difficult low because of expression amount, cost height will obtain a large amount of HCV E2 albumen and still have certain difficulty.In escherichia expression system, breeding is fast, cost is low, but owing to the equiprobable reason of extracellular domain carboxylic end strong-hydrophobicity, and E2 that all the time can not expression of HCV total length structure can not obtain the complete E2 expressing protein of total length.Usually all must remove the hydrophobic region (662-746 amino acids) of carboxylic end, removed the E2 albumen of carboxylic end hydrophobic region (as people J.Hepatology 1997 26:1179-1186 such as H ü ssy R, people Biochem.Biophy.Res.Commun.1992 183:925-930 such as Mita E) as only expressing the 384-661 amino acids.Do not see the report that the strong hydrophobic region of single expression envelope protein E2 extracellular domain carboxylic end is arranged, do not see the report that successful expression coding total length envelope protein E2 is arranged yet.
Summary of the invention
Core of the present invention has been to break through HCV envelope protein E2 extracellular domain carboxylic end parts and complete HCV envelope protein E2 can not obtain total length expressed restriction always in intestinal bacteria, the ammonia end of coding extracellular domain carboxylic end parts in 1b hypotype HCV envelope protein raq gene, that is the middle section of HCV is introduced the phase shift mutation of 4 amino-acid residues of a coding, do not change the basic structure and the sequence of extracellular domain carboxylic end parts and E2 complete genome in the raq gene, at expression in escherichia coli, can obtain the expression of HCV E2 extracellular domain carboxylic end parts or HCV E2 albumen total length.
The invention provides and a kind ofly can in intestinal bacteria, obtain total length expressed HCV envelope protein raq gene, its nucleotide sequence sees in the sequence table＜210〉1, the ammonia end of its extracellular domain carboxylic end parts in 1b hypotype HCV envelope protein raq gene, it is the middle section of envelope protein E2, introduce the phase shift mutation of 4 amino-acid residues of a coding coding the 568th to the 571st, also be about to the phase shift mutation that original aminoacid sequence PCNI changes into RVTS.[this expression of gene clone pQE8/E2 (385-730) m has changed among the intestinal bacteria TG-1 and has deposited to contain the E2 extracellular domain carboxylic end parts (encoding 567-730) of this phase shift mutation and closely complete E2m (coding 385-730 position) gene, be TG-1/pQE8E2m, by Beijing China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, on March 4th, 2002, deposit number CGMCC No.0725], in coli expression system, break through original E2 extracellular domain carboxylic end parts and raq gene and can not in intestinal bacteria, obtain total length expressed restriction, can obtain the expression of total length E2 extracellular domain carboxylic end parts (567-730) or nearly complete E2 albumen (385-730) efficiently.This E2 albumen can utilize Ni easily by merge the 6xHis sequence that has on expression vector
2+The E2 extracellular domain carboxylic end parts of medium enrichment of-NTA affinity and purifying HCV (567-730) or nearly complete E2 albumen (385-730).Total length expressed E2 albumen (its aminoacid sequence see in the sequence table＜210〉2) has antigenicity and the immunogenicity of good E2, can with the E2 antibody response that exists among the HCV patients serum, can be applicable to enzyme-linked immunosorbent assay to E2 antibody.
The present invention also provide this can be in intestinal bacteria the application of proteins encoded in the medicine of preparation, diagnosis, prevention and treatment hepatitis C of total length expressed HCV envelope protein raq gene.
This total length expressed E2 protein immune animal (mouse or rabbit) can produce anti-E2 antibody, the preparation polyvalent antibody can with multiple different system (as vaccinia virus recombinant, mammalian cell, intestinal bacteria etc.) the E2 albumen test of Biao Daing, can be used in each system expressing the detection of E2, as immunoblotting assay etc.
This invention provides a kind of condition, and promptly can study E2 extracellular domain carboxylic end parts separately or intactly study HCV E2 property of protein and the Molecular Virology of HCV E2, and the research of carrying out complete HCV E2 antigen and antibody effect and character and application thereof.
An energy provided by the present invention obtains the gene of the total length expressed nearly complete envelope protein E2 of hepatitis C virus or the extracellular domain carboxylic end parts and the proteins encoded thereof of raq gene in the intestinal bacteria system, be the phase shift mutation that the middle section of complete raq gene has been introduced 4 aminoacid sequences of a coding at the ammonia end of extracellular domain carboxylic end hydrophobic region in this gene, concrete grammar comprises:
1. the structure [pQE8/E2 (567-700)] that contains the cloning by expression of hepatitis C virus envelope protein E2 extracellular domain carboxylic end parts (567-700);
2. the clone [pQE8/E2 (567-700) m] who contains 4 the amino-acid residue phase shift mutations of encoding at E2 extracellular domain carboxylic end parts (567-700) ammonia end;
3.E2 extracellular domain carboxylic end parts (567-730) ammonia end contains the structure [pQE8/E2 (567-730) m] of the cloning by expression of phase shift mutation;
4. the structure [pQE8/E2 (385-730) m] of the complete HCV envelope protein raq gene that contains phase shift mutation (coding 385-730) cloning by expression and do not contain the structure [pQE8/e2 (385-730)] of cloning by expression of the raq gene (385-730 encodes) of phase shift mutation;
5. contain the evaluation of the nearly complete E2 of HCV (385-730) m that the complete E2 of HCV has a phase shift mutation total length expressed and expression product character in intestinal bacteria.
Description of drawings
The present invention further sets forth by the following drawings and embodiment, but does not limit the scope of the invention.
Description of drawings of the present invention is as follows:
Fig. 1 is the structure synoptic diagram that contains HCV E2 extracellular domain carboxylic end parts (567-700) cloning by expression pQE8/E2 (567-700).
Various nomenclatures:
Envelope protein E2, the numeral that is marked on the below is this position encoded amino acid whose sequence number of E2; A-restriction enzyme ApaI; H-HindIII; B BamHI; S-SmaI; Lig-connects; The left side is denoted as reorganization back plasmid title, and the right side is labeled as the preceding original plasmid title of reorganization;
Fig. 2 is the 568th synoptic diagram to 571 amino acids sequence generation phase shift mutations of E2 extracellular domain carboxylic end parts (567-700) coding in cloning by expression pQE8/E2 (567-700) m.
The sequence number of top this site coded amino acid of figure shift E2: P-proline(Pro); The C-halfcystine; The acid of N-N; The I-Isoleucine; The R-arginine; The V-Xie Ansuan; The T-Threonine; The S-Serine; The 568-571 amino acids residue of phase shift mutation has taken place in codon below setting-out representative relatively;
Fig. 3 is that clone pQE8/E2 (567-730) m that contains the HCV E2 extracellular domain carboxylic end parts (567-730 position) of phase shift mutation makes up synoptic diagram.
Sp-restriction enzyme SphI; Sa-SalI; H-HindIII; Lig-connects.
Envelope protein E2, the numeral that is marked on the below is this position encoded amino acid whose sequence number of E2; The left side is denoted as reorganization back plasmid title, and the right side is labeled as the preceding original plasmid title of reorganization;
Fig. 4 is that the nearly complete E2 of HCV (385730) m cloning by expression pQE8/E2 (385-730) m that contains phase shift mutation makes up synoptic diagram.
The left side is denoted as reorganization back plasmid title, and the right side is labeled as the preceding original plasmid title of reorganization;
Fig. 5 be the expression product of the cloning by expression pQE8/E2 (567-700), pQE8/E2 (567-700) m that contain E2 extracellular domain carboxylic end parts, pQE8/E2 (567-730) m and the cloning by expression pQE8/E2 (385-730) that contains complete E2 with the expression product electrophoresis of pQE8/E2 (385-730) m after coomassie brilliant blue staining identify.
A-pQE8/E2(567-700)；B-pQE8/E2(567-700)m；
C-pQE8/E2(567-730)m；D-pQE8/E2(385-730)；
E-pQE8/E2(385-730)m。
The estimated molecular weight size of chart display: A-17.8KD; B-17.8KD;
C-23.2KD；D-41.6KD；E-41.6KD；
Fig. 6 is the immunoblotting assay of the total length expressed product E2 of complete envelope protein E2 (384-730) m, ECL development photo.
A-HCV infected person anteserum S94; B-E2 polyvalent antibody RE2116;
Fig. 7 is that Ni column purification and the coomassie brilliant blue staining behind the electrophoresis of the total length expressed product E2 of HCV E2 (384-730) m identified.
Sample before the A-purifying; Sample behind the B-purifying;
Fig. 8 be by the polyvalent antibody of E2 (384-730) m preparation can with the immunoblotting assay of the E2 specific combination in different recombinant expression systems source.
The E2 that A-vaccinia virus recombinant vv/E2 expresses;
The E2 that B-intestinal bacteria TG-1/pQE8/E2 (385-730) m expresses.
Embodiment
Embodiment 1 contains the structure of HCV E2 extracellular domain carboxylic end parts (567-700) cloning by expression
It is original plasmid people JMed Virol 1993 40:254-360 such as () Wang Y that employing contains 1b hypotype HCV HC-C2 strain (Wang Y, GenBank Accession No.D13094) cDNA clone pUC19/E1E2 or pTAg/E1E2.Used restriction enzyme, ligase enzyme etc. all are Roche company or Chinese T aKaRa company product in the gene recombination.Plasmid preparation provides reagent and method to carry out by German QIAGEN company.Plasmid reorganization and bacterium conversion are carried out with reference to the ordinary method that people's such as Sambrook J " molecular cloning " provides.Used recipient bacterium is intestinal bacteria TG-1.
Because of the 565th the former ApaI of containing enzyme in raq gene cut recognition site, and the HindIII recognition site on the 700th external carrier, scabble after cutting with the ApaI enzyme earlier, again with downcutting behind the HindIII enzymolysis, tell and contain E2 (567-700) fragment, insertion is through SmaI and HindIII double enzymolysis transition plasmid pUC19, be built into pUC19/E2 (567-700), return with BamHI and HindIII again and cut out E2 (567-700) fragment, insert expression vector through same enzymolysis, its cloning site ammonia end contains BamHI and the HindIII site on the pQE8 (QIAGEN company) of 6xHis (6x Histidine) sign, is built into the cloning by expression pQE8/E2 (567-700) that contains E2 extracellular domain carboxylic end parts (567-700).(see figure 1).
Expression plasmid has all passed through sequencing (being finished by Shanghai Bo Ya company).
Embodiment 2 can contain clone pQE8/E2 (567-700) m of phase shift mutation at total length expressed HCV E2 extracellular domain carboxylic end parts (567-700) the ammonia end of intestinal bacteria.
The constructed clone pQE8/E2 (567-700) that contains HCV E2 extracellular domain carboxylic end parts (567-700) can not obtain to estimate the expression (seeing A among embodiment 5 Fig. 5) of total length E2 (567-700) in intestinal bacteria in embodiment 1.PQE8/E2 in selection (567-700) m can obtain the expression (seeing embodiment 5, B among Fig. 5) of total length E2 (567-700) m in intestinal bacteria.Lacked a C through encode the 1st of the codon of the 567th amino-acid residue of the ammonia end that shows E2 in pQE8/E2 (567-700) m behind the sequencing, and have more a G coding the 3rd of the 571st amino acids residue codon, but do not change from 571 later amino-acid sequences, and in preceding 4 amino acids sequences phase shift mutation has taken place only, the encode aminoacid sequence of 567-571 position of original E2 extracellular domain carboxylic end parts is changed into the RVTS (see figure 2) by original PCNI.Contain the raq gene sequence of phase shift mutation and corresponding aminoacid sequence thereof to see respectively in the sequence table＜210〉1 and＜210〉2.
Embodiment 3 contains the structure of HCV E2 extracellular domain carboxylic end parts (567-730 position) cloning by expression pQE8/E2 (567-730) m of phase shift mutation
For extending to, the carboxylic end of the E2 extracellular domain carboxylic end parts that will express is close to complete the 730th, utilization contains the expression plasmid pEH (people such as Li Yingchun of HCV E2 carboxylic end to 730, Chinese science C collects, 1998 28 (3), 204-210), flat with mending behind the SalI enzymolysis earlier, cut out with the SphI enzymolysis again and contain the E2 fragment and be inserted at the pQE8/E2 described in the embodiment 2 (567-700) m plasmid and behind the HindIII enzymolysis, mend earlier in the flat expression vector after going this fragment with SphI enzymolysis branch again, promptly be built into pQE8/E2 (567-730) the m cloning by expression of expression of HCV E2 extracellular domain carboxylic end parts (567-730) m, see Fig. 3.
Embodiment 4 contains the structure of near complete HCV E2 (385-730) m cloning by expression pQE8/E2 (385-730) m of phase shift mutation
In order to obtain the cloning by expression of closely complete HCV E2 (385-730), with the plasmid pEH (people such as Li Yingchun who contains complete HCV E2, Chinese science C collects, 1998 28 (3), 204-210) be template, with the reverse primer P2 5 '-CTTTTAAGCTTAGGTATGGTGGTG-3 ' that introduces BamHI recognition site forward primer P1 5 '-GGGGATCCAACACCTACGTGACGGGG-3 ' and introducing HindIII recognition site, adopt round pcr to amplify HCV E2 (385-730) fragment, through being inserted into elder generation behind BamHI and the HindIII enzymolysis on the expression vector pQE8 behind BamHI and the HindIII double enzymolysis, be built into pQE8/E2 (385-730) cloning by expression (seeing Fig. 4 A) of expression of HCV E2 (385-730) then.
Be template with embodiment 3 described cloning by expression pQE8/E2 (567-730) m in addition, reverse primer P4 5 '-CAAGCTAGCTTGGATTCTCACC-3 ' with forward primer P3 5 '-GTCGGGCCCCCGTGTAACATCG-3 ' that introduces the ApaI recognition site and introducing NheI recognition site, adopt round pcr to amplify the ApaI-NheI fragment that contains phase shift mutation, again through being inserted into behind ApaI and the NheI double enzymolysis earlier after enzyme is cut equally, and divide pQE8/E2 (385-730) expression plasmid go after the original ApaI-NheI fragment, reconstruct into the nearly complete E2 of expression of HCV, wherein the ammonia end of extracellular domain carboxylic end parts contains cloning by expression pQE8/E2 (385-730) m (seeing Fig. 4 B) of phase shift mutation.
Embodiment 5 contain the cloning by expression pQE8/E2 (567-700) of E2 extracellular domain carboxylic end parts and cloning by expression pQE8/E2 (567-700) m, pQE8/E2 (567-730) m that E2 extracellular domain carboxylic end parts contains phase shift mutation and contain the cloning by expression pQE8/E2 (385-730) of nearly complete E2 and contain nearly complete E2, wherein extracellular domain contains the expression of cloning by expression pQE8/E2 (385-730) m of phase shift mutation and the dyeing of expression product is identified.
The TG-1 that contains each recombinant plasmid through final concentration 1mM IPTG inducing culture, collects thalline, again with PBS damping fluid (pH8.0) extracting that contains 8M urea/20mM mercaptoethanol.Extract the Laemmli of standard (Laemmli K etc., Nature 1970; 223:680) behind the SDS-PAGE electrophoresis, adopt Xylene Brilliant Cyanine G G-250 dyeing, the results are shown in Figure 5.
The expressing protein A of recombinant clone, E2 (567-700); B, E2 (567-700) m; C, E2 (567-730) m and D, E2 (385-730); E, the molecular weight that E2 (385-730) m is inferred by coded amino acid is respectively 17.8kDa, 17.8kDa, 23.2kDa and 41.6kDa, 41.6kDa.But contain the clone pQE8/E2 (567-700) of extracellular domain carboxylic end parts and the cloning by expression pQE8/E2 (385-730) of complete E2 and can not obtain in intestinal bacteria all to estimate that the expression product of total length maybe can only obtain to see A and D among Fig. 5 less than total length expressed product.Having recombinant expressed clone pQE8/E2 (385-730) m that ammonia end in E2 extracellular domain carboxylic end parts contains reorganization E2 extracellular domain carboxylic end parts cloning by expression pQE8/E2 (567-700) m, pQE8/E2 (567-730) m of phase shift mutation and the nearly complete E2 that extracellular domain contains phase shift mutation only could have the expression of full-length proteins in intestinal bacteria, see B among Fig. 5, C, E, and have respectively and the molecular weight estimated is 17.8kDa, the protein expression that 23.2kDa and 41.6kDa conform to.
Its antigenic immunoblotting assay of HCV E2 (385-730) m that embodiment 6 is total length expressed
Total length expressed E2 (385-730) m is through transferring to (Wattman company product) on the cellulose nitrate film behind the electrophoresis, used one anti-has a hepatitis C patients serum S94 (Wang Yu in the immunoblotting assay, Medical School of Peking University provides) or be used in polyvalent antibody RE2116 (people such as Liu Jing, the Biotechnol Appl Biochem 2001 of E2 (450-565) protein Preparation of expression in escherichia coli; 34:109-119), two anti-horseradish peroxidase connection bonded albumin A (the Sigma company products that adopt, 1: 1000) or the horseradish peroxidase of the anti-rabbit Ig of pig connect compound (Dako company product, 1: 1000), the ECL reagent and the method that provide by Amersham company, produce fluorescence and compressing tablet, the results are shown in Figure 6.The result shows with the HCV patients serum (Fig. 6 A) of containing E2 antibody or with expressing polyvalent antibody (Fig. 6 B) combination with it specifically that E2 prepares, and be the special band of 41.6kDa place generation at the E2 envelope protein molecular weight of the total length of estimating, this illustrates at the E2 of expression in escherichia coli (385-730) though process saccharification processing of m, but still has good antigenicity, can be by the E2 antibody recognition in the hepatitis C patients serum with by the E2 antibody recognition in the polyvalent antibody of E2 preparation, the total length E2 of expression (385-730) m has good antigenicity.
HCV E2 (385-730) m that embodiment 7 is total length expressed uses Ni
2+The purifying of-NTA metal chelate affinity chromatography technology
Through the centrifugal collection thalline of the TG-1/pQE8/E2 of IPTG inducing culture (385-730) m, be suspended from PBS damping fluid (pH8.0) extracting that contains 8M urea/20mM mercaptoethanol, centrifugal collection supernatant is used Ni under the sex change condition
2+-NTA sepharose metallic chelate affinity medium (QIAGEN company product) combination, with PBS damping fluid (pH6.3) washing that contains 8M urea/20mM mercaptoethanol, again with PBS damping fluid (pH4.3) wash-out, the collection that contain 8M urea/20mM mercaptoethanol, on each sample behind the sample electrophoresis, and dyeing, behind known quantity bovine serum albumin (BioRad company) electrophoresis, protein content is relatively calculated in optical density scanning.Purification result is seen Fig. 7, and wherein A is a sample before the purifying.Can get total length expressed E2 (385-730) m albumen through single step purification and account for more than 30% of whole expressing proteins under the sex change condition, recovery rate is the E2 albumen 1mg above (Fig. 7 B) that every liter of culture finally can get purifying.
HCV E2 (385-730) the m albumen of embodiment 8 purifying is used for the enzyme-linked immunoassay (ELISA) of HCV patients serum E2 antibody specific combination
The characteristic of E2 antibody specific combination in E2 (385-730) former performance of utilize expressing of m good resistance and the serum detects the E2 antibody in the clinical serum with E2 (385-730) m of purifying.By 0.15 μ g E2 (385-730) m, 37 ℃ of insulations are after 1 hour by every hole bag, and 4 ℃ are spent the night, and with the new-born calf serum sealing of PBS/1% bovine serum albumin BSA (w/v)/2% deactivation, HCVEIA 4.0 test kits and the method that provides by UBI company carried out then.Serum dilutes at 1: 20, and 37 ℃ are incubated 1 hour, and goat-anti people Ig antibody-horseradish peroxidases that the two anti-UBI of being provide connect compound, dilution in 1: 150, and 37 ℃ were reacted 30 minutes, and O-Phenylene Diamine (OPD) develops the color, and 492nm measures absorption value.
Select for use 100 parts of health to help blood person's serum (total negative antibody), 158 parts of total antibody positive serum of HCV, 20 parts of hepatitis B patient serum and 20 parts of non-A-E hepatitis serum are measured, and the results are shown in Table 1.As can be seen from Table 1, wrap with E2 (385-730) m that E2 antibody has specificity preferably in the determined human serum, 20 parts of hepatitis B patient serum and 20 parts of non-A-E hepatitis patients serums all are negative.The positive rate of E2 antibody is 56% in the total antibody positive serum of HCV patient, and health helps has 3 parts to detect the E2 antibody positive among the blood person, with confirming to have probably E2 antibody behind the immunoblotting assay.The result shows, with E2 (385-730) the m bag quilt of expressing, can detect E2 antibody in the human serum effectively with the ELISA method.
Table 1 utilizes E2 (385-730) m to detect HCV E2 antibody in the human serum
The total antibody E2 of the HCV of the antibody colony that surveys (sample number) antibody
The positive umber positive rate of positive umber positive rate health helps blood person (100) 0 0% 3
*3%
*The non-A-E hepatitis patient of hepatitis B patient (20) 0 0% 0 0% (20) 0 0% 0 0% hepatitis C patients (158) 158 100% 89 56%
Embodiment 9 is by the E2 envelope protein in the different recombinant expressed sources of polyvalent antibody energy specific recognition of HCV E2 (385-730) the m preparation of purifying
Select the female new zealand rabbit of 1-1.5 kilogram (available from the Shanghai Experimental Animal Center) for use, the E2 of purifying (385-730) m albumen is earlier with after isopyknic Fo Shi Freund's complete adjuvant (all adjuvants all are GibcoBRL company product) mixes, subcutaneous injection behind neck, each immune 300 μ g, strengthen once after 4 weeks, used albumen mixes with isopyknic Freund during reinforcement.The antiserum(antisera) antibody titers of blood sampling preparation at last reaches 3.2 * 10
4Polyvalent antibody is applied to immunoblotting assay, the results are shown in Figure 8.The E2 antibody capable in the polyvalent antibody and the E2 in different recombinant expressed sources---Tathagata is from recombinating vaccinia virus (Fig. 8 A) and without the special combination of HCV envelope protein E2 of the intestinal bacteria system expressions such as (Fig. 8 B) of saccharification processing.A [sees that people such as Li Yingchun China science C collects for the cell pyrolysis liquid of the recombinant vaccinia virus infection HeLa cell that contains HCV E2 (384-730), 1998 28 (3), 204-210)], the saccharification E2 of visible vaccinia virus recombinant system expression among the figure, molecular weight is about 55kDa.B is for containing the intestinal bacteria TG-1 expression product of HCV E2 (385-730) m, and visible E2 is the specific band of 41.6kDa without E2 (385-730) the m molecular weight of saccharification processing in intestinal bacteria among the figure.
SEQUENCE LISTING＜110〉＜120〉E2＜160〉2＜170〉PatentIn version 3.1＜210〉1＜211〉1038＜212〉DNA＜213〉 ( Hepatitis C Virus )＜400〉1acc tac gtg acg ggg ggg gcg gct gca cgc ggg gcc tcc ggg atc acg 48agc ctc ttt tca cgt ggt ccg tct cag aaa atc cag ctt gtg aac acc 96aat ggc agc cgg cac atc aac ggg act gcc ctg aac tgc aat gac tcc 144ttc aac act ggg ttc ctc gcc gcg ctg ttc tac gcg cac agg ttc aac 192tcg tcc gga tgc cca gag cgc atg gcc agc tgc cgc tcc att gac aag 240ttc gac cag gga cgg ggt ccc atc act tat tat cag ggt gac agc ccg 288gac cag agg cct tat tgc tgg cac tac cca cct cga ccg tgt ggt ata 336gtg ccc gcg tcg gag gtg tgt ggt cca gtg tat tgt ttc acc cca agc 384ccc gtt gtg gtg ggg acg acc gat cgc ctc ggc gtc cct aca tat aac 432tgg ggg gaa aat gag aca gac gtg ctg ctc ctt aac aac acg cgg ccg 480ccg caa ggc aac tgg ttc ggc tgt acg tgg atg aat acc acc ggg ttc 528acc aag acg ttc ggg ggc ccc cgt gta aca tcg ggg ggg gcc ggc aac 576aac acc ttg act tgc ccc acg gac tgc ttc cgg aag cac ccc gag gcc 624act tac aca aaa tgt ggt tcg ggg cct tgg ttg aca cct agg tgc tta 672gtt gac tat cca tac agg ctc tgg cat tac ccc tgc act gtt aac ttc 720acc atc ttt aag gtt agg atg tat gtg ggg ggc gtg gag cac agg ctc 768gac gcc gca tgc aac tgg act cga gga gaa cgt tgc gcc ttg gag gac 816agg gat aga tca gag ctc agc ccg ctg ctg ctg tct aca aca gag tgg 864cag ata ctg ccc tgt tcc ttc acc acc cta ccg gcc ctg tct act ggt 912ttg atc cat ctc cac cgg aac atc gtg gac gtg caa tac cta tac ggt 960ata agg tca gca gtt gtc tcc ttt gcc atc aaa tgg gag tat gtc ctg 1008ttg ctt ttc ctt ctc ctg gca gac gcg cgc 1038＜210〉2＜211〉346＜212〉PRT＜213〉 ( Hepatitis C Virus )＜400〉2Thr Tyr Val Thr Gly Gly Ala Ala Ala Arg Gly Ala Ser Gly Ile Thr1 5 10 15Ser Leu Phe Ser Arg Gly Pro Ser Gln Lys Ile Gln Leu Val Asn Thr
20 25 30Asn?Gly?Ser?Arg?His?Ile?Asn?Gly?Thr?Ala?Leu?Asn?Cys?Asn?Asp?Ser
35 40 45Phe?Asn?Thr?Gly?Phe?Leu?Ala?Ala?Leu?Phe?Tyr?Ala?His?Arg?Phe?Asn
50 55 60Ser?Ser?Gly?Cys?Pro?Glu?Arg?Met?Ala?Ser?Cys?Arg?Ser?Ile?Asp?Lys65 70 75 80Phe?Asp?Gln?Gly?Arg?Gly?Pro?Ile?Thr?Tyr?Tyr?Gln?Gly?Asp?Ser?Pro
85 90 95Asp?Gln?Arg?Pro?Tyr?Cys?Trp?His?Tyr?Pro?Pro?Arg?Pro?Cys?Gly?Ile
100 105 110Val?Pro?Ala?Ser?Glu?Val?Cys?Gly?Pro?Val?Tyr?Cys?Phe?Thr?Pro?Ser
115 120 125Pro?Val?Val?Val?Gly?Thr?Thr?Asp?Arg?Leu?Gly?Val?Pro?Thr?Tyr?Asn
130 135 140Trp?Gly?Glu?Asn?Glu?Thr?Asp?Val?Leu?Leu?Leu?Ash?Asn?Thr?Arg?Pro145 150 155 160Pro?Gln?Gly?Asn?Trp?Phe?Gly?Cys?Thr?Trp?Met?Asn?Thr?Thr?Gly?Phe
165 170 175Thr?Lys?Thr?Phe?Gly?Gly?Pro?Arg?Val?Thr?Ser?Gly?Gly?Ala?Gly?Asn
180 185 190Asn?Thr?Leu?Thr?Cys?Pro?Thr?Asp?Cys?Phe?Arg?Lys?His?Pro?Glu?Ala
195 200 205Thr?Tyr?Thr?Lys?Cys?Gly?Ser?Gly?Pro?Trp?Leu?Thr?Pro?Arg?Cys?Leu
210 215 220Val?Asp?Tyr?Pro?Tyr?Arg?Leu?Trp?His?Tyr?Pro?Cys?Thr?Val?Asn?Phe225 230 235 240Thr?Ile?Phe?Lys?Val?Arg?Met?Tyr?Val?Gly?Gly?Val?Glu?His?Arg?Leu
245 250 255Asp?Ala?Ala?Cys?Asn?Trp?Thr?Arg?Gly?Glu?Arg?Cys?Ala?Leu?Glu?Asp
260 265 270Arg?Asp?Arg?Ser?Glu?Leu?Ser?Pro?Leu?Leu?Leu?Ser?Thr?Thr?Glu?Trp
275 280 285Gln?Ile?Leu?Pro?Cys?Ser?Phe?Thr?Thr?Leu?Pro?Ala?Leu?Ser?Thr?Gly
290 295 300Leu?Ile?His?Leu?His?Arg?Asn?Ile?Val?Asp?Val?Gln?Tyr?Leu?Tyr?Gly305 310 315 320Ile?Arg?Ser?Ala?Val?Val?Ser?Phe?Ala?Ile?Lys?Trp?Glu?Tyr?Val?Leu
325 330 335Leu?Leu?Phe?Leu?Leu?Leu?Ala?Asp?Ala?Arg
340 345
Claims (3)
1. an energy total length expressed hepatitis C virus envelope protein raq gene in intestinal bacteria, its nucleotides sequence classify as in the sequence table＜and 210〉1.
2. the proteins encoded of a raq gene according to claim 1, its aminoacid sequence be in the sequence table＜210〉2.
3. the application of hepatitis C envelope protein E2 as claimed in claim 2 in the medicine of preparation, diagnosis, prevention and treatment hepatitis C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN02112089.7A CN1273482C (en) | 2002-06-14 | 2002-06-14 | Hepatitis C virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN02112089.7A CN1273482C (en) | 2002-06-14 | 2002-06-14 | Hepatitis C virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1465700A true CN1465700A (en) | 2004-01-07 |
| CN1273482C CN1273482C (en) | 2006-09-06 |
Family
ID=34141838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN02112089.7A Expired - Fee Related CN1273482C (en) | 2002-06-14 | 2002-06-14 | Hepatitis C virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1273482C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100395339C (en) * | 2006-02-28 | 2008-06-18 | 中国人民解放军第二军医大学 | Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion |
| CN103435690A (en) * | 2013-09-13 | 2013-12-11 | 武汉大学 | DNA (deoxyribonucleic acid) vaccine of HCV (hepatitis C virus) and preparation method thereof |
| CN104974246A (en) * | 2014-04-01 | 2015-10-14 | 中国科学院上海巴斯德研究所 | Total-human monoclonal antibody for neutralizing hepatitis C virus and application of total-human monoclonal antibody |
| CN105330730A (en) * | 2014-07-29 | 2016-02-17 | 中国科学院上海巴斯德研究所 | Preparation and application of hepatitis C virus recombinant protein |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103354748B (en) * | 2010-08-04 | 2016-09-28 | 麦克法兰博尼特医学健康研究公司 | The hepatitis C virus protein modified |
-
2002
- 2002-06-14 CN CN02112089.7A patent/CN1273482C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100395339C (en) * | 2006-02-28 | 2008-06-18 | 中国人民解放军第二军医大学 | Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion |
| CN103435690A (en) * | 2013-09-13 | 2013-12-11 | 武汉大学 | DNA (deoxyribonucleic acid) vaccine of HCV (hepatitis C virus) and preparation method thereof |
| CN103435690B (en) * | 2013-09-13 | 2016-02-10 | 武汉大学 | DNA vaccination of a kind of hepatitis C virus and preparation method thereof |
| CN104974246A (en) * | 2014-04-01 | 2015-10-14 | 中国科学院上海巴斯德研究所 | Total-human monoclonal antibody for neutralizing hepatitis C virus and application of total-human monoclonal antibody |
| CN105330730A (en) * | 2014-07-29 | 2016-02-17 | 中国科学院上海巴斯德研究所 | Preparation and application of hepatitis C virus recombinant protein |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1273482C (en) | 2006-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20040247617A1 (en) | Fusion antigen used as vaccine | |
| CN106995488A (en) | The H1N1 influenza antigens of the width reactivity of calculation optimization | |
| CN101076591A (en) | Novel human virus causing respiratory tract infection and uses thereof | |
| CN101062410A (en) | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof | |
| LiHua et al. | Evaluation of an outer membrane protein as a vaccine against Edwardsiella anguillarum in Japanese eels (Anguilla japonica) | |
| CN100412088C (en) | Enzyme-linked immunosorbent test detecting reagent kit for non-structure protein of pig and cow foot and mouth disease virus | |
| CN104628871B (en) | A kind of preparation for recombinating bursal disease protein engineering vaccine | |
| CN1465700A (en) | Hepatitis c virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use | |
| Li et al. | The development of classical swine fever marker vaccines in recent years | |
| Harasawa | Adventitious pestivirus RNA in live virus vaccines against bovine and swine diseases | |
| CN105906717A (en) | Preparation method and application of Brucella multi-epitope fusion protein vaccine | |
| AU718740B2 (en) | Polyvalent anti-dengue vaccine | |
| CN1176945C (en) | Plasmodium fused antigen and its preparing process and usage | |
| KR101127926B1 (en) | Iridovirus antigenic peptide and vaccine comprising the same | |
| CN109021115A (en) | A kind of pig circular ring virus trivalent subunit vaccine | |
| CN100335636C (en) | Thioredoxin gene of Schistosoma japonicum (Chinese Mainland Strain) and its cloning expression method and application | |
| CN1737147B (en) | Heat shock protein 65- multiple epitope hepatitis B virus core antigen recombinant protein (HSP65-HBcAg) | |
| Inchauspé | DNA vaccine strategies for hepatitis C | |
| CN102212120A (en) | Salmonella paratyphi A PagC subunit vaccine and preparation method thereof | |
| CN103361364A (en) | Eimeria tenella eIF3d gene and application thereof | |
| CA2370278C (en) | Chimeric lyssavirus nucleic acids and polypeptides | |
| CN101845084B (en) | Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen | |
| CN1831009A (en) | Prawn white-spots syndrom virus (WSSV) membrane protein and its antibody | |
| CN105727278B (en) | A kind of sore mouth virus recombinant protein antigen vaccine and preparation method thereof | |
| EP1578791B1 (en) | Inclusion bodies for the oral vaccination of animals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI INST. OF LIFE SCIENCE, CAS Free format text: FORMER OWNER: INSTITUTE OF BIOCHEMISTRY AND CELL BIOLOGY, SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES Effective date: 20090626 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20090626 Address after: No. 320, Yueyang Road, Shanghai Patentee after: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES Address before: No. 320, Yueyang Road, Shanghai Patentee before: Institute of Biochemistry and cell biology, Shanghai Institute of life sciences, Chinese Academy of Sciences |
|
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Shenzhen Feipeng Biological Co.,Ltd. Assignor: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES Contract fulfillment period: 2009.6.17 to 2014.6.17 Contract record no.: 2009310000125 Denomination of invention: Hepatitis c virus tunic protein E2 gene capable of being total length expressed in E. coli, its coding protein and use Granted publication date: 20060906 License type: Exclusive license Record date: 20090713 |
|
| LIC | Patent licence contract for exploitation submitted for record |
Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2009.6.17 TO 2014.6.17; CHANGE OF CONTRACT Name of requester: SHENZHEN CITY FAPON BIOTECH CO., LTD. Effective date: 20090713 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060906 Termination date: 20170614 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |