CN1493595A - Polypeptide-human tumour protein 20 and polynucleotide for coding said polypeptide - Google Patents
Polypeptide-human tumour protein 20 and polynucleotide for coding said polypeptide Download PDFInfo
- Publication number
- CN1493595A CN1493595A CNA021377324A CN02137732A CN1493595A CN 1493595 A CN1493595 A CN 1493595A CN A021377324 A CNA021377324 A CN A021377324A CN 02137732 A CN02137732 A CN 02137732A CN 1493595 A CN1493595 A CN 1493595A
- Authority
- CN
- China
- Prior art keywords
- polynucleotide
- polypeptide
- sequence
- human tumour
- white
- 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.)
- Pending
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 85
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 83
- 108091033319 polynucleotide Proteins 0.000 title claims abstract description 80
- 102000040430 polynucleotide Human genes 0.000 title claims abstract description 80
- 239000002157 polynucleotide Substances 0.000 title claims abstract description 80
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 77
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 76
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 76
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 claims description 65
- 101710089165 Protein white Proteins 0.000 claims description 49
- 235000018102 proteins Nutrition 0.000 claims description 38
- 239000012634 fragment Substances 0.000 claims description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 13
- 108091008819 oncoproteins Proteins 0.000 claims description 13
- 102000027450 oncoproteins Human genes 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 3
- 238000013459 approach Methods 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 17
- 201000010099 disease Diseases 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 11
- 239000005557 antagonist Substances 0.000 abstract description 10
- 230000009471 action Effects 0.000 abstract description 2
- 201000011510 cancer Diseases 0.000 abstract description 2
- 238000012270 DNA recombination Methods 0.000 abstract 1
- 208000031886 HIV Infections Diseases 0.000 abstract 1
- 208000037357 HIV infectious disease Diseases 0.000 abstract 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 45
- 108020004414 DNA Proteins 0.000 description 36
- 239000002299 complementary DNA Substances 0.000 description 28
- 238000005516 engineering process Methods 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 20
- 239000000047 product Substances 0.000 description 20
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 description 17
- 101150078498 MYB gene Proteins 0.000 description 15
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 15
- 125000003729 nucleotide group Chemical group 0.000 description 15
- 238000009396 hybridization Methods 0.000 description 13
- 238000003752 polymerase chain reaction Methods 0.000 description 13
- 239000000523 sample Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 108010087776 Proto-Oncogene Proteins c-myb Proteins 0.000 description 7
- 102000009096 Proto-Oncogene Proteins c-myb Human genes 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 108700021654 myb Genes Proteins 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 230000008034 disappearance Effects 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 241000282326 Felis catus Species 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000008827 biological function Effects 0.000 description 5
- 230000002759 chromosomal effect Effects 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 230000008521 reorganization Effects 0.000 description 5
- 102000000584 Calmodulin Human genes 0.000 description 4
- 108010041952 Calmodulin Proteins 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 4
- 108090000994 Catalytic RNA Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000007901 in situ hybridization Methods 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 108091092562 ribozyme Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 206010027336 Menstruation delayed Diseases 0.000 description 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 108020005091 Replication Origin Proteins 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 108060003552 hemocyanin Proteins 0.000 description 3
- 210000003917 human chromosome Anatomy 0.000 description 3
- 210000004754 hybrid cell Anatomy 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 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
- 241000196324 Embryophyta Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 241000287828 Gallus gallus Species 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
- 102100034711 Myb-related protein A Human genes 0.000 description 2
- 101710115158 Myb-related protein A Proteins 0.000 description 2
- 102100034670 Myb-related protein B Human genes 0.000 description 2
- 101710115153 Myb-related protein B Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 102220369446 c.1274G>A Human genes 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000014107 chromosome localization Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003053 immunization Effects 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
- 239000002502 liposome Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 230000002969 morbid Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 102220004457 rs11567847 Human genes 0.000 description 2
- 102220023257 rs387907546 Human genes 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 230000017105 transposition Effects 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 230000004304 visual acuity Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 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
- 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
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000053602 DNA Human genes 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
- 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
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000305071 Enterobacterales Species 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
- 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 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
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710147844 Myb protein Proteins 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
- 229930193140 Neomycin Natural products 0.000 description 1
- 108010054076 Oncogene Proteins v-myb Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- BFDMCHRDSYTOLE-UHFFFAOYSA-N SC#N.NC(N)=N.ClC(Cl)Cl.OC1=CC=CC=C1 Chemical compound SC#N.NC(N)=N.ClC(Cl)Cl.OC1=CC=CC=C1 BFDMCHRDSYTOLE-UHFFFAOYSA-N 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 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
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000022131 cell cycle Effects 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
- 230000001143 conditioned effect Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 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
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development 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
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 125000000487 histidyl group Chemical class [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001114 immunoprecipitation 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
- 238000002513 implantation Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 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
- 238000002955 isolation 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
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 238000010841 mRNA extraction Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000001167 myeloblast Anatomy 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 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
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000014621 translational initiation 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
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 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
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
A novel polypeptide-human tumor protein 20, the polynucleotide for coding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide in treating diseases, such as cancer, HIV infection, immunopathy, etc. the antagonist of said polypeptide and its treating action, and the application of said polynucleotide are disclosed.
Description
Technical field
The invention belongs to biological technical field, specifically, the invention describes a kind of polypeptide-human oncoprotein 20, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.
Background technology
The growth of cell, differentiation and propagation have constituted the important step of cell life cycle, and wherein any one link goes wrong and all can cause the variation of cells survival state, thereby influence the health of whole machine body.Therefore, seem at this gene that wherein plays regulating effect in any link and be even more important.The myb gene all plays regulating and controlling effect in the growth of cell, differentiation, death, be a crucial gene.
The myb gene is the oncogene v-myb that finds in chicken the earliest, and its corresponding gene in normal cell is c-myb.Several new myb genes had been found afterwards again successively, A-myb and B-myb.They all comprise three similar structures: the DNA calmodulin binding domain CaM of a N end, one is positioned at the transcriptional activity structure at center and the pair regulation and control zone of a C-terminal.
The structure similar to the myb gene all has discovery in many biologies, for example in plant, yeast and mould.Generally expressing in proliferating cells of myb gene is higher.And in the cell that breaks up end and withdraw from from the cell cycle, the myb gene is in closing condition.B-myb expresses in sophisticated whole process at the zygote implantation, and that C-myb expresses in hemocytopoietic organ, unstriated muscle and mucous membrane of rectum is higher, wide expression in the mitotically active tissue of A-myb when fetal development, but after birth, just only in lymph and germinal tissue, expressed.
The myb gene also plays an important role in the adjusting of cytodifferentiation.C-Myb is regulated by negative in hematopoietic cell differentiation and sophisticated process.If c-Myb overexpression then be suppressed at the atomization of immature marrow and erythron.The possible mechanism that c-Myb regulates cytodifferentiation is: c-Myb regulates the gene group of a complexity, and this gene group plays important regulating effect again in cell differentiation procedure then.
Except the effect in cell proliferation and differentiation, Myb albumen still is medullary cell and the existence of T cell in growth course and dead regulatory factor.Discover, in the chicken myeloblast, close the Myb-Ets fusion rotein and can cause necrocytosis.Overexpression c-Myb albumen can protect the CTLL-2T cell not because of iuntercellular Jie element apoptosis, and in the T cell, c-Myb has the effect of important anti-apoptotic at least in this explanation.
The proteic overexpression of Myb can make the excessive propagation of cell, suppresses cytodifferentiation and death, produces tumour, for example lymphoma, leukemia etc.In the hematopoietic cell of liver, the c-Myb overexpression can cause liver cancer.In addition, the meeting on the low side of the disappearance of Myb or expression level causes cell proliferation rate to reduce, and produces various disorder of development, also can cause cell resistance to be subjected to ectocine in addition and dead surge capability reduces.
The based composition of the proteic DNA calmodulin binding domain CaM of sMyb is very conservative, in all Myb albumen discovery is arranged all, and its structure is:
Its conserved sequence is: W-[ST]-x (2)-E-[DE]-x (2)-[LIV]
Polypeptide of the present invention contains the conservative characteristic structure of Myb albumen, and the analysis by express spectra is found, polypeptide expression of the present invention illustrates that than the expression amount height in normal liver tissue it and liver cancer have confidential relation in liver cancer tissue, and is consistent with the function of Myb.In sum, polypeptide of the present invention is a kind of new Myb albumen, has similar biological function, and name is people Myb protein 20.
Because white 20 albumen of human tumour protein play an important role in the body critical function as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify white 20 albumen of human tumour protein of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new person's oncoprotein 20 protein coding genes also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.
Summary of the invention
An object of the present invention is to provide isolating new polypeptide-human oncoprotein 20 with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of this polypeptide of coding.
Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the human tumour protein white 20 of encoding.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the human tumour protein white 20 of encoding.
Another object of the present invention provides the method for producing human tumour protein white 20.
Another object of the present invention provides the antibody at polypeptide-human oncoprotein 20 of the present invention.
The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: have＜polypeptide or its examples of conservative variations, bioactive fragment or the derivative of 210〉2 aminoacid sequences.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.
The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:
(a) coding has＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences;
(b) with polynucleotide (a) complementary polynucleotide;
(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 70% homogeny.
More preferably, the sequence of these polynucleotide is be selected from down group a kind of: (a) have＜210〉1 in the sequence of 137-1564 position; (b) have＜210〉1 in the sequence of 1-2381 position.
The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.
The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
The invention provides a kind of polypeptide-human oncoprotein 20, it is basically by＜210〉aminoacid sequence shown in 2 forms.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology 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 glycosylated, 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 analogue of human tumour protein white 20.As used herein, term " fragment ", " derivative " and " analogue " are meant biological function or the active polypeptide that keeps human tumour protein of the present invention white 20 identical basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.The invention provides isolating nucleic acid (polynucleotide), substantially by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.Polynucleotide sequence of the present invention comprises＜210〉1 nucleotide sequence.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 846 bases, its open reading frame (58-594) 178 amino acid of having encoded.This polypeptide has Myb DNA calmodulin binding domain CaM and repeats pulsating characteristic sequence, and deducibility goes out this human tumour protein white 20 and has the 26S Proteasome Structure and Function that Myb DNA calmodulin binding domain CaM repeats the segment representative.
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＜210〉1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has＜210〉2 protein or polypeptide, but with the differentiated nucleotide sequence of coding region sequence shown in＜210〉1.
The polynucleotide of the mature polypeptide of coding＜210〉2 comprise: the encoding sequence that has only 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 " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, 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 the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.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 adds and to use 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 95%, be more preferably 97% and just hybridize when above.And, the polypeptide of interfertile polynucleotide encoding and＜210〉and the mature polypeptide shown in 2 has identical biological function and activity.
The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding human tumour protein white 20.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding human tumour protein white 20 of the present invention can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.
Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.
In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of the transcript of mensuration human tumour protein white 20; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.
In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.
In (4) kind method, detect the protein product of white 20 genetic expressions of human tumour protein and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to polynucleotide 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 gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
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 the carrier of the present invention or oncoprotein 20 encoding sequences of directly choosing, and the method that produces polypeptide of the present invention through recombinant technology.
Among the present invention, the polynucleotide sequence of coding human tumour protein white 20 can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " 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 carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor 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) with 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 may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains the encode dna sequence dna of human tumour protein white 20 and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, 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; The P of lambda particles phage
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte 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 that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, 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.
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 etc.
Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.
Among the present invention, the polynucleotide of coding human tumour protein white 20 or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to 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; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.
Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.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.Alternative 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, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.
By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce white 20 (Science, 1984 of human tumour protein of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of everybody oncoprotein 20 of coding of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). in suitable medium, cultivate host cell;
(3). separation, protein purification from substratum or cell.
In step (2), 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.
In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular 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.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, 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 present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) human tumour protein white 20.Agonist improves human tumour protein white 20 biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can in the presence of medicine, the film preparation of mammalian cell or expressing human oncoprotein 20 be cultivated with the human tumour protein white 20 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of human tumour protein white 20 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of human tumour protein white 20 can combine and eliminate its function with human tumour protein white 20, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.
In screening during as the compound of antagonist, human tumour protein white 20 can be added during bioanalysiss measure, determine to interactional influence between human tumour protein white 20 and its acceptor whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.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 white 20 bonded peptide molecules of human tumour protein obtains.During screening, generally tackle white 20 molecules of human tumour protein and carry out mark.
The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody at white 20 antigenic determinants of human tumour protein.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
The method of the white 20 direct injection immune animals of the available human tumour protein of the production of polyclonal antibody (as rabbit, mouse, rat etc.) obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of monoclonal antibody of preparation human tumour protein white 20 include but not limited to hybridoma technology (Kohler andMilstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody of anti-human tumour protein white 20.
The antibody of anti-human tumour protein white 20 can be used in the immunohistochemistry technology, detects the human tumour protein white 20 in the biopsy specimen.
With the also available labelled with radioisotope of the white 20 bonded monoclonal antibodies of human tumour protein, 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 also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of white 20 high-affinities of human tumour protein 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 white 20 positive cells of human tumour protein.
The disease that antibody among the present invention can be used for treating or prevention and human tumour protein white 20 are relevant.The antibody that gives suitable dosage can stimulate or block the generation or the activity of human tumour protein white 20.
The invention still further relates to the diagnostic testing process of quantitative and white 20 levels of detection and localization human tumour protein.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.White 20 levels of the human tumour protein that is detected in the test can and be used to the disease of diagnosing human tumour protein white 20 to work with the importance of the human tumour protein that lays down a definition white 20 in various diseases.
Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.
The polynucleotide of coding human tumour protein white 20 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 nothing expression of human tumour protein white 20 or the unusual/non-activity expression.It is white 20 that the gene therapy vector (as virus vector) of reorganization can be designed for the human tumour protein of expressing variation, to suppress white 20 activity of endogenic human tumour protein.For example, a kind of human tumour protein of variation white 20 can be that the human tumour protein that shortens, lacked signal conduction function territory is white 20, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of white 20 expression of human tumour protein or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the polynucleotide of coding human tumour protein white 20 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding human tumour protein white 20 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding human tumour protein white 20 can be packaged in the liposome and be transferred in the cell in addition.
Polynucleotide import 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.
Suppress the oligonucleotide (comprising sense-rna and DNA) of the white 20mRNA of human tumour protein 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.
The polynucleotide of coding human tumour protein white 20 can be used for the diagnosis with the relative disease of human tumour protein white 20.The unconventionality expression of the expression that the polynucleotide of coding human tumour protein white 20 can be used for detecting human tumour protein white 20 people's oncoprotein 20 whether or under morbid state.As the dna sequence dna of the human tumour protein of encoding white 20 can be used for biopsy specimen is hybridized to judge the expression situation of human tumour protein white 20.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.Personnel selection oncoprotein 20 special primers carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect human tumour protein white 20.
The sudden change that detects white 20 genes of human tumour protein also can be used for diagnosing the relevant disease of human tumour protein white 20.The form of white 20 sudden changes of human tumour protein comprises that the point mutation compared with white 20 dna sequence dnas of normal wild type human tumour protein, 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).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.
In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.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.
The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).
The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).
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 Inheritancein Man (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.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Human tumour protein white 20 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's human tumour protein white 20 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
Description of drawings
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 polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating human tumour protein white 20.20kDa is proteinic molecular weight.The arrow indication is isolated protein band.
Embodiment
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 Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of human tumour protein white 20
Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0417h12 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 0417h12 clone is 846bp (as＜210〉1 shown in), from 58bp to 594bp the open reading frame (ORF) of a 846bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-0417h12, encoded protein matter called after human tumour protein white 20.
Embodiment 2: with the gene of RT-PCR method clones coding human tumour protein white 20
Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:
Primer1：5’-GGAGTGACCCGGCGTGGCTACTAGG-3’(<210>3)
Primer2：5’-CCAAGTAAGTGATTTTATTATTATC-3’(<210>4)
Primer1 for to be positioned at＜the forward sequence that begins of 1bp of 210〉15 ' end;
Primer2 be＜210〉1 in 3 ' end reverse sequence.
The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl
2, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows the dna sequence dna and＜210 of PCR product〉1-846bp shown in 1 is identical.
Embodiment 3:Northern blotting analyst oncoprotein 20 expression of gene:
Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α-
32P dATP prepares by random priming
32The dna probe of P-mark.Used dna probe is white 20 coding region sequences (58bp to 594bp) of the human tumour protein of pcr amplification.Probe (about 2 * 10 with the 32P-mark
6Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH
2PO
4(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.
Embodiment 4: the vivoexpression of recombination human tumor protein 20, separation and purifying
According to＜210〉1 and coding region sequence, design a pair of specificity amplification primer, sequence is as follows:
Primer3：5’-CCCCATATGATGTCGAGTTTCTCTAGGGCGCCCC-3’(<210>5)
Primer4：5’-CCCAAGCTTTTATAGCCGATAATCTTCAGGTGGA-3’(<210>6)
5 ' end of these two sections primers contains NdeI and HindIII restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NdeI and HindIII restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0417h12 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0417h12 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantagepolymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with NdeI and HindIII, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0417h12) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0417h12) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein human tumour protein white 20 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 1) at the 20kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.
White 20 production of antibodies of embodiment 5 anti-human tumour proteins
Synthesize following human tumour protein white 20 specific polypeptide with Peptide synthesizer (PE company product): NH2-Met-Ser-Ser-Phe-Ser-Arg-Ala-Pro-Gln-Gln-Trp-Ala-Thr-Phe-Ala-COOH
(SEQ?ID?NO：7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with human tumour protein white 20 specifically.
Sequence table
＜110〉Bode Gene Development Co., Ltd., Shanghai
The polynucleotide of＜a 120〉peptide species--human tumour protein white 20 and this peptide species of coding
<130>0417h12
<160>6
<170>PatentIn?version3.1
<210>1
<211>660
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(58)..(594)
<223>
<400>1
ggagtgaccc?ggcgtggcta?ctaggagaag?gacgtacggt?cctgctagta?gaggaat 57
atg?tcg?agt?ttc?tct?agg?gcg?ccc?cag?caa?tgg?gcc?act?ttt?gct?aga 105
Met?Ser?Ser?Phe?Ser?Arg?Ala?Pro?Gln?Gln?Trp?Ala?Thr?Phe?Ala?Arg
1 5 10 15
ata?tgg?tat?ctc?tta?gat?ggg?aaa?atg?cag?cca?cct?ggc?aaa?ctt?gct 153
Ile?Trp?Tyr?Leu?Leu?Asp?Gly?Lys?Met?Gln?Pro?Pro?Gly?Lys?Leu?Ala
20 25 30
gct?atg?gca?tct?ata?aga?ctt?cag?gga?tta?cat?aaa?cct?gtg?tac?cat 201
Ala?Met?Ala?Ser?Ile?Arg?Leu?Gln?Gly?Leu?His?Lys?Pro?Val?Tyr?His
35 40 45
gca?ctg?agt?gac?tgt?ggg?gat?cat?gtt?gtt?ata?atg?aac?aca?aga?cac 249
Ala?Leu?Ser?Asp?Cys?Gly?Asp?His?Val?Val?Ile?Met?Asn?Thr?Arg?His
50 55 60
att?gca?ttt?tct?gga?aac?aaa?tgg?gaa?caa?aaa?gta?tac?tct?tcg?cat 297
Ile?Ala?Phe?Ser?Gly?Asn?Lys?Trp?Glu?Gln?Lys?Val?Tyr?Ser?Ser?His
65 70 75 80
act?ggc?tac?cca?ggt?gga?ttt?aga?caa?gta?aca?gct?gct?cag?ctt?cac 345
Thr?Gly?Tyr?Pro?Gly?Gly?Phe?Arg?Gln?Val?Thr?Ala?Ala?Gln?Leu?His
85 90 95
ctg?agg?gat?cca?gtg?gca?att?gta?aaa?cta?gct?att?tat?ggc?atg?ctg 393
Leu?Arg?Asp?Pro?Val?Ala?Ile?Val?Lys?Leu?Ala?Ile?Tyr?Gly?Met?Leu
100 105 110
cca?aaa?aac?ctt?cac?aga?aga?aca?atg?atg?gaa?agg?ttg?cat?ctt?ttt 441
Pro?Lys?Asn?Leu?His?Arg?Arg?Thr?Met?Met?Glu?Arg?Leu?His?Leu?Phe
115 120 125
cca?gat?gag?tat?att?cca?gaa?gat?att?ctt?aag?aat?tta?gta?gag?gag 489
Pro?Asp?Glu?Tyr?Ile?Pro?Glu?Asp?Ile?Leu?Lys?Asn?Leu?Val?Glu?Glu
130 135 140
ctt?cct?caa?cca?cga?aaa?ata?cct?aaa?cgt?cta?gat?gag?tac?aca?caa 537
Leu?Pro?Gln?Pro?Arg?Lys?Ile?Pro?Lys?Arg?Leu?Asp?Glu?Tyr?Thr?Gln
145 150 155 160
gaa?gaa?ata?gac?gcc?ttc?cca?aga?ttg?tgg?act?cca?cct?gaa?gat?tat 585
Glu?Glu?Ile?Asp?Ala?Phe?Pro?Arg?Leu?Trp?Thr?Pro?Pro?Glu?Asp?Tyr
165 170 175
cgg?cta?taa?gagaataaga?attgcagaaa?ataacagtga?agtgattgaa 634
Arg?Leu
actttcttct?gatgagtttc?tctaac 660
<210>2
<211>178
<212>PRT
<213>Homo?sapiens
<400>2
Met?Ser?Ser?Phe?Ser?Arg?Ala?Pro?Gln?Gln?Trp?Ala?Thr?Phe?Ala?Arg
1 5 10 15
Ile?Trp?Tyr?Leu?Leu?Asp?Gly?Lys?Met?Gln?Pro?Pro?Gly?Lys?Leu?Ala
20 25 30
Ala?Met?Ala?Ser?Ile?Arg?Leu?Gln?Gly?Leu?His?Lys?Pro?Val?Tyr?His
35 40 45
Ala?Leu?Ser?Asp?Cys?Gly?Asp?His?Val?Val?Ile?Met?Asn?Thr?Arg?His
50 55 60
Ile?Ala?Phe?Ser?Gly?Asn?Lys?Trp?Glu?Gln?Lys?Val?Tyr?Ser?Ser?His
65 70 75 80
Thr?Gly?Tyr?Pro?Gly?Gly?Phe?Arg?Gln?Val?Thr?Ala?Ala?Gln?Leu?His
85 90 95
Leu?Arg?Asp?Pro?Val?Ala?Ile?Val?Lys?Leu?Ala?Ile?Tyr?Gly?Met?Leu
100 105 110
Pro?Lys?Asn?Leu?His?Arg?Arg?Thr?Met?Met?Glu?Arg?Leu?His?Leu?Phe
115 120 125
Pro?Asp?Glu?Tyr?Ile?Pro?Glu?Asp?Ile?Leu?Lys?Asn?Leu?Val?Glu?Glu
130 135 140
Leu?Pro?Gln?Pro?Arg?Lys?Ile?Pro?Lys?Arg?Leu?Asp?Glu?Tyr?Thr?Gln
145 150 155 160
Glu?Glu?Ile?Asp?Ala?Phe?Pro?Arg?Leu?Trp?Thr?Pro?Pro?Glu?Asp?Tyr
165 170 175
Arg?Leu
<210>3
<211>25
<212>DNA
<213>Homo?sapiens
<400>3
ggagtgaccc?ggcgtggcta?ctagg 25
<210>4
<211>25
<212>DNA
<213>Homo?sapiens
<400>4
ccaagtaagt?gattttatta?ttatc 25
<210>5
<211>34
<212>DNA
<213>Homo?sapiens
<400>5
ccccatatga tgtcgagttt ctctagggcg?cccc 34
<210>6
<211>34
<212>DNA
<213>Homo?sapiens
<400>6
cccaagcttt?tatagccgat?aatcttcagg?tgga 34
Claims (10)
1, a kind of isolated polypeptide-human tumour protein is white 20, it is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉2 or the active fragments of its polypeptide, analogue or derivative.
2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in＜210〉2.
3, polypeptide as claimed in claim 2 is characterized in that it comprises and has＜polypeptide of the aminoacid sequence shown in 210〉2.
4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:
(a) coding have＜210〉2 shown in the polynucleotide of the polypeptide of aminoacid sequence or its fragment, analogue, derivative;
(b) with polynucleotide (a) complementary polynucleotide; Or
(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.
5, polynucleotide as claimed in claim 4, it is characterized in that described polynucleotide comprise coding and have＜210〉2 shown in the polynucleotide of aminoacid sequence.
6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 58-594 position sequence or＜210〉1 in the sequence of 1-846 position.
7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.
8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:
(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or
(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.
9, a kind of preparation method with the white 20 active polypeptide of human tumour protein is characterized in that described method comprises:
(a) under expressing human oncoprotein 20 conditions, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have human tumour protein white 20 active polypeptide.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with the white 20 specificity bonded antibody of human tumour protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021377324A CN1493595A (en) | 2002-10-30 | 2002-10-30 | Polypeptide-human tumour protein 20 and polynucleotide for coding said polypeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021377324A CN1493595A (en) | 2002-10-30 | 2002-10-30 | Polypeptide-human tumour protein 20 and polynucleotide for coding said polypeptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1493595A true CN1493595A (en) | 2004-05-05 |
Family
ID=34231682
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA021377324A Pending CN1493595A (en) | 2002-10-30 | 2002-10-30 | Polypeptide-human tumour protein 20 and polynucleotide for coding said polypeptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1493595A (en) |
-
2002
- 2002-10-30 CN CNA021377324A patent/CN1493595A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1296974A (en) | Polypeptide-human histone H2A.21 and polynucleotide for coding said polypeptide | |
| CN1297902A (en) | Human reticulin 80 as one new kind of polypeptide and polynucleotides encoding this polypeptide | |
| CN1493595A (en) | Polypeptide-human tumour protein 20 and polynucleotide for coding said polypeptide | |
| CN1296975A (en) | Polypeptide-human ribosomal protein L23 and polynucleotide for coding said polypeptide | |
| CN1297907A (en) | Human acetylglactoside transferase 45 as one new kind of polypeptide and polynucleotides encoding this polypeptide | |
| CN1493594A (en) | Polypeptide-human signal peptide secretory protein I 10 and polynucleolide for coding said polypeptide | |
| CN1297917A (en) | Human zinc-finger protein 38 as one new kind of polypeptide and polynucleotides encoding this polypeptide | |
| CN1510051A (en) | Polypeptide-human cytochrome C oxidase COII protein9 and polynucleotide for encoding said polypeptide | |
| CN1519253A (en) | Polypeptide-EDF-1, endothelial cell differentiation factor 14.75 of human and polynucleotide for encoding polypeptide | |
| CN1303933A (en) | Novel polypeptide-human muscle BOP protein 41 and polynucleotide coding said polypeptide | |
| CN1296952A (en) | Polypeptide-complement receptor 222 and polynucleotide for coding said polypeptide | |
| CN1297915A (en) | Human zinc-finger protein 46 as one new kind of polypeptide and polynucleotides encoding this polypeptide | |
| CN1493591A (en) | Polypeptide-human protein synthetic protein L14, 22 and nucleotide for coding said polypeptide | |
| CN1493585A (en) | Polypeptide-human tubulin monomer releasing factor 28 and polynucleotide for coding said polypeptide | |
| CN1493596A (en) | Polypeptide-human programming cell death protein 22 and polynucleotide for coding said polypeptide | |
| CN1493690A (en) | Polypeptide-human protein metabolic enzyme 29 and polynucleotide for coding said polypeptide | |
| CN1477195A (en) | A polypeptide-human methionyl-tRNA synthetase 29 and polynucleotide for coding said polypeptide | |
| CN1303930A (en) | Novel polypeptide-zinc finger protein 57 and polynucleotide coding said polypeptide | |
| CN1493587A (en) | Polypeptide-human protein regulatory protein s9 and polynucleotide for coding said polypeptide | |
| CN1493592A (en) | Polypeptide-respiratory protein 18 and polynucleotide for coding said polypeptide | |
| CN1519250A (en) | Polypeptide-human ring finger protein 27.17 and polynucleotide for encoding polypeptide | |
| CN1510042A (en) | Polypeptide-Human PAX protein 12 and polynucleotide for coding this polypeptide | |
| CN1493588A (en) | Polyepeptide-human transcription regulate and control protein 35 and polynucleotide for coding said polypeptide | |
| CN1477194A (en) | A polypeptide-human transpasase 105 and polynucleotide coding said polypetide | |
| CN1352087A (en) | New polypeptide-human glycoprotein 42 and polynucleotide for encoding such polypeptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |