CN1299831A - New interferon-like protein and its code sequence and use - Google Patents
New interferon-like protein and its code sequence and use Download PDFInfo
- Publication number
- CN1299831A CN1299831A CN 99124272 CN99124272A CN1299831A CN 1299831 A CN1299831 A CN 1299831A CN 99124272 CN99124272 CN 99124272 CN 99124272 A CN99124272 A CN 99124272A CN 1299831 A CN1299831 A CN 1299831A
- Authority
- CN
- China
- Prior art keywords
- ifl
- polypeptide
- sequence
- people
- polynucleotide
- 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 43
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 21
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 claims abstract description 143
- 102100040019 Interferon alpha-1/13 Human genes 0.000 claims abstract description 118
- 238000000034 method Methods 0.000 claims abstract description 48
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 47
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 47
- 239000002157 polynucleotide Substances 0.000 claims abstract description 47
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 89
- 229920001184 polypeptide Polymers 0.000 claims description 83
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 83
- 239000012634 fragment Substances 0.000 claims description 27
- 235000018102 proteins Nutrition 0.000 claims description 18
- 150000007523 nucleic acids Chemical class 0.000 claims description 14
- 239000002773 nucleotide Substances 0.000 claims description 13
- 125000003729 nucleotide group Chemical group 0.000 claims description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 239000000523 sample Substances 0.000 claims description 10
- 238000009396 hybridization Methods 0.000 claims description 9
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 239000000556 agonist Substances 0.000 claims description 5
- 239000005557 antagonist Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 3
- 238000002493 microarray Methods 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 19
- 201000010099 disease Diseases 0.000 abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 14
- 102000014150 Interferons Human genes 0.000 abstract description 9
- 108010050904 Interferons Proteins 0.000 abstract description 9
- 206010028980 Neoplasm Diseases 0.000 abstract description 9
- 229940079322 interferon Drugs 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000009385 viral infection Effects 0.000 abstract description 5
- 230000036737 immune function Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 50
- 235000001014 amino acid Nutrition 0.000 description 29
- 150000001413 amino acids Chemical class 0.000 description 29
- 108020004414 DNA Proteins 0.000 description 21
- 239000002299 complementary DNA Substances 0.000 description 19
- 239000000047 product Substances 0.000 description 19
- 238000011282 treatment Methods 0.000 description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 description 15
- 108010047761 Interferon-alpha Proteins 0.000 description 14
- 102000006992 Interferon-alpha Human genes 0.000 description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 13
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 239000013604 expression vector Substances 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 9
- 230000008521 reorganization Effects 0.000 description 9
- 108091034117 Oligonucleotide Proteins 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 108091026890 Coding region Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 230000004853 protein function Effects 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 108090000994 Catalytic RNA Proteins 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 210000005260 human cell Anatomy 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 108091092562 ribozyme Proteins 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102100026720 Interferon beta Human genes 0.000 description 3
- 108090000467 Interferon-beta Proteins 0.000 description 3
- 206010027336 Menstruation delayed Diseases 0.000 description 3
- 241000276498 Pollachius virens Species 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108020005091 Replication Origin Proteins 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 108091092724 Noncoding DNA Proteins 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 210000004754 hybrid cell Anatomy 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000007365 immunoregulation Effects 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 239000002516 radical scavenger Substances 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 description 1
- UZGKAASZIMOAMU-UHFFFAOYSA-N 124177-85-1 Chemical compound NP(=O)=O UZGKAASZIMOAMU-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- RDIKFPRVLJLMER-BQBZGAKWSA-N Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)N RDIKFPRVLJLMER-BQBZGAKWSA-N 0.000 description 1
- 206010059313 Anogenital warts Diseases 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- LJUOLNXOWSWGKF-ACZMJKKPSA-N Asn-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N LJUOLNXOWSWGKF-ACZMJKKPSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- NUSWUSKZRCGFEX-FXQIFTODSA-N Glu-Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O NUSWUSKZRCGFEX-FXQIFTODSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 1
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- CZCIKBSVHDNIDH-NSHDSACASA-N N(alpha)-methyl-L-tryptophan Chemical compound C1=CC=C2C(C[C@H]([NH2+]C)C([O-])=O)=CNC2=C1 CZCIKBSVHDNIDH-NSHDSACASA-N 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- CZCIKBSVHDNIDH-UHFFFAOYSA-N Nalpha-methyl-DL-tryptophan Natural products C1=CC=C2C(CC(NC)C(O)=O)=CNC2=C1 CZCIKBSVHDNIDH-UHFFFAOYSA-N 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- WEMYTDDMDBLPMI-DKIMLUQUSA-N Phe-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N WEMYTDDMDBLPMI-DKIMLUQUSA-N 0.000 description 1
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- FZXOPYUEQGDGMS-ACZMJKKPSA-N Ser-Ser-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZXOPYUEQGDGMS-ACZMJKKPSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 208000031673 T-Cell Cutaneous Lymphoma Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 208000005485 Thrombocytosis Diseases 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- ARJASMXQBRNAGI-YESZJQIVSA-N Tyr-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N ARJASMXQBRNAGI-YESZJQIVSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 201000007241 cutaneous T cell lymphoma Diseases 0.000 description 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 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 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- -1 methane amide Chemical class 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 201000005962 mycosis fungoides Diseases 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- DCWXELXMIBXGTH-QMMMGPOBSA-N phosphonotyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-QMMMGPOBSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种新的细胞因子－干扰素样蛋白(IFL蛋白),编码IFL蛋白的多核苷酸和经重组技术产生这种IFL蛋白的方法。IFL蛋白是干扰素(IFN α/β)的同源蛋白。本发明还公开了编码这种IFL蛋白的多核苷酸的用途。本发明还公开了此IFL蛋白用于治疗多种疾病的方法,如用于病毒感染、肿瘤、免疫功能低下等疾病。本发明还提供了含IFL蛋白的药物组合物。The invention provides a new cytokine-interferon-like protein (IFL protein), a polynucleotide encoding the IFL protein and a method for producing the IFL protein through recombinant technology. The IFL protein is a homologous protein of interferon (IFN α/β). The invention also discloses the application of the polynucleotide encoding the IFL protein. The invention also discloses a method for using the IFL protein to treat various diseases, such as virus infection, tumor, low immune function and other diseases. The invention also provides a pharmaceutical composition containing the IFL protein.
Description
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding human cell factor-interferon-like protein (interferon-like protein abbreviates " IFL " as), and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of new and immune related cytokine, it and IFN α/β homology.
Cytokine is an interactional main medium between the cells in vivo, immunne response, inflammatory reaction, hemopoietic function at body, and even the embryo take place, grow etc. all respects all play keying action (Pan et al.FEBS Lett 1998,431:351).It is antiviral that Interferon, rabbit is that gang has, and influences cell growth, differentiation and regulate the albumen of immunologic function isoreactivity.Be find the earliest, study at most, first cloning, first is used for the cytokine of clinical treating disease.People are divided into α, β, three types of γ with Interferon, rabbit at present.Wherein interferon-alpha (IFN-α) has hypotypes different more than 20 kinds at least, is made of the about 19KD of molecular weight 166-172 amino acid.
That the main function of IFN-α comprises is antiviral, inhibition of cell proliferation and immunomodulatory: 1. antivirus action: IFN-α is except the direct inducing anti-disease poison state, can also produce antiviral effect widely by activation NK cell, scavenger cell and other immunologic function of enhancing.2. inhibition of cell proliferation effect: can influence cell proliferation and differentiation, also have the effect of oncogene expression before regulating; 3. immunoregulation effect: mainly show the adjusting of lymphocyte and scavenger cell and induce the MHC developed by molecule.
IFN-α is the cytokine of the widest treatment tumour of clinical application, its curative effect to hematopoietic system cancer and lymphoma patient is comparatively sure, and the scheme that is used for the treatment of hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, non-Hodgkin lymphoma, cutaneous T cell lymphoma and thrombocytosis and polycyth(a)emia is comparatively ripe.Simultaneously, the scheme that application IFN-α carries out treatment of solid tumors also begins in clinical application, comprises treatment gastroenteric tumor, renal cell carcinoma.Malignant melanoma and ovarian cancer etc.
IFN-α can also be used for the treatment of hepatitis and virus disease.For the efficient of chronic hepatitis B treatment is 30-50%, but patient's long-term remission, and HBsAg turns out cloudy.IFN-α also has certain curative effect to pointed condyloma, and can carry out topical therapeutic.IFN-α also can be used for treating some parillomarvirus infections.
IFN-α is that first is applied to the gene engineering product into bed in the world, and the annual sales volume is 1,500,000,000 dollars now.Commercial IFN-α preparation has Intro A (the humanIFN-2b of reorganization), Roferon (the humanIFN-2a of reorganization) and Welferon (natural humanIFN-) etc.Domestic also have different IFN-α preparation production, comprises the humanIFN-D of reorganization, the humanIFN-A of reorganization etc.
The biologic activity of IFN-β and IFN-α are basic identical, also have antiviral, inhibition of cell proliferation and immunoregulation effect, and the treatment that is used for multiple sclerosis must be ratified in FDA.
Therefore, in order to treat illnesss such as virus infection, tumour, immunologic hypofunction better, this area presses for the new relevant cell factor of exploitation.
Before the present invention, the amino acid or the nucleotide sequence of new interferon protein involved in the present invention still do not disclosed.
The purpose of this invention is to provide a kind of new human cell factor-IFL albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.IFL albumen of the present invention is a kind of IFN homologous protein.
Another object of the present invention provides and contains the new proteic pharmaceutical composition of people IFL.
The new Interferon, rabbit IFL that provides of the present invention belongs to the new subtype of α/interferon-, has antiviral and antitumor action, aspect the treatments such as viral infection and cancer application prospect is being arranged.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is the cDNA sequence of people IFL of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is represented with capitalization.
Fig. 2 is the proteic full length amino acid sequence of people IFL of the present invention.
Fig. 3 is people IFL albumen of the present invention and the proteic amino acid sequence homology comparison diagram of human interferon IFN-B.The top sequence is people IFL, and the below sequence is a people IFN-B albumen.Identical amino acid marks with " | " between two sequences, and similar amino acid marks with ": " or ". ".
Fig. 4 is the hydrophobicity graphic representation of the proteic Kyte-doolitte hydrophobicity analysis of people IFL of the present invention.
In the present invention, term " IFL albumen ", " IFL polypeptide " or " IFL interferon " are used interchangeably, and all refer to have the human cell factor IFL of amino acid sequence of the mature form (SEQ ID NO:3) of the total length form (SEQ ID NO:2) of human cell factor IFL amino acid sequence or no signal peptide. They comprise the cell factor IFL that contains or do not contain initial methionine.
As used herein, " separation " refers to that material separates (if natural material, original environment namely is natural environment) from its original environment. Do not have separation and purification such as polynucleotide and polypeptide under the natural state in the active somatic cell, but same polynucleotide or polypeptide as from natural state with in other materials that exist separately, then for separation and purification.
As used herein, " IFL albumen or the polypeptide of separation " refers to that the IFL polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the purified technology of protein purifying IFL albumen of standard. Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel. The purity of IFL polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptide, synthetic polypeptide, preferred recombinant polypeptide. Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis, or uses recombinant technique to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). The host used according to the recombinant production scheme, polypeptide of the present invention can be the sugar baseization, maybe can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the similar thing of people IFL albumen. As used herein, term " fragment ", " derivative " refer to basically keep the identical biology function of natural human IFL albumen of the present invention or active polypeptide with " similar thing ". Polypeptide fragment of the present invention, derivative or similar thing can be that (ⅰ) has one or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ⅱ) in one or more amino acid residues, has a polypeptide of substituted radical, or (ⅲ) mature polypeptide and another compound (such as the compound that prolongs the polypeptide half-life, polyethylene glycol for example) merges formed polypeptide, or (ⅳ) additional amino acid sequence is fused to this peptide sequence and the polypeptide that forms (such as leading sequence or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion albumen of the formation of antigen I gG fragment). According to the instruction of this paper, these fragments, derivative and similar thing belong to the known scope of those skilled in the art.
In the present invention, term " people IFL polypeptide " refers to have the SEQ ID NO.2 of people IFL protein active or the polypeptide of 3 sequences. This term also comprises having and variant forms people IFL albumen identical function, SEQ ID NO.2 or 3 sequences. These variant forms comprise (but being not limited to): some (are generally 1-50, better ground 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and C end and/or one of the terminal interpolation of N or several individual (being generally in 20, be in 10, more preferably be in 5 goodly) amino acid. For example, in the art, when replacing with the close or similar amino acid of performance, usually can not change the function of protein. Again such as, add the functions that or several amino acid also can not change protein usually in that C end and/or N are terminal. This term also comprises active fragment and the reactive derivative of people IFL albumen.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people IFL DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people IFL polypeptide to obtain.The present invention also provides other polypeptide, as comprises people IFL polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people IFL polypeptide.Usually, this fragment have people IFL peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of people IFL albumen or polypeptide.The difference of these analogues and natural human IFL polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " people IFL albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2 or 3, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2 or 3, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of coding SEQ ID NO:2 or 3 mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding IFL.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People IFL Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCP can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or IFL albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the IFL polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people IFL polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people IFL polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people IFL DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people IFL albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism IFL protein function as pharmacological agent IFL protein function.The peptide molecule that can suppress or stimulate people IFL protein function that can be used for seeking therapeutic value with the recombinant human IFL protein screening peptide library of expressing.
On the other hand, the present invention also comprises people IFL DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people IFL gene product or fragment.Preferably, refer to that those can combine with people IFL gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people IFL, comprise that also those do not influence the antibody of people IFL protein function.The present invention also comprise those can with modify or without the people IFL gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people IFL gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human IFL albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people IFL protein function and the antibody that does not influence people IFL protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people IFL gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people IFL gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-people IFL can be used in the immunohistochemistry technology, detects the people IFL albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people IFL, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people IFL albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people IFL or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people IFL albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people IFL protein positive.
The production of polyclonal antibody can choose IFL albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with IFL albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of virus infection, tumour and immunocompromised aspect.When using IFL albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α, TNF-β etc.
The present invention also provides a kind of pharmaceutical composition, and it contains IFL polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the IFL albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people IFL also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of IFL of the proteic nothing expression of IFL or unusual/non-activity.The IFL albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic IFL protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the IFL transgenosis to cell.The method that structure carries the recombinant viral vector of IFL gene is found in existing document (Sambrook, et al.).Recombinant human IFL gene can be packaged in the liposome in addition, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people IFL mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people IFL obtains.During screening, must carry out mark to people IFL protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people IFL protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people IFL protein level that is detected in the test can be with laying down a definition the importance of people IFL albumen in various diseases and be used to the disease of diagnosing IFL albumen to work.
Whether having the proteic method of IFL in a kind of detection test sample is to utilize the proteic specific antibody of IFL to detect, and it comprises: sample is contacted with the IFL protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample IFL albumen.
The proteic polynucleotide of IFL can be used for the diagnosis and the treatment of IFL protein related diseases.Aspect diagnosis, the proteic polynucleotide of IFL can be used for detecting the proteic expression of IFL IFL abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of IFL as the IFL dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of IFL albumen and also can detect the proteic transcription product of IFL.
The sudden change that detects the IFL gene also can be used for the disease of diagnosing IFL albumen relevant.The form of IFL protein mutation comprises that the point mutation compared with normal wild type IFL dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of IFL prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance inMan (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or 3.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1186 bases, and its open reading frame is positioned at the 46-669 position, and the coding total length is 207 amino acid whose people IFL albumen (SEQ ID NO:2).Wherein, amino acid/11-27 is a signal peptide.Sophisticated peptide molecule is formed (SEQID NO:2) by 180 amino-acid residues, is a novel cytokine that does not appear in the newspapers, and belongs to Interferon, rabbit family.IFL of the present invention provides new treatment approach for diseases such as treatment tumour, virus infection, immunologic hypofunctions, thereby has great application prospect.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of new people IFL albumen cDNA
Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poiy (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is inserted into cDNA fragment orientation on the multiple clone site of carrier with SuperScript II clone's test kit (available from Gibco), transforms DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as new people IFL albumen, the people IFL protein gene that its encoding gene called after is new.
Sequence SEQ ID NO:1 total length is 1186bp (SEQ ID NO:1 and Fig. 1), comprises 3 ' end non-coding region of 5 of 45bp ' end non-coding region and 517bp, and coding contains 207 amino acid whose full-length polypeptides (SEQ ID NO:2 and Fig. 2).
Contain the signal peptide of being made up of 27 amino acid according to Kyte-Doolitte hydrophobicity analysis and GCG software prediction N end, this shows that new people IFL albumen is secretory protein.Sophisticated new people IFL albumen is formed (SEQ ID NO:3 and Fig. 2) by 180 amino acid, and calculating not in theory, the molecular weight of glycosylated ripe molecule is 25kD.
They are different with known for the BLAST analysis revealed, and protein level and humanIFN-/β tool homology especially has 36% consistence with IFN-β, 45% similarity (Fig. 3), and this prompting IFL is a kind of new interferon molecule.Embodiment 2: with the new proteic encoding sequence of people IFL of RT-PCR method clone
Be in the total RNA of logarithmic phase person monocytic cell strain THP-1 with Trizol (Gibco company) extraction, get 6mg cell total rna and 0.5 μ g Oligo-dT
12-
18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing
2O dilutes.The used primer of the people IFL albumen that pcr amplification is new is as follows: adopted primer is arranged: 5 ' (SEQ ID NO:4), and antisense primer 5 ' (SEQ ID NO:5).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 1-1160bp shown in the SEQ ID NO:1 are identical.Recombinant expressed and active detection of the people IFL albumen eucaryon that embodiment 3 is new
Used carrier for expression of eukaryon pcDNA3.1/neo (Invitrogen company) in the structure.It contains CMV promotor, SV40 replication origin, neomycin resistance gene, can carry out the transient expression and the stably express of goal gene.The two ends in exogenous gene cloning site are respectively the poiyA signals that contains T7 promotor and Trobest BGH, and available T7 and BGH universal primer directly check order to main foreign gene.Pcr amplification product with embodiment 2 is a template, the performing PCR amplifying target genes.Amplimer is as follows:
Upstream primer: gGAATTCCATGAGCACCAAACCTGAT (SEQ ID NO:6)
Downstream primer: gGGATCCACAA TCTCTGTTAA TTCTT (SEQ ID NO:7)
The PCR product behind EcoR I and the BamH I double digestion, directly is connected into the carrier for expression of eukaryon pcDNA3.1/Myc-His (-) that same enzyme is cut behind column purification, its gene order is through T7 and BGH primer order-checking conclusive evidence.Recombinant plasmid dna is mixed by a certain percentage room temperature effect 45 minutes with liposome; 293 cells are washed twice back and are mixed with DNA, collect culture supernatant in transfection 48-72 hour and carry out the activity detection.
Adopt micro-50% cytopathic-effect inhibition assay, earlier sample was carried out doubling dilution by 1: 2, get 100ul respectively and add 96 hole trace detection plates, each extent of dilution is established three multiple holes, is standard substance with humanIFN-(Sigma).Establish cell contrast and virus control hole simultaneously; It is 1.5-2.0 * 10 that every hole adds concentration
5Human fibroblasts's strain Wish cell suspension 100ul of/ml, in 37 ℃, 5%CO
2The middle cultivation after 24 hours abandoned supernatant, and except that the cell contrast, every hole adds the VCV (50-100TCID of 100ul
50), continue to cultivate 24-48 hour.When the cell well-grown of cell control group, and the virus control group overwhelming majority is observed also record result when dead.
The result shows, can detect the interferon activity of 10-24U/ml in the cells and supernatant of IFL gene transfection.The proteic recombinant expressed and purifying of people IFL that embodiment 4 is new
In this embodiment, be template with the pcr amplification product among the embodiment 2, increase with the PCR Oligonucleolide primers of 5 ' and 3 following ' end of sequence, obtain people IFL DNA as inserting fragment.
5 ' end Oligonucleolide primers sequence of using in the PCR reaction is:
5′-CGGGATCC-CTG?GAC?TGTAACTTACTG-3′(SEQ?ID?NO:8)
This primer contains the restriction enzyme site of BamH I restriction enzyme, is the part encoding sequence that is begun by ripe first amino acid of molecule of coding IFL after this restriction enzyme site
3 ' end primer sequence is:
5′-gGAATTCTTAT?TTCCTCCTGA?ATAGA-3′(SEQ?ID?NO:9)，
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people IFL of EcoR I restriction enzyme.
People IFL albumen cDNA PCR product purification after BamH I/EcoR I enzyme cut and recombinate according to a conventional method with plasmid pUC18 again and be converted into the competence bacillus coli DH 5 alpha, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The people IFL albumen cDNA BamH I/EcoR I endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia company), forms carrier pGEX-2T-IFL, transform people DH5 α then.
Choosing the positive DH5 α clone who expresses IFL is inoculated in 100ml2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mM IPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na
2HPO
4, 1.8mM KH
2PO
4PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10 mM gsh, 50 mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeats wash-out 2-3 time, and obtain N and hold the people IFL fusion rotein that contains GST, obtain IFL albumen after thrombin (Sigma) enzyme cuts except that GST, molecular weight is about 25kD.
Carry out the n terminal amino acid sequential analysis with the Edams hydrolysis method, confirm that its N terminal sequence conforms to the N terminal sequence shown in the SEQ ID NO:3.Embodiment 5 resists the generation of new people IFL protein antibodies
The new people IFL albumen of reorganization that obtains among the embodiment 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of the new people IFL gene translation product of external precipitation with it.Found that antibody can combine with albumen of the present invention specifically.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table (1) general information: (ⅱ) denomination of invention: new interferon-like protein, its encoding sequence and purposes (ⅲ) sequence number: the information of 8 (2) SEQ ID NO:1: (ⅰ) sequence signature:
(A) length: 1186bp
(B) type: nucleic acid
(C) chain: two strands
( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO:1:aggccctcga cccacgcgtc cgggattttt tagcttgcaa aaaaaATGAG CACCAAACCT 60GATATGATTC AAAAGTGTTT GTGGCTTGAG ATCCTTATGG GTATATTCAT TGCTGGCACC 120CTATCCCTGG ACTGTAACTT ACTGAACGTT CACCTGAGAA GAGTCACCTG GCAAAATCTG 180AGACATCTGA GTAGTATGAG CAATTCATTT CCTGTAGAAT GTCTACGAGA AAACATAGCT 240TTTGAGTTGC CCCAAGAGTT TCTGCAATAC ACCCAACCTA TGAAGAGGGA CATCAAGAAG 300GCCTTCTATG AAATGTCCCT ACAGGCCTTC AACATCTTCA GCCAACACAC CTTCAAATAT 360TGGAAAGAGA GACACCTCAA ACAAATCCAA ATAGGACTTG ATCAGCAAGC AGAGTACCTG 420AACCAATGCT TGGAGGAAGA CGAGAATGAA AATGAAGACA TGAAAGAAAT GAAAGAGAAT 480GAGATGAAAC CCTCAGAAGC CAGGGTCCCC CAGCTGAGCA GCCTGGAACT GAGGAGATAT 540TTCCACAGGA TAGACAATTT CCTGAAAGAA AAGAAATACA GTGACTGTGC CTGGGAGATT 600GTCCGAGTGG AAATCAGAAG ATGTTTGTAT TACTTTTACA AATTTACAGC TCTATTCAGG 660AGGAAATAAg aatcatctac cttcaagcaa gaattaacag agattgtggc tacgcaaatg 720caccaaaaaa gggtgaaata tatctgaaat gtacctggtt ctgcccttgg aagccacttc 780ctgctcatgc cactaacagc atgctgccaa actgttcaga ttcaagatta ttccaagcgc 840agggcccaaa tgttatagcc aaagaaagtc ttatgataaa agtgaggcaa atttcagcca 900agaagttaga agagatgttt aaaagaacaa gaacaaattg tggatcatgg tatatgcagg 960ctatcagcag aaggatcaga caataaaatg agttagtgca aaccatttag taaaaataac 1020tatcagcaga gttgttccag attaaaaata gtactacaag cttgtaaagg agttaggaca 1080tgcaagctac tgagcataaa atatatactt gctatttttc atgactttct ctaataaagt 1140ctttgactgt tctctctaat aaaaaaaaaa aaaaaaaaaa aaaaaa 1186 ( 2 ) SEQ ID NO:2： ( ⅰ ) ：
(A) length: 207 amino acid
(B) type: amino acid
(D) topological structure: linear (ⅱ) molecule type: polypeptide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:MSTKPDMIQK CLWLEILMGI FIAGTLSLDC NLLNVHLRRV TWQNLRHLSS 50MSNSFPVECL RENIAFELPQ EFLQYTQPMK RDIKKAFYEM SLQAFNIFSQ 100HTFKYWKERH LKQIQIGLDQ QAEYLNQCLE EDENENEDMK EMKENEMKPS 150EARVPQLSSL ELRRYFHRID NFLKEKKYSD CAWEIVRVEI RRCLYYFYKF 200TALFRRK 207 (2) SEQ ID NO:3: (ⅰ) sequence signature:
(A) length: 180 amino acid
(B) type: amino acid
(D) topological structure: linearity, (ⅱ) molecule type: polypeptide, (ⅹ ⅰ) sequence description: SEQ ID NO:3:LDCNLLNVHL RRVTWQNLRH LSSMSNSFPV ECLRENIAFE LPQEFLQYTQ 50PMKRDIKKAF YEMSLQAFNI FSQHTFKYWK ERHLKQIQIG LDQQAEYLNQ 100CLEEDENENE DMKEMKENEM KPSEARVPQL SSLELRRYFH RIDNFLKEKK 150YSDCAWEIVR VEIRRCLYYF YKFTALFRRK 180, (2) information of SEQ ID NO:4, (ⅰ) sequence feature
(A) length: 30 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:AGGCCCTCGA CCCACGCGTC CGGGATTTTT 30 (2) SEQ ID NO:5
(A) length: 28 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:5:ATTAGAGAGA ACAGTCAAAG ACTTTATT 28 (2) SEQ ID NO:6
(A) length: 26 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:6:gGAATTCCAT GAGCACCAAA CCTGAT 26 (2) SEQ ID NO:7
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:7:gGGATCCACA ATCTCTGTTA ATTCTT 26 (2) SEQ ID NO:8
(A) length: 26 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:8:CGGGATCCCT GGACTGTAAC TTACTG 26 (2) SEQ ID NO:9
(A) length: 26 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: SEQ ID NO:9:gGAATTCTTAT TTCCTCCTGA ATAGA 26
Claims (14)
1. an isolating people IFL polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 or 3 aminoacid sequences.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 or 3 aminoacid sequences.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:
(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or 3.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) has the sequence of 46-669 position among the SEQ ID NO:1;
(b) has the sequence of 127-669 position among the SEQ ID NO:1;
(c) has the sequence of 1-1186 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. preparation method with polypeptide of people IFL protein-active is characterized in that this method comprises:
(a) under the proteic condition of suitable expressing human IFL, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with people IFL protein-active.
9. energy and the described people IFL of claim 1 protein-specific bonded antibody.
10. nucleic acid molecule, it contains a successive 10-1186 Nucleotide in the described polynucleotide of claim 3.
11. whether there is the proteic method of IFL in the test sample, it is characterized in that, comprising:
The described antibody of sample and claim 9 is contacted,
Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample IFL albumen.
12. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people IFL protein-active, and perhaps screening suppresses the antagonist of people IFL protein-active or is used to the peptide finger print identification.
13. the purposes of a nucleic acid molecule as claimed in claim 10 is characterized in that, it is used as primer and is used for pcr amplification reaction, perhaps is used for hybridization as probe, perhaps is used to make gene chip or microarray.
14. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991242726A CN1171901C (en) | 1999-12-16 | 1999-12-16 | Novel interferon-like protein, its coding sequence and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991242726A CN1171901C (en) | 1999-12-16 | 1999-12-16 | Novel interferon-like protein, its coding sequence and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1299831A true CN1299831A (en) | 2001-06-20 |
| CN1171901C CN1171901C (en) | 2004-10-20 |
Family
ID=5283356
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991242726A Expired - Lifetime CN1171901C (en) | 1999-12-16 | 1999-12-16 | Novel interferon-like protein, its coding sequence and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1171901C (en) |
-
1999
- 1999-12-16 CN CNB991242726A patent/CN1171901C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CN1171901C (en) | 2004-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1477117A (en) | Novel human phosphotidylethanolamine binding protein, its coding sequence and application | |
| CN1223607C (en) | Human calcium ion/calmodulin dependent protein kinase II inhibitory protein and its application | |
| CN1371916A (en) | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use | |
| CN1343725A (en) | Human angiogenin-like protein and coding sequence and application thereof | |
| CN1171901C (en) | Novel interferon-like protein, its coding sequence and use | |
| CN1477118A (en) | Novel DnaJ/Hsp40 molecule DC-DJIII, its coding sequence and application | |
| CN1299827A (en) | New human CD20 homolgous molecule and its code sequence and use | |
| CN100408597C (en) | Human calcium ion / calmodulin deopendent protein kinase II inhibitory protein alpha, its coded sequence and use | |
| CN1385441A (en) | Novel human lymphokine, its coding sequence and use | |
| CN1299823A (en) | New human cell factor and its code sequence and use | |
| CN1475502A (en) | New withered induced protein, its coded sequence and use | |
| CN1325874A (en) | Human C-type lectin-like receptor and its coding sequence and application | |
| CN1477193A (en) | Novel human Rab GTP enzyme, its coding sequence and application | |
| CN1299828A (en) | New cell factor acceptor and its code sequence and use | |
| CN1477119A (en) | Novel Human cysteine proteinase inhibitor sample molecule, its coding sequence and application | |
| CN1208344C (en) | Novel human cell endocytic regulatory protein, its coding sequence and use | |
| CN1477115A (en) | Novel human cyclin, its coding sequence and application | |
| CN1475500A (en) | New type human marrow differentiation tagged object its coded sequence and use | |
| CN1475501A (en) | New type human bone marrow substrate cell source 1 type phosphoric acid enzyme inhibition factor, its coded sequence and use | |
| CN1477116A (en) | Novel human secretion-related membrane protion, its coding sequence and its application | |
| CN1299832A (en) | New human cell cycle regulating protein and its code sequence and use | |
| CN1475568A (en) | New type human death resist protein, its coded sequence and use | |
| CN1238379C (en) | New osteoclast formation inhibiting factor and its coding sequence and use | |
| CN1477114A (en) | Immunoglobulin sample receptor originated from human dendritic cell, its coding sequence and application | |
| CN1377891A (en) | Zinc finger protein from dendritic cell, its coding sequence and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SECOND MILITARY MEDICAL UNIVERSITY, PLA Free format text: FORMER OWNER: HUACHEN BIOLOGICAL TECHNOLOGY INST., SHANGHAI Effective date: 20041210 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20041210 Address after: 200433 No. 800 Xiang Yin Road, Shanghai Patentee after: Army Medical Univ. No.2, Chinese PLA Address before: 200433 No. 6, building 1, foundation department, No. 800 Xiang Yin Road, Shanghai Patentee before: Huachen Biological Technology Inst., Shanghai |
|
| CX01 | Expiry of patent term |
Granted publication date: 20041020 |
|
| CX01 | Expiry of patent term |