CN112481303B - Il15ra基因人源化非人动物及其构建方法和应用 - Google Patents
Il15ra基因人源化非人动物及其构建方法和应用 Download PDFInfo
- Publication number
- CN112481303B CN112481303B CN202110173729.9A CN202110173729A CN112481303B CN 112481303 B CN112481303 B CN 112481303B CN 202110173729 A CN202110173729 A CN 202110173729A CN 112481303 B CN112481303 B CN 112481303B
- Authority
- CN
- China
- Prior art keywords
- il15ra
- human
- gene
- seq
- humanized
- 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.)
- Active
Links
- 101001003140 Homo sapiens Interleukin-15 receptor subunit alpha Proteins 0.000 title claims abstract description 244
- 238000010276 construction Methods 0.000 title claims abstract description 20
- 239000002773 nucleotide Substances 0.000 claims abstract description 97
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 97
- 102100020789 Interleukin-15 receptor subunit alpha Human genes 0.000 claims abstract description 92
- 102000052224 human IL15RA Human genes 0.000 claims abstract description 69
- 230000008685 targeting Effects 0.000 claims abstract description 30
- 239000013598 vector Substances 0.000 claims abstract description 27
- 206010061218 Inflammation Diseases 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 13
- 238000012216 screening Methods 0.000 claims abstract description 9
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 238000011160 research Methods 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 50
- 238000000034 method Methods 0.000 claims description 32
- 150000001413 amino acids Chemical class 0.000 claims description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 21
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 18
- 241001465754 Metazoa Species 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 14
- 238000006467 substitution reaction Methods 0.000 claims description 12
- 230000014509 gene expression Effects 0.000 claims description 9
- 230000001105 regulatory effect Effects 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 238000001727 in vivo Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 230000019491 signal transduction Effects 0.000 claims description 6
- 230000000857 drug effect Effects 0.000 claims description 5
- 238000011156 evaluation Methods 0.000 claims description 5
- 150000007523 nucleic acids Chemical group 0.000 claims description 5
- 210000005260 human cell Anatomy 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 230000002829 reductive effect Effects 0.000 claims description 3
- 238000012356 Product development Methods 0.000 claims description 2
- 230000000259 anti-tumor effect Effects 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
- 230000001900 immune effect Effects 0.000 claims description 2
- 230000037189 immune system physiology Effects 0.000 claims description 2
- 230000002906 microbiologic effect Effects 0.000 claims description 2
- 230000000144 pharmacologic effect Effects 0.000 claims description 2
- 238000010200 validation analysis Methods 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 28
- 230000004054 inflammatory process Effects 0.000 abstract description 11
- 230000009466 transformation Effects 0.000 abstract description 4
- 239000002547 new drug Substances 0.000 abstract description 3
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 230000006801 homologous recombination Effects 0.000 abstract 1
- 238000002744 homologous recombination Methods 0.000 abstract 1
- 238000012827 research and development Methods 0.000 abstract 1
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 41
- 241000699666 Mus <mouse, genus> Species 0.000 description 37
- 210000004027 cell Anatomy 0.000 description 35
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 23
- 241000699670 Mus sp. Species 0.000 description 20
- 108700024394 Exon Proteins 0.000 description 18
- 238000003780 insertion Methods 0.000 description 16
- 230000037431 insertion Effects 0.000 description 16
- 238000010171 animal model Methods 0.000 description 15
- 239000012634 fragment Substances 0.000 description 12
- 241000283984 Rodentia Species 0.000 description 11
- 238000005215 recombination Methods 0.000 description 11
- 230000006798 recombination Effects 0.000 description 11
- 108020004999 messenger RNA Proteins 0.000 description 10
- 238000011161 development Methods 0.000 description 9
- 101001019594 Mus musculus Interleukin-15 receptor subunit alpha Proteins 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000011577 humanized mouse model Methods 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000007704 transition Effects 0.000 description 7
- 102000003812 Interleukin-15 Human genes 0.000 description 6
- 108090000172 Interleukin-15 Proteins 0.000 description 6
- 238000002105 Southern blotting Methods 0.000 description 6
- 210000001671 embryonic stem cell Anatomy 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 108010031719 prolyl-serine Proteins 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 241001529936 Murinae Species 0.000 description 5
- 238000012239 gene modification Methods 0.000 description 5
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- GWFSQQNGMPGBEF-GHCJXIJMSA-N Ala-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N GWFSQQNGMPGBEF-GHCJXIJMSA-N 0.000 description 3
- GRIFPSOFWFIICX-GOPGUHFVSA-N Ala-His-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O GRIFPSOFWFIICX-GOPGUHFVSA-N 0.000 description 3
- YVTHEZNOKSAWRW-DCAQKATOSA-N Arg-Lys-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O YVTHEZNOKSAWRW-DCAQKATOSA-N 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- KSMSFCBQBQPFAD-GUBZILKMSA-N Cys-Pro-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 KSMSFCBQBQPFAD-GUBZILKMSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- STHSGOZLFLFGSS-SUSMZKCASA-N Gln-Thr-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STHSGOZLFLFGSS-SUSMZKCASA-N 0.000 description 3
- GCYFUZJHAXJKKE-KKUMJFAQSA-N Glu-Arg-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GCYFUZJHAXJKKE-KKUMJFAQSA-N 0.000 description 3
- FKGNJUCQKXQNRA-NRPADANISA-N Glu-Cys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCC(O)=O FKGNJUCQKXQNRA-NRPADANISA-N 0.000 description 3
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 3
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 3
- PZVGOVRNGKEFCB-KKHAAJSZSA-N Thr-Asn-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N)O PZVGOVRNGKEFCB-KKHAAJSZSA-N 0.000 description 3
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 3
- 108010047495 alanylglycine Proteins 0.000 description 3
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 108010040030 histidinoalanine Proteins 0.000 description 3
- 108040002039 interleukin-15 receptor activity proteins Proteins 0.000 description 3
- 102000008616 interleukin-15 receptor activity proteins Human genes 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 108010077112 prolyl-proline Proteins 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 3
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 2
- OKIKVSXTXVVFDV-MMWGEVLESA-N Ala-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N OKIKVSXTXVVFDV-MMWGEVLESA-N 0.000 description 2
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 2
- AJBVYEYZVYPFCF-CIUDSAMLSA-N Ala-Lys-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O AJBVYEYZVYPFCF-CIUDSAMLSA-N 0.000 description 2
- QDGMZAOSMNGBLP-MRFFXTKBSA-N Ala-Trp-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N QDGMZAOSMNGBLP-MRFFXTKBSA-N 0.000 description 2
- ZEAYJGRKRUBDOB-GARJFASQSA-N Arg-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZEAYJGRKRUBDOB-GARJFASQSA-N 0.000 description 2
- ZZZWQALDSQQBEW-STQMWFEESA-N Arg-Gly-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZZZWQALDSQQBEW-STQMWFEESA-N 0.000 description 2
- BSYKSCBTTQKOJG-GUBZILKMSA-N Arg-Pro-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BSYKSCBTTQKOJG-GUBZILKMSA-N 0.000 description 2
- WLVLIYYBPPONRJ-GCJQMDKQSA-N Asn-Thr-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O WLVLIYYBPPONRJ-GCJQMDKQSA-N 0.000 description 2
- JJQGZGOEDSSHTE-FOHZUACHSA-N Asp-Thr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JJQGZGOEDSSHTE-FOHZUACHSA-N 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 102000016607 Diphtheria Toxin Human genes 0.000 description 2
- 108010053187 Diphtheria Toxin Proteins 0.000 description 2
- LVSYIKGMLRHKME-IUCAKERBSA-N Gln-Gly-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N LVSYIKGMLRHKME-IUCAKERBSA-N 0.000 description 2
- KHNJVFYHIKLUPD-SRVKXCTJSA-N Gln-Leu-Met Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCC(=O)N)N KHNJVFYHIKLUPD-SRVKXCTJSA-N 0.000 description 2
- XXCDTYBVGMPIOA-FXQIFTODSA-N Glu-Asp-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O XXCDTYBVGMPIOA-FXQIFTODSA-N 0.000 description 2
- ZHNHJYYFCGUZNQ-KBIXCLLPSA-N Glu-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O ZHNHJYYFCGUZNQ-KBIXCLLPSA-N 0.000 description 2
- YLEIWGJJBFBFHC-KBPBESRZSA-N Gly-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 YLEIWGJJBFBFHC-KBPBESRZSA-N 0.000 description 2
- NGRPGJGKJMUGDM-XVKPBYJWSA-N Gly-Val-Gln Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O NGRPGJGKJMUGDM-XVKPBYJWSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 2
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 description 2
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- SACHLUOUHCVIKI-GMOBBJLQSA-N Ile-Arg-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N SACHLUOUHCVIKI-GMOBBJLQSA-N 0.000 description 2
- FADXGVVLSPPEQY-GHCJXIJMSA-N Ile-Cys-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N FADXGVVLSPPEQY-GHCJXIJMSA-N 0.000 description 2
- AKOYRLRUFBZOSP-BJDJZHNGSA-N Ile-Lys-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N AKOYRLRUFBZOSP-BJDJZHNGSA-N 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 108010025815 Kanamycin Kinase Proteins 0.000 description 2
- 102000017578 LAG3 Human genes 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 2
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- WXHHTBVYQOSYSL-FXQIFTODSA-N Met-Ala-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O WXHHTBVYQOSYSL-FXQIFTODSA-N 0.000 description 2
- UYAKZHGIPRCGPF-CIUDSAMLSA-N Met-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCSC)N UYAKZHGIPRCGPF-CIUDSAMLSA-N 0.000 description 2
- JPCHYAUKOUGOIB-HJGDQZAQSA-N Met-Glu-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPCHYAUKOUGOIB-HJGDQZAQSA-N 0.000 description 2
- 208000034578 Multiple myelomas Diseases 0.000 description 2
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 2
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 2
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 2
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 description 2
- LXVLKXPFIDDHJG-CIUDSAMLSA-N Pro-Glu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O LXVLKXPFIDDHJG-CIUDSAMLSA-N 0.000 description 2
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 2
- WLJYLAQSUSIQNH-GUBZILKMSA-N Pro-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@@H]1CCCN1 WLJYLAQSUSIQNH-GUBZILKMSA-N 0.000 description 2
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 2
- FDMKYQQYJKYCLV-GUBZILKMSA-N Pro-Pro-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 FDMKYQQYJKYCLV-GUBZILKMSA-N 0.000 description 2
- SXJOPONICMGFCR-DCAQKATOSA-N Pro-Ser-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O SXJOPONICMGFCR-DCAQKATOSA-N 0.000 description 2
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 2
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- -1 SEQ ID NO: 2 amino acid Chemical class 0.000 description 2
- BTPAWKABYQMKKN-LKXGYXEUSA-N Ser-Asp-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BTPAWKABYQMKKN-LKXGYXEUSA-N 0.000 description 2
- BQWCDDAISCPDQV-XHNCKOQMSA-N Ser-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CO)N)C(=O)O BQWCDDAISCPDQV-XHNCKOQMSA-N 0.000 description 2
- HMRAQFJFTOLDKW-GUBZILKMSA-N Ser-His-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O HMRAQFJFTOLDKW-GUBZILKMSA-N 0.000 description 2
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 2
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 2
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 2
- NVNPWELENFJOHH-CIUDSAMLSA-N Ser-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)N NVNPWELENFJOHH-CIUDSAMLSA-N 0.000 description 2
- CUXJENOFJXOSOZ-BIIVOSGPSA-N Ser-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CO)N)C(=O)O CUXJENOFJXOSOZ-BIIVOSGPSA-N 0.000 description 2
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 2
- LKJCABTUFGTPPY-HJGDQZAQSA-N Thr-Pro-Gln Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O LKJCABTUFGTPPY-HJGDQZAQSA-N 0.000 description 2
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 2
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 2
- YXONONCLMLHWJX-SZMVWBNQSA-N Trp-Glu-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O)=CNC2=C1 YXONONCLMLHWJX-SZMVWBNQSA-N 0.000 description 2
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 2
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 2
- RWOKVQUCENPXGE-IHRRRGAJSA-N Tyr-Ser-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RWOKVQUCENPXGE-IHRRRGAJSA-N 0.000 description 2
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 2
- YDPFWRVQHFWBKI-GVXVVHGQSA-N Val-Glu-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N YDPFWRVQHFWBKI-GVXVVHGQSA-N 0.000 description 2
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 2
- SDSCOOZQQGUQFC-GVXVVHGQSA-N Val-His-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N SDSCOOZQQGUQFC-GVXVVHGQSA-N 0.000 description 2
- WANVRBAZGSICCP-SRVKXCTJSA-N Val-Pro-Met Chemical compound CSCC[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C)C(O)=O WANVRBAZGSICCP-SRVKXCTJSA-N 0.000 description 2
- PMKQKNBISAOSRI-XHSDSOJGSA-N Val-Tyr-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N PMKQKNBISAOSRI-XHSDSOJGSA-N 0.000 description 2
- ZMJPCIAEJKVKMQ-UHFFFAOYSA-M [4-[[4-[benzyl(methyl)amino]phenyl]-[4-(dimethylamino)phenyl]methylidene]cyclohexa-2,5-dien-1-ylidene]-dimethylazanium;chloride Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)N(C)CC=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 ZMJPCIAEJKVKMQ-UHFFFAOYSA-M 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000013373 clone screening Methods 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 2
- 108010018006 histidylserine Proteins 0.000 description 2
- 108040006849 interleukin-2 receptor activity proteins Proteins 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 108010016686 methionyl-alanyl-serine Proteins 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 2
- 108010053725 prolylvaline Proteins 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WZUMSFQGYWBRNX-AVGNSLFASA-N (2s)-6-amino-2-[[(2s)-2-[[(2s)-2-[(2-aminoacetyl)amino]-3-hydroxypropanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)CN)CC1=CN=CN1 WZUMSFQGYWBRNX-AVGNSLFASA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- YEELWQSXYBJVSV-UWJYBYFXSA-N Ala-Cys-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YEELWQSXYBJVSV-UWJYBYFXSA-N 0.000 description 1
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 1
- LBFXVAXPDOBRKU-LKTVYLICSA-N Ala-His-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LBFXVAXPDOBRKU-LKTVYLICSA-N 0.000 description 1
- ZBLQIYPCUWZSRZ-QEJZJMRPSA-N Ala-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 ZBLQIYPCUWZSRZ-QEJZJMRPSA-N 0.000 description 1
- YCRAFFCYWOUEOF-DLOVCJGASA-N Ala-Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 YCRAFFCYWOUEOF-DLOVCJGASA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- NHWYNIZWLJYZAG-XVYDVKMFSA-N Ala-Ser-His Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N NHWYNIZWLJYZAG-XVYDVKMFSA-N 0.000 description 1
- NCQMBSJGJMYKCK-ZLUOBGJFSA-N Ala-Ser-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O NCQMBSJGJMYKCK-ZLUOBGJFSA-N 0.000 description 1
- WQKAQKZRDIZYNV-VZFHVOOUSA-N Ala-Ser-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WQKAQKZRDIZYNV-VZFHVOOUSA-N 0.000 description 1
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 1
- 201000003076 Angiosarcoma Diseases 0.000 description 1
- SGYSTDWPNPKJPP-GUBZILKMSA-N Arg-Ala-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SGYSTDWPNPKJPP-GUBZILKMSA-N 0.000 description 1
- YHQGEARSFILVHL-HJGDQZAQSA-N Arg-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N)O YHQGEARSFILVHL-HJGDQZAQSA-N 0.000 description 1
- ULBHWNVWSCJLCO-NHCYSSNCSA-N Arg-Val-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N ULBHWNVWSCJLCO-NHCYSSNCSA-N 0.000 description 1
- RCFGLXMZDYNRSC-CIUDSAMLSA-N Asn-Lys-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O RCFGLXMZDYNRSC-CIUDSAMLSA-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
- OOXUBGLNDRGOKT-FXQIFTODSA-N Asn-Ser-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OOXUBGLNDRGOKT-FXQIFTODSA-N 0.000 description 1
- XAJRHVUUVUPFQL-ACZMJKKPSA-N Asp-Glu-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XAJRHVUUVUPFQL-ACZMJKKPSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 208000036170 B-Cell Marginal Zone Lymphoma Diseases 0.000 description 1
- 208000025324 B-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 206010004593 Bile duct cancer Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 238000011752 CBA/J (JAX™ mouse strain) Methods 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 208000005243 Chondrosarcoma Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- XGIAHEUULGOZHH-GUBZILKMSA-N Cys-Arg-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)N XGIAHEUULGOZHH-GUBZILKMSA-N 0.000 description 1
- UVZFZTWNHOQWNK-NAKRPEOUSA-N Cys-Ile-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UVZFZTWNHOQWNK-NAKRPEOUSA-N 0.000 description 1
- UEHCDNYDBBCQEL-CIUDSAMLSA-N Cys-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N UEHCDNYDBBCQEL-CIUDSAMLSA-N 0.000 description 1
- 238000011765 DBA/2 mouse Methods 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 208000006168 Ewing Sarcoma Diseases 0.000 description 1
- 102100027280 Fanconi anemia group A protein Human genes 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-N 0.000 description 1
- DXVOKNVIKORTHQ-GUBZILKMSA-N Glu-Pro-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O DXVOKNVIKORTHQ-GUBZILKMSA-N 0.000 description 1
- SWDNPSMMEWRNOH-HJGDQZAQSA-N Glu-Pro-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWDNPSMMEWRNOH-HJGDQZAQSA-N 0.000 description 1
- YZACQYVWLCQWBT-BQBZGAKWSA-N Gly-Cys-Arg Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YZACQYVWLCQWBT-BQBZGAKWSA-N 0.000 description 1
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 1
- MYXNLWDWWOTERK-BHNWBGBOSA-N Gly-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN)O MYXNLWDWWOTERK-BHNWBGBOSA-N 0.000 description 1
- 208000001258 Hemangiosarcoma Diseases 0.000 description 1
- 208000035186 Hemolytic Autoimmune Anemia Diseases 0.000 description 1
- 208000027761 Hepatic autoimmune disease Diseases 0.000 description 1
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000914673 Homo sapiens Fanconi anemia group A protein Proteins 0.000 description 1
- 101001055157 Homo sapiens Interleukin-15 Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 206010021245 Idiopathic thrombocytopenic purpura Diseases 0.000 description 1
- IXEFKXAGHRQFAF-HVTMNAMFSA-N Ile-Glu-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N IXEFKXAGHRQFAF-HVTMNAMFSA-N 0.000 description 1
- JNLSTRPWUXOORL-MMWGEVLESA-N Ile-Ser-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N JNLSTRPWUXOORL-MMWGEVLESA-N 0.000 description 1
- COWHUQXTSYTKQC-RWRJDSDZSA-N Ile-Thr-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N COWHUQXTSYTKQC-RWRJDSDZSA-N 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 108010053727 Interleukin-15 Receptor alpha Subunit Proteins 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 102220470475 L-seryl-tRNA(Sec) kinase_C57L_mutation Human genes 0.000 description 1
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 description 1
- 208000018142 Leiomyosarcoma Diseases 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- POJPZSMTTMLSTG-SRVKXCTJSA-N Leu-Asn-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N POJPZSMTTMLSTG-SRVKXCTJSA-N 0.000 description 1
- IASQBRJGRVXNJI-YUMQZZPRSA-N Leu-Cys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)NCC(O)=O IASQBRJGRVXNJI-YUMQZZPRSA-N 0.000 description 1
- YVKSMSDXKMSIRX-GUBZILKMSA-N Leu-Glu-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O YVKSMSDXKMSIRX-GUBZILKMSA-N 0.000 description 1
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 1
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 1
- JDBQSGMJBMPNFT-AVGNSLFASA-N Leu-Pro-Val Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O JDBQSGMJBMPNFT-AVGNSLFASA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- IPSDPDAOSAEWCN-RHYQMDGZSA-N Lys-Met-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IPSDPDAOSAEWCN-RHYQMDGZSA-N 0.000 description 1
- VWPJQIHBBOJWDN-DCAQKATOSA-N Lys-Val-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O VWPJQIHBBOJWDN-DCAQKATOSA-N 0.000 description 1
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- ULNXMMYXQKGNPG-LPEHRKFASA-N Met-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N ULNXMMYXQKGNPG-LPEHRKFASA-N 0.000 description 1
- VSJAPSMRFYUOKS-IUCAKERBSA-N Met-Pro-Gly Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O VSJAPSMRFYUOKS-IUCAKERBSA-N 0.000 description 1
- QQPMHUCGDRJFQK-RHYQMDGZSA-N Met-Thr-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QQPMHUCGDRJFQK-RHYQMDGZSA-N 0.000 description 1
- QYIGOFGUOVTAHK-ZJDVBMNYSA-N Met-Thr-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QYIGOFGUOVTAHK-ZJDVBMNYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699729 Muridae Species 0.000 description 1
- 101000746372 Mus musculus Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 241000699669 Mus saxicola Species 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- MHHQQZIFLWFZGR-DCAQKATOSA-N Pro-Lys-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O MHHQQZIFLWFZGR-DCAQKATOSA-N 0.000 description 1
- CGSOWZUPLOKYOR-AVGNSLFASA-N Pro-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 CGSOWZUPLOKYOR-AVGNSLFASA-N 0.000 description 1
- POQFNPILEQEODH-FXQIFTODSA-N Pro-Ser-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O POQFNPILEQEODH-FXQIFTODSA-N 0.000 description 1
- LNICFEXCAHIJOR-DCAQKATOSA-N Pro-Ser-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LNICFEXCAHIJOR-DCAQKATOSA-N 0.000 description 1
- KWMZPPWYBVZIER-XGEHTFHBSA-N Pro-Ser-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWMZPPWYBVZIER-XGEHTFHBSA-N 0.000 description 1
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 1
- FIODMZKLZFLYQP-GUBZILKMSA-N Pro-Val-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FIODMZKLZFLYQP-GUBZILKMSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- WDXYVIIVDIDOSX-DCAQKATOSA-N Ser-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CCCN=C(N)N WDXYVIIVDIDOSX-DCAQKATOSA-N 0.000 description 1
- WSTIOCFMWXNOCX-YUMQZZPRSA-N Ser-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N WSTIOCFMWXNOCX-YUMQZZPRSA-N 0.000 description 1
- JFWDJFULOLKQFY-QWRGUYRKSA-N Ser-Gly-Phe Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JFWDJFULOLKQFY-QWRGUYRKSA-N 0.000 description 1
- ZFVFHHZBCVNLGD-GUBZILKMSA-N Ser-His-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFVFHHZBCVNLGD-GUBZILKMSA-N 0.000 description 1
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 1
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 1
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 description 1
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 102000039471 Small Nuclear RNA Human genes 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 208000021712 Soft tissue sarcoma Diseases 0.000 description 1
- 208000027520 Somatoform disease Diseases 0.000 description 1
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- PXQUBKWZENPDGE-CIQUZCHMSA-N Thr-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)O)N PXQUBKWZENPDGE-CIQUZCHMSA-N 0.000 description 1
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 1
- GFRIEEKFXOVPIR-RHYQMDGZSA-N Thr-Pro-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O GFRIEEKFXOVPIR-RHYQMDGZSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- XEVHXNLPUBVQEX-DVJZZOLTSA-N Thr-Trp-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)NCC(=O)O)N)O XEVHXNLPUBVQEX-DVJZZOLTSA-N 0.000 description 1
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 1
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 1
- KZTLZZQTJMCGIP-ZJDVBMNYSA-N Thr-Val-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KZTLZZQTJMCGIP-ZJDVBMNYSA-N 0.000 description 1
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- UOXPLPBMEPLZBW-WDSOQIARSA-N Trp-Val-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 UOXPLPBMEPLZBW-WDSOQIARSA-N 0.000 description 1
- TYFLVOUZHQUBGM-IHRRRGAJSA-N Tyr-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TYFLVOUZHQUBGM-IHRRRGAJSA-N 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- PFMAFMPJJSHNDW-ZKWXMUAHSA-N Val-Cys-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N PFMAFMPJJSHNDW-ZKWXMUAHSA-N 0.000 description 1
- XXWBHOWRARMUOC-NHCYSSNCSA-N Val-Lys-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N)C(=O)O)N XXWBHOWRARMUOC-NHCYSSNCSA-N 0.000 description 1
- JAKHAONCJJZVHT-DCAQKATOSA-N Val-Lys-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N JAKHAONCJJZVHT-DCAQKATOSA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- OFTXTCGQJXTNQS-XGEHTFHBSA-N Val-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N)O OFTXTCGQJXTNQS-XGEHTFHBSA-N 0.000 description 1
- JAIZPWVHPQRYOU-ZJDVBMNYSA-N Val-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O JAIZPWVHPQRYOU-ZJDVBMNYSA-N 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 201000000448 autoimmune hemolytic anemia Diseases 0.000 description 1
- 201000003710 autoimmune thrombocytopenic purpura Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000026900 bile duct neoplasm Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 238000013377 clone selection method Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 201000003444 follicular lymphoma Diseases 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 102000056003 human IL15 Human genes 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000032832 immune response to tumor cell Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 210000000428 immunological synapse Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 206010024627 liposarcoma Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 201000007924 marginal zone B-cell lymphoma Diseases 0.000 description 1
- 208000021937 marginal zone lymphoma Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000002956 necrotizing effect Effects 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 208000027753 pain disease Diseases 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 208000031223 plasma cell leukemia Diseases 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 206010042863 synovial sarcoma Diseases 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 238000010809 targeting technique Methods 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0387—Animal model for diseases of the immune system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Endocrinology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Rheumatology (AREA)
- Plant Pathology (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种IL15RA基因人源化改造的非人动物的构建方法、一种人源化IL15RA蛋白、一种人源化IL15RA基因、一种IL15RA基因的靶向载体和其在生物医药领域的应用,利用同源重组的方式将部分编码人IL15RA蛋白的核苷酸序列导入非人动物基因组中,该非人动物体内能正常表达人源化IL15RA蛋白,可以用于人IL15RA信号机理研究、炎症、肿瘤及自身免疫性疾病药物筛选,对免疫靶点的新药研发具有重要的应用价值。
Description
技术领域
本发明属于动物基因工程和基因遗传修饰领域,具体地说,涉及一种IL15RA基因人源化的非人动物及其构建方法和在生物医药领域的应用。
背景技术
人IL15具有4-螺旋束机构,主要在单核细胞和树突状细胞合成。IL-15主要通过和IL-15R三聚体复合物结合发挥作用,其中,β链和IL2Rβ相同而γ链和IL2、4、7、9和21的Rγ共用,而α链(CD215)是IL-15特异性的受体亚基。IL15RA在人和小鼠中广泛表达,独立于IL-2R/IL-15Rβ-γc。它与IL-15具有高亲和力(Kd>10–11M)结合,并在细胞表面保留IL-15。IL-15Ra通过与附近的效应NK细胞和T细胞上的IL-2R/IL-15Rβ-γc形成免疫突触。IL-15Ra结合IL15, 通过激活JAK和STAT信号通路调节下游信号表达。肿瘤的发生发展和转移过程离不开淋巴细胞介导的免疫应答和信号转导,而IL15Ra增强T细胞分行增值和B细胞分泌抗体等特性,对于增强对肿瘤细胞的免疫应答起关键作用。
随着基因工程技术的不断发展和成熟,用人类基因替代或置换动物的同源性基因已经实现,通过这种方式开发人源化实验动物模型是动物模型未来的发展方向。其中基因人源化动物模型,即,利用基因编辑技术,用人源正常或突变基因替换动物基因组的同源基因,可建立更接近人类生理或疾病特征的正常或突变基因动物模型。基因人源化动物不但本身具有重要应用价值,如通过基因人源化可改进和提升细胞或组织移植人源化动物模型,更重要的是,由于人类基因片段的插入,动物体内可表达或部分表达人源蛋白,可作为仅能识别人蛋白氨基酸序列的药物的靶点,为在动物水平进行抗人抗体及其它药物的筛选提供了可能。然而,由于动物与人类在生理学及病理学方面存在差异,加上基因(即遗传因子)的复杂性,如何能构建出“有效”的人源化动物模型用于新药研发仍是最大的挑战(Scheer N, Snaith M, Wolf CR, Seibler J. Generation and utility ofgenetically humanized mouse models, Drug Discov Today; 18(23-24):1200-11,2013)。
鉴于IL15RA在肿瘤、自身免疫性疾病等多种疾病发生过程中的广泛参与性以及靶向该信号通路的巨大应用价值,为了使临床前期的试验更有效并使研发失败最小化,本领域仍急需开发人源化IL15RA信号通路相关的非人动物模型。
发明内容
本发明的第一方面,提供了一种IL15RA基因人源化改造的非人动物的构建方法,所述的非人动物的基因组中包括编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列,所述的构建方法包括用包含编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列替换至非人动物IL15RA基因座。
优选的,所述的非人动物的基因组中包括SEQ ID NO:5所示核苷酸序列,所述的构建方法包括用包含SEQ ID NO:5所示核苷酸序列替换至非人动物IL15RA基因座。
优选的,所述的插入或替换至非人动物IL15RA基因座为插入或替换非人动物IL15RA基因中编码SEQ ID NO:1第40至211位的核苷酸序列,或者插入或替换SEQ ID NO:31所示序列相同的核苷酸序列。
优选的,所述的构建方法包括插入、翻转、敲除或替换。
优选的,所述的非人动物的基因组中包含人IL15RA核苷酸序列的2号外显子的部分、3号至5号外显子的全部和6号外显子的部分,进一步优选的,包含2-3号内含子和/或5-6号内含子,更优选的,包含2-6号外显子之间的任一内含子;其中,所述人IL15RA核苷酸序列的2号外显子的部分至少包含2号外显子中去除编码人IL15RA蛋白胞外区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列,6号外显子的部分至少包含6号外显子中编码人IL15RA蛋白跨膜区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列。
优选的,所述的构建方法包括用包含人IL15RA核苷酸序列的2号至6号外显子的全部或部分核苷酸序列插入或替换到非人动物IL15RA基因座上,进一步优选的,用包含人IL15RA核苷酸序列的2号外显子的部分、3号至5号外显子的全部和6号外显子的部分核苷酸序列插入或替换到非人动物IL15RA基因座上,更优选的,包含2-3号内含子和/或5-6号内含子,更进一步优选的,包含2-6号外显子之间的任一内含子;其中,所述人IL15RA基因的2号外显子的部分至少包含2号外显子中去除编码人IL15RA蛋白胞外区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列,6号外显子的部分至少包含6号外显子中编码人IL15RA蛋白跨膜区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列。
在本发明的一个具体实施方式中,所述的构建方法包括用包含编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列或者包含SEQ ID NO:5所示核苷酸序列替换至非人动物IL15RA基因的相应区域。
优选的,所述的构建方法包括用包含人IL15RA核苷酸序列的2号至6号外显子全部或部分替换非人动物IL15RA核苷酸序列的2号至6号外显子的全部或部分;其中,所述非人动物IL15RA核苷酸序列包含编码非人动物2号外显子的部分、3号至5号外显子的全部、6号外显子的部分核苷酸序列,优选的,包含2-3号内含子和/或5-6号内含子,进一步优选的,包含2-6号外显子之间的任一内含子,其中,所述非人动物IL15RA核苷酸序列的2号外显子的部分至少包含2号外显子中去除编码IL15RA蛋白胞外区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列,6号外显子的部分至少包含6号外显子中编码IL15RA蛋白跨膜区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列。
优选的,所述的构建方法包括用包含所述人源化IL15RA基因的核苷酸序列插入或替换到非人动物IL15RA基因座上。
优选的,所述的构建方法包括用包含编码所述人源化IL15RA蛋白的核苷酸序列插入或替换到非人动物IL15RA基因座上。优选的,所述的插入或替换位点为IL15RA基因的内源调控元件之后。
优选的,所述的插入为首先破坏非人动物内源IL15RA基因的编码框,随后进行插入操作,或者所述的插入步骤既可在内源IL15RA基因处造成移码突变又可以实现插入人源序列的步骤。
优选的,所述的非人动物中人源化IL15RA基因是纯合或杂合的。
优选的,所述非人动物的基因组中至少一个染色体上包含人源化IL15RA基因。
优选的,所述的非人动物中至少一个细胞表达人或人源化IL15RA蛋白。
优选的,使用基因编辑技术进行IL15RA基因人源化改造的非人动物的构建,所述的基因编辑技术包括利用胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶或其他分子生物学技术。
本发明所述的非人动物为啮齿类动物;优选的,所述的啮齿类动物为大鼠或小鼠。
优选的,使用靶向载体进行IL15RA基因人源化改造的非人动物的构建,所述靶向载体包含编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列或SEQ ID NO:5所示核苷酸序列,任选地,所述的靶向载体还包含与待改变的转换区5’端同源的DNA片段,即5’臂,和/或,所述的靶向载体还包含与待改变的转换区3’端同源的DNA片段,即3’臂,进一步优选的,所述的5’臂选自非人动物IL15RA基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的5’臂与NCBI登录号为NC_000068.8至少具有90%同源性的核苷酸;进一步优选的,所述5’臂序列与SEQ ID NO:3至少具有90%同源性,或者如SEQ ID NO:3所示;优选的,所述的3’臂选自非人动物IL15RA基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的3’臂与NCBI登录号为NC_000068.8至少具有90%同源性的核苷酸;进一步优选的,所述的3’臂序列与SEQ ID NO:4至少具有90%同源性,或者如SEQ ID NO:4所示。
优选的,所述的靶向载体包含人IL15RA的2号至6号外显子的全部或部分核苷酸序列;进一步优选的,包含2号外显子的部分、3号至5号外显子的全部和6号外显子的部分,更优选的,包含2-3号内含子和/或5-6号内含子,更进一步优选的,包含2-6号外显子之间的任一内含子,其中,所述人IL15RA的核苷酸序列的2号外显子的部分至少包含2号外显子中去除编码人IL15RA蛋白胞外区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列,6号外显子的部分至少包含6号外显子中编码人IL15RA蛋白跨膜区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列。
优选的,所述的待改变的转换区位于非人动物IL15RA基因座上。进一步优选的,位于非人动物IL15RA基因的2号外显子至6号外显子上。
在本发明的一个具体实施方式中,所述的构建方法包括将上述靶向载体导入非人动物细胞中,培养该细胞(优选为胚胎干细胞),然后将培养后的细胞移植至雌性非人动物输卵管内,允许其发育,鉴定筛选获得非人动物。
优选的,所述的非人动物体内表达人或人源化IL15RA蛋白,包含与SEQ ID NO:2第37至211位或SEQ ID NO:22具有至少70%、80%、85%、90%、95%或至少99%同一性的氨基酸序列或者包含与SEQ ID NO:2第37至211位或SEQ ID NO:22所示氨基酸序列一致的氨基酸序列,同时内源IL15RA蛋白的表达降低或缺失。
优选的,所述的人源化IL15RA蛋白包含人IL15RA蛋白的胞外区和/或跨膜区的全部或部分,进一步优选的,包含胞外区的部分和跨膜区的部分,更优选的,所述的胞外区的部分包含N端去除0-10(例如0、1、2、3、4、5、6、7、8、9、10)个氨基酸的人IL15RA蛋白胞外区,所述的跨膜区的部分包含N端0-10(例如0、1、2、3、4、5、6、7、8、9、10)个氨基酸的人IL15RA蛋白跨膜区。
优选的,所述的人源化IL15RA蛋白还包含非人动物IL15RA蛋白的部分,优选为非人动物IL15RA蛋白的信号肽、胞外区、跨膜区和/或胞质区。
在本发明的一个具体实施方式中,所述的人源化IL15RA蛋白包含下列组中的一种:
a)SEQ ID NO:22或SEQ ID NO:2第37至211位所示氨基酸序列的部分或全部;
b)与SEQ ID NO:22或SEQ ID NO:2第37至211位所示氨基酸的序列同一性程度为至少大约为90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
c)与SEQ ID NO:22或SEQ ID NO:2第37至211位所示的氨基酸的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸;
d)具有SEQ ID NO:22或SEQ ID NO:2第37至211位所示的,包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
优选的,所述的非人动物的基因组中包含人源化IL15RA基因,所述的人源化IL15RA基因编码人源化IL15RA蛋白。
优选的,所述的人源化IL15RA基因包含SEQ ID NO:5所示的核苷酸序列,进一步优选的,所述的非人动物中包含的IL15RA基因转录的mRNA序列包含SEQ ID NO:8所示的核苷酸序列。
在本发明的一个具体实施方式中,所述的人源化IL15RA基因包含下列组中的一种:
a)人源化IL15RA基因的mRNA序列为SEQ ID NO:8所示的序列的部分或全部;
b)人源化IL15RA基因的mRNA序列与SEQ ID NO:8所示的核苷酸序列的部分或全部的同一性程度为至少大约为90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
c)人源化IL15RA基因的mRNA序列与SEQ ID NO:8所示的核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸;
d)人源化IL15RA基因的mRNA序列具有SEQ ID NO:8所示的核苷酸序列所示的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
本发明的第二方面,提供了一种IL15RA基因人源化改造的非人动物,所述的非人动物采用上述构建方法获得。
本发明的第三方面,提供了一种靶向载体,所述的靶向载体包含人IL15RA核苷酸序列的部分,优选的,所述的靶向载体包含编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列或者SEQ ID NO:5所示核苷酸序列,任选地,所述的靶向载体还包含与待改变的转换区5’端同源的DNA片段,即5’臂,和/或,所述的靶向载体还包含与待改变的转换区3’端同源的DNA片段,即3’臂,优选的,所述的5’臂选自非人动物IL15RA基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的5’臂与NCBI登录号为NC_000068.8至少具有90%同源性的核苷酸;进一步优选的,所述5’臂序列与SEQ ID NO:3至少具有90%同源性,或者如SEQ ID NO:3所示;优选的,所述的3’臂选自非人动物IL15RA基因基因组DNA的100-10000个长度的核苷酸;优选的,所述的3’臂与NCBI登录号为NC_000068.8至少具有90%同源性的核苷酸;进一步优选的,所述的3’臂序列与SEQ ID NO:4至少具有90%同源性,或者如SEQ ID NO:4所示。
优选的,所述的人IL15RA核苷酸序列的部分包含人IL15RA的2号至6号外显子的全部或部分核苷酸序列;进一步优选的,包含2号外显子的部分、3号至5号外显子的全部和6号外显子的部分,更优选的,包含2-3号内含子和/或5-6号内含子,更进一步优选的,包含2-6号外显子之间的任一内含子,其中,所述人IL15RA的核苷酸序列的2号外显子的部分至少包含2号外显子中去除编码人IL15RA蛋白胞外区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列,6号外显子的部分至少包含6号外显子中编码人IL15RA蛋白跨膜区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列。
优选的,所述的待改变的转换区位于非人动物IL15RA基因座上,进一步优选的,所述的待改变的转换区位于非人动物IL15RA基因2号至6号外显子上。
本发明所述的非人动物为啮齿类动物;优选的,所述的啮齿类动物为大鼠或小鼠。
优选的,所述的靶向载体还包含标记基因,进一步优选的,所述标记基因为负筛选标记的编码基因,更进一步优选的,所述负筛选标记的编码基因为白喉毒素A亚基的编码基因(DTA)。
在本发明的一个具体实施方式中,所述的靶向载体中还包括阳性克隆筛选的抗性基因,进一步优选的,所述阳性克隆筛选的抗性基因为新霉素磷酸转移酶编码序列Neo。
在本发明的一个具体实施方式中,所述的靶向载体中还包括特异性重组系统,进一步优选的,所述特异性重组系统为Frt重组位点(也可选择常规的LoxP重组系统),所述的特异性重组系统为具有两个Frt重组位点,分别连接在抗性基因的两侧。
本发明的第四方面,提供了一种包含上述靶向载体的细胞。
本发明的第五方面,提供了上述靶向载体,或者上述的细胞在IL15RA基因修饰中的应用,优选的,所述的应用包括但不限于翻转、敲除、插入或替换。
本发明的第六方面,涉及一种IL15RA基因改造的人源化细胞,所述的人源化IL15RA基因改造细胞的基因组中包括人IL15RA基因的2号外显子至6号外显子。优选的,所述的人IL15RA基因编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列或包含SEQ ID NO:5所示核苷酸序列,其通过内源性IL15RA调控元件调控;该人源化IL15RA基因改造细胞体内表达人或人源化IL15RA蛋白,同时内源IL15RA蛋白的表达降低或缺失。优选的,所述的人IL15RA基因通过内源性IL15RA调控元件调控。
本发明的第七方面,涉及一种IL15RA基因缺失的细胞,所述的IL15RA基因缺失的细胞缺失内源IL15RA基因的2号外显子至6号外显子。
本发明的第八方面,涉及一种荷瘤动物模型的制备方法,所述的动物模型的制备方法包括通过上述的人源化IL15RA基因改造非人动物制备荷瘤动物模型的步骤。
优选的,所述的荷瘤动物模型的制备方法还包括在上述人源化基因改造非人动物或其后代植入肿瘤细胞的步骤。
本发明的第九方面,提供了一种上述的制备方法获得的荷瘤动物模型。
本发明的第十方面,涉及一种细胞或细胞系或原代细胞培养物,所述细胞或细胞系或原代细胞培养物来源于上述的非人动物或上述的荷瘤动物模型。
本发明的第十一方面,涉及一种组织或器官或其培养物,所述组织或器官或其培养物来源于上述的非人动物或上述的荷瘤动物模型。
优选的,所述的组织或器官或其培养物为脾脏、肿瘤或其培养物。
本发明的第十二方面,提供了一种人源化IL15RA蛋白,所述的人源化IL15RA蛋白包含人IL15RA蛋白的全部或部分,所述的人源化IL15RA蛋白包含与SEQ ID NO:2第37至211位或SEQ ID NO:22具有至少70%、80%、85%、90%、95%或至少99%同一性的氨基酸序列或者包含与SEQ ID NO:2第37至211位或SEQ ID NO:22所示氨基酸序列一致的氨基酸序列。
优选的,所述的人源化IL15RA蛋白包含人IL15RA蛋白的胞外区和/或跨膜区的全部或部分,进一步优选的,包含胞外区的部分和跨膜区的部分,更优选的,所述的胞外区的部分包含N端去除0-10(例如0、1、2、3、4、5、6、7、8、9、10)个氨基酸的人IL15RA蛋白胞外区,所述的跨膜区的部分包含N端0-10(例如0、1、2、3、4、5、6、7、8、9、10)个氨基酸的人IL15RA蛋白跨膜区。
优选的,所述的人源化IL15RA蛋白还包含非人动物IL15RA蛋白的部分,优选为非人动物IL15RA蛋白的信号肽、胞外区、跨膜区、胞质区。
优选的,所述的人源化IL15RA蛋白包含人IL15RA基因的2号外显子至6号外显子编码的氨基酸序列,和非人动物IL15RA蛋白的氨基酸序列。
在本发明的一个具体实施方式中,所述的人源化IL15RA蛋白包含下列组中的一种:
a)SEQ ID NO:22或SEQ ID NO:2第37至211位所示氨基酸序列的部分或全部;
b)与SEQ ID NO:22或SEQ ID NO:2第37至211位所示氨基酸的序列同一性程度为至少大约为90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%;
c)与SEQ ID NO:22或SEQ ID NO:2第37至211位所示的氨基酸的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸;
d)具有SEQ ID NO:22或SEQ ID NO:2第37至211位所示的,包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明的第十三方面,提供了一种编码上述人源化IL15RA蛋白的人源化IL15RA基因,所述的人源化IL15RA基因包含人IL15RA基因的2号外显子至6号外显子,和非人动物IL15RA基因的核苷酸序列。
优选的,所述的人源化IL15RA基因包含SEQ ID NO:5所示的核苷酸序列。
优选的,所述的人源化IL15RA基因转录的mRNA序列包含SEQ ID NO:8所示的核苷酸序列。
在本发明的一个具体实施方式中,所述的人源化IL15RA基因中包含的人IL15RA核苷酸序列的部分选自下列组中的一种:
(A)包含SEQ ID NO:5所示核苷酸序列的全部或部分;
(B)包含与SEQ ID NO:5所示核苷酸序列的同一性至少为75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%的核苷酸序列;
(C)包含与SEQ ID NO:5所示核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列;
(D)具有SEQ ID NO:5所示核苷酸序列的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
在本发明的一个具体实施方式中,所述的人源化IL15RA基因的核苷酸序列转录的mRNA选自下列组中的一种:
(a)包含SEQ ID NO:8所示核苷酸序列的全部或部分;
(b)包含与SEQ ID NO:8所示核苷酸序列的同一性至少为75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或至少99%的核苷酸序列;
(c)包含与SEQ ID NO:8所示的核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列;或
(d)包含SEQ ID NO:8所示的核苷酸序列所示的,包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
本发明的第十四方面,涉及一种表达上述的人源化IL15RA蛋白的构建体。
本发明的第十五方面,涉及一种包含上述构建体的细胞。
本发明的第十六方面,涉及一种包含上述细胞的组织。
本发明的第十七方面,提供了一种多基因修饰的非人动物的构建方法,所述的构建方法包括:
(a)应用上述的构建方法制备获得非人动物;
(b)将步骤(a)制备获得的非人动物与除IL15RA外的其他基因修饰的动物交配、体外授精或直接进行基因编辑,并进行筛选,得到多基因人源化修饰的非人动物。
优选的,所述多基因人源化修饰的非人动物为双基因人源化非人动物、三基因人源化非人动物、四基因人源化非人动物、五基因人源化非人动物、六基因人源化非人动物、七基因人源化非人动物、八基因人源化非人动物或九基因人源化非人动物。
优选的,所述的除IL15RA外的其他基因修饰的动物选自基因PD-1、PD-L1、CTLA4、OX40、LAG3、TIM3或CD73等修饰的动物中的一种或两种以上的组合。
本发明的第十八方面,涉及了一种上述的非人动物、上述的荷瘤动物模型、上述的细胞或细胞系或原代细胞培养物、上述的组织或器官或其培养物、上述的人源化IL15RA蛋白或上述的人源化IL15RA基因在制备治疗或预防肿瘤的药物中的应用。
本发明的第十九方面,涉及一种上述的非人动物、上述的荷瘤动物模型、上述的细胞或细胞系或原代细胞培养物、上述的组织或器官或其培养物、上述的人源化IL15RA蛋白或上述的人源化IL15RA基因在IL15RA基因或蛋白中相关研究中的应用,所述的应用包括:
A)涉及人类细胞的免疫过程的产品开发,制造或筛选人类抗体中的应用;
B)作为药理学、免疫学、微生物学和医学研究的模型系统中的应用;
C)涉及人类细胞的免疫过程的生产和利用动物实验疾病模型,用于病原学研究、用于开发诊断策略或用于开发治疗策略中的应用;
D)在体内研究人IL15RA信号通路调节剂的筛选、药效检测、评估疗效、验证或评价;或者,
E)研究IL15RA基因功能,研究人IL15RA抗体,研究针对人IL15RA靶位点的药物、药效,研究免疫相关疾病药物以及抗肿瘤或炎症药物方面的用途。
优选的,所述应用包括在制备药物组合物或者检测试剂盒中的用途。
优选的,所述应用不是疾病的诊断和治疗方法。
本发明所述的“肿瘤”包括但不限于淋巴瘤、B细胞肿瘤、T细胞肿瘤、骨髓/单核细胞肿瘤、非小细胞肺癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、胃癌、膀胱癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤。其中,所述的白血病选自急性淋巴细胞性(成淋巴细胞性)白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病、以及慢性骨髓性白血病;所述淋巴瘤选自霍奇金淋巴瘤和非霍奇金淋巴瘤,包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤、和瓦尔登斯特伦巨球蛋白血症;所述肉瘤选自骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、及软骨肉瘤。在本发明的一个具体实施方式中,所述的肿瘤选自B细胞肿瘤、T细胞肿瘤、骨髓/单核细胞肿瘤。优选包括B或T细胞急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、非霍奇金淋巴瘤(NHL)和多发性骨髓瘤(MM)、鼻咽癌、肺癌。
本发明所述的“免疫相关疾病”包括但不限于过敏、哮喘、心肌炎、肾炎、肝炎、系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺功能亢进、原发性血小板减少性紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、自身免疫性肝病、糖尿病、疼痛或神经障碍等。在本发明的一个具体实施方式中。所述的免疫相关疾病为类风湿性关节炎。
本发明所述的“炎症”包括急性炎症,也包括慢性炎症。具体的,包括但不限于变质性炎症、渗出性炎症(浆液性炎、纤维素性炎、化脓性炎、出血性炎、坏死性炎、卡他性炎)、增生性炎症、特异性炎症(结核、梅毒、麻疯、淋巴肉芽肿等)。
本发明所述的IL15RA基因人源化的非人动物体内可以正常表达人或人源化IL15RA蛋白。可用于针对人IL15RA靶位点的药物筛选、药效评估、免疫相关疾病和肿瘤治疗,可以加快新药研发过程、节约时间和成本。对于研究IL15RA蛋白功能及相关疾病药物筛选提供了有效的保障。
本发明所述的“全部或部分”,“全部”为整体,“部分”为整体中的局部,或者组成整体的个体。
本发明所述的“人源化IL15RA蛋白”,包含来源于人IL15RA蛋白的部分和非人IL15RA蛋白的部分。其中,所述的“人IL15RA蛋白”同“人IL15RA蛋白的全部”,即其氨基酸序列与人IL15RA蛋白的全长氨基酸序列一致。所述的“人IL15RA蛋白的部分”,为连续或间隔的5-267个(优选为10-175个)氨基酸序列与人IL15RA蛋白的氨基酸序列一致或与人IL15RA蛋白的氨基酸序列具有70%以上同源性。
本发明所述的“人IL15RA蛋白的跨膜区的全部”或“人IL15RA蛋白的胞外区的全部”,代表其氨基酸序列分别与人IL15RA蛋白的胞外区的全长氨基酸序列一致。
本发明所述的“人IL15RA蛋白的胞外区的部分”,为连续或间隔5-175个(优选为5-169个)氨基酸序列与人IL15RA蛋白的胞外区氨基酸序列一致,或与人IL15RA蛋白的胞外区氨基酸序列具有70%以上同源性。
本发明所述的“人IL15RA蛋白的跨膜区的部分”,为连续或间隔5-23个(优选为5-6个)氨基酸序列与人IL15RA蛋白的跨膜区氨基酸序列一致,或与人IL15RA蛋白的胞外区氨基酸序列具有70%以上同源性。
本发明所述的“人源化IL15RA基因”,包含来源于人IL15RA核苷酸序列的部分和非人IL15RA基因的部分。其中,所述的“人IL15RA核苷酸序列”同“人IL15RA核苷酸序列的全部”,即其核苷酸序列与人IL15RA核苷酸序列的全长核苷酸序列一致。所述的“人IL15RA核苷酸序列的部分”为连续或间隔的20-29845bp(优选为20-3757bp或20-525bp)个核苷酸序列与人IL15RA核苷酸序列一致或与人IL15RA核苷酸序列具有70%以上同源性。
本发明所述的“xx号至xxx号外显子”或“xx号至xxx号外显子的全部”包含外显子及其期间的内含子的核苷酸序列,例如所述的“2号至6号外显子”包含2号外显子、2-3号内含子、3号外显子、3-4号内含子、4号外显子、4-5号内含子、5号外显子、5-6号内含子、6号外显子的全部核苷酸序列。
本发明所述的“x-xx号内含子”表示x号外显子与xx号外显子之间的内含子。例如“2-3号内含子”表示2号外显子与3号外显子之间的内含子。
本发明所述的“外显子的部分”表示连续或间隔几个、几十个或几百个核苷酸序列与全部的外显子核苷酸序列一致。例如人IL15RA核苷酸序列的2号外显子的部分,包含连续或间隔的5-195bp个,优选10-175bp个核苷酸序列与人IL15RA核苷酸序列的2号外显子核苷酸序列一致。在本发明的一个具体实施方式中,所述的“人源化IL15RA基因”中包含的“2号外显子的部分”至少包括2号外显子中去除编码人IL15RA蛋白胞外区N端1-10(例如1、2、3、4、5、6、7、8、9、10)个氨基酸的核苷酸序列。
本发明所述的“基因座”广义上讲代表基因在染色体上所占的位置,狭义上讲代表某一基因上的一段DNA片段,即可以是一个基因也可以是一个基因的一部分。例如所述的“IL15RA基因座”表示IL15RA基因1号至7号外显子上的任选一段的DNA片段。优选为1号外显子、2号外显子、3号外显子、4号外显子、5号外显子、6号外显子、7号外显子或其期间的内含子中的任一个或两个或多个的组合,或一个或两个或多个的全部或部分,更优选为IL15RA基因的2号至6号外显子上。
本发明所述的“核苷酸序列”包含天然的或经过修饰的核糖核苷酸序列、脱氧核糖核苷酸序列。优选为DNA、cDNA、pre-mRNA、mRNA、rRNA、hnRNA、miRNAs、scRNA、snRNA、siRNA、sgRNA、tRNA。
本发明所述“治疗(treating)”(或“治疗(treat)”或“治疗(treatment)”)表示减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性,但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。术语“治疗(treating)”等是指在疾病已开始发展后改善疾病或病理状态的体征、症状等等的治疗干预。
本发明所述“同源性”,是指在使用蛋白序列或核苷酸序列的方面,本领域技术人员可以根据实际工作需要对序列进行调整,使使用序列与现有技术获得的序列相比,具有(包括但不限于)1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%的同一性。
本领域的技术人员能够确定并比较序列元件或同一性程度,以区分另外的小鼠和人序列。
在一个方面,所述非人动物是哺乳动物。在一个方面,所述非人动物是小型哺乳动物,例如跳鼠科或鼠总科超家族。在一个实施方式中,所述基因修饰的动物是啮齿动物。在一个实施方式中,所述啮齿动物选自小鼠、大鼠和仓鼠。在一个实施方式中,所述啮齿动物选自鼠家族。在一个实施方式中,所述基因修饰的动物来自选自丽仓鼠科(例如小鼠样仓鼠)、仓鼠科(例如仓鼠、新世界大鼠和小鼠、田鼠)、鼠总科(真小鼠和大鼠、沙鼠、刺毛鼠、冠毛大鼠)、马岛鼠科(登山小鼠、岩小鼠、有尾大鼠、马达加斯加大鼠和小鼠)、刺睡鼠科(例如多刺睡鼠)和鼹形鼠科(例如摩尔大鼠、竹大鼠和鼢鼠)家族。在一个特定实施方式中,所述基因修饰的啮齿动物选自真小鼠或大鼠(鼠总科)、沙鼠、刺毛鼠和冠毛大鼠。在一个实施方式中,所述基因修饰的小鼠来自鼠科家族成员。在一个实施方式中,所述动物是啮齿动物。在一个特定实施方式中,所述啮齿动物选自小鼠和大鼠。在一个实施方式中,所述非人动物是小鼠。
在一个特定实施方式中,所述非人动物是啮齿动物,其为选自BALB/c、A、A/He、A/J、A/WySN、AKR、AKR/A、AKR/J、AKR/N、TA1、TA2、RF、SWR、C3H、C57BR、SJL、C57L、DBA/2、KM、NIH、ICR、CFW、FACA、C57BL/A、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/6、C57BL/6J、C57BL/6ByJ、C57BL/6NJ、C57BL/10、 C57BL/10ScSn、C57BL/10Cr和C57BL/Ola的C57BL、C58、CBA/Br、CBA/Ca、CBA/J、CBA/st、CBA/H品系的小鼠。
除非特别说明,本发明的实践将采取细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术。这些技术在以下文献中进行了详细的解释。例如:Molecular Cloning A Laboratory Manual,2ndEd.,ed. By Sambrook,FritschandManiatis (Cold Spring Harbor Laboratory Press:1989);DNA Cloning,Volumes I and II (D.N.Glovered.,1985);Oligonucleotide Synthesis (M.J.Gaited.,1984);Mullisetal. U.S. Pat.No.4,683,195;Nucleic Acid Hybridization(B.D.Hames& S.J.Higginseds.1984);Transcription And Translation (B.D.Hames&S.J.Higginseds.1984);Culture Of Animal Cells (R.I.Freshney,AlanR.Liss,Inc.,1987);Immobilized Cells And Enzymes (IRL Press,1986);B.Perbal,A PracticalGuide To Molecular Cloning(1984);the series,Methods In ENZYMOLOGY (J.Abelsonand M.Simon,eds.inchief,Academic Press,Inc.,New York),specifically,Vols. 154and 155 (Wuetal.eds.) and Vol.185,″Gene Expression Technology″ (D.Goeddel,ed.);Gene Transfer Vectors For Mammalian Cells (J.H.Miller and M.P.Caloseds.,1987,Cold Spring Harbor Laboratory);Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker,eds.,Academic Press,London,1987);HandbookOf Experimental Immunology,Volumes V (D.M.Weir and C.C.Blackwell,eds.,1986);and Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.,1986)。
以上只是概括了本发明的一些方面,不是也不应该认为是在任何方面限制本发明。
本说明书提到的所有专利和出版物都是通过参考文献作为整体而引入本发明的。本领域的技术人员应认识到,对本发明可作某些改变并不偏离本发明的构思或范围。
下面的实施例进一步详细说明本发明,不能认为是限制本发明或本发明所说明的具体方法的范围。
附图说明
以下,结合附图来详细说明本发明的实施例,其中:
图1:人和小鼠IL15RA基因结构对比示意图(非按比例);
图2:人源化IL15RA基因座示意图(非按比例);
图3:IL15RA基因打靶策略示意图(非按比例);
图4:重组后ES细胞Southern blot结果,其中WT为野生型对照;
图5:IL15RA人源化小鼠FRT重组过程示意图(非按比例);
图6:F1代小鼠PCR结果,其中WT为野生型对照,H2O为水对照,PC为阳性对照;
图7:流式检测结果,其中WT为野生型C57BL/6小鼠,H/H为IL15RA人源化纯合子小鼠;
图8:RT-PCR结果示意图,其中,+/+为C57BL/6野生型小鼠,H/H为IL15RA人源化纯合子小鼠,GAPDH为内参,H2O为水对照。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
在下述每一实施例中,设备和材料是从以下所指出的几家公司获得:
PE Mouse IgG2b, κ Isotype CtrlAntibody购自Biolegend,货号:400314;
Mouse IL-15R alpha APC-conjugated Antibody购自R&D,货号:FAB5511A-100;
Brilliant Violet 510™ anti-mouse CD45Antibody购自Biolegend,货号:103138;
Brilliant Violet 605™anti-mouse CD11cAntibody购自Biolegend,货号:117334;
PE anti-human CD215 (IL-15Rα) Antibody购自Biolegend,货号:330207;
APC Rat IgG2b, K Isotype CtrlAntibody购自Biolegend,货号:400612。
实施例1 IL15RA基因人源化小鼠的制备
本实施例对非人动物(如小鼠)进行改造,使该非人动物体内包含编码人源化IL15RA蛋白的核苷酸序列,得到经遗传修饰的非人动物体内可表达人源化IL15RA蛋白。小鼠IL15RA基因(NCBI Gene ID:16169,Primary source:MGI:104644,UniProt ID:Q60819,位于2号染色体NC_000068.8的第11709992位至第11738796位,基于转录本NM_008358.2及其编码蛋白NP_ 032384.1(SEQ ID NO:1))和人IL15RA基因(NCBI Gene ID:3601,Primarysource:HGNC:5978,UniProt ID:Q13261,位于10号染色体NC_000010.11的第5948897-5978741位,基于转录本NM_002189.4及其编码蛋白NP_002189.4(SEQ ID NO:2))。对比示意图如图1所示。
为了达到本发明的目的,可在小鼠内源IL15RA基因座引入编码人IL15RA蛋白的基因序列,使得该小鼠表达人或人源化IL15RA蛋白。具体来说,可以通过基因编辑技术在小鼠内源IL15RA基因座上用人IL15RA基因的核苷酸序列(例如DNA序列、cDNA序列等)替换小鼠相应序列,如将至少包含小鼠IL15RA基因的2号外显子的部分序列至6号外显子的部分序列用对应的人DNA序列替换,得到人源化IL15RA基因座(示意图如图2所示),实现对小鼠IL15RA基因的人源化改造。
进一步设计如图3所示的打靶策略示意图,图中显示了靶向载体上含有小鼠IL15RA基因上游和下游的同源臂序列,以及包含人IL15RA序列的A片段。其中,上游同源臂序列(5’同源臂,SEQ ID NO:3)与NCBI登录号为NC_000068.8第11717833至11723094位核苷酸序列相同,下游同源臂序列(3’同源臂,SEQ ID NO:4)与NCBI登录号为NC_000068.8第11735827至11739583位核苷酸序列相同;人IL15RA序列(SEQ ID NO:5)与NCBI登录号为NC_000010.11的第5956438至5966319位核苷酸序列相同。
靶向载体上还包括用于阳性克隆筛选的抗性基因,即新霉素磷酸转移酶编码序列Neo,并在抗性基因的两侧装上两个同向排列的位点特异性重组系统Frt重组位点,组成Neo盒(Neo cassette)。其中,Neo盒5’端与鼠的连接设计为5’-CATGTCAGCCTTGATTCTGTATTTCTAATAGCAGAACATATGGAATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGGT-3’(SEQ ID NO:6),其中,序列“GCAGAA”的最后一个“A”是鼠的最后一个核苷酸,序列“CATATG”的第一个“G”是Neo盒的第一个核苷酸;Neo盒3’端与鼠的连接设计为5’-TCTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCATGACACAGCGCAGCTCACTCACCAGTTCCCTCCACTTCCTGACATTTCAGT-3’(SEQ ID NO:7)内,其中序列“GGATCC”的最后一个“C”是Neo盒的最后一个核苷酸,序列“ATGACA”的第一个“A”是鼠的第一个核苷酸。此外,还在靶向载体3’同源臂下游构建了具有负筛选标记的编码基因(白喉毒素A亚基的编码基因(DTA))。改造后的人源化小鼠IL15RA的mRNA序列如SEQ ID NO:8所示,表达的蛋白序列如SEQ ID NO:22所示。
靶向载体构建可采用常规方法进行,如酶切连接等。构建好的靶向载体通过酶切进行初步验证后,再送测序公司进行测序验证。将测序验证正确的靶向载体电穿孔转染入C57BL/6小鼠的胚胎干细胞中,利用阳性克隆筛选标记基因对得到的细胞进行筛选,并利用PCR(PCR引物详见表1)和Southern Blot技术进行检测确认外源基因的整合情况,筛选出正确的阳性克隆细胞,经PCR鉴定为阳性的克隆再进行Southern Blot(分别用HpaI或NdeI或HindIII消化细胞DNA并使用3个探针进行杂交,酶、探针及目的片段长度如表2所示)检测,Southern Blot检测结果如图4所示,表明12个经PCR验证为阳性的胚胎干细胞(ES-01至ES-12)均为阳性的克隆,且无随机插入。
表1 PCR检测引物序列及目的片段长度
表2 Southern Blot酶和探针表
Southern Blot检测包括如下探针引物:
5’探针(5’Probe):
5’Probe-F:5’-tcctatcaggcagggttcacaaggt-3’(SEQ ID NO:13),
5’Probe-R:5’-aggagcctaagagtcccttcctcac-3’ (SEQ ID NO:14);
3’探针(3’Probe):
3’Probe-F:5’-cagatccccagccttttgcaacatc-3’ (SEQ ID NO:15),
3’Probe-R:5’-tcaagaacccagaatgaatttgcagt-3’(SEQ ID NO:16);
Neo探针(Neo Probe):
Neo Probe-F:5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO:17),
Neo Probe-R:5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO:18)。
将筛选出的正确阳性克隆细胞(黑色鼠)按照本领域已知的技术导入已分离好的囊胚中(白色鼠),得到的嵌合囊胚转移至培养液中短暂培养后移植至受体母鼠(白色鼠)的输卵管,可生产F0代嵌合体鼠(黑白相间)。将F0代嵌合鼠与野生型鼠回交获得F1代鼠,再将F1代杂合小鼠互相交配即可获得F2代纯合子鼠。还可将阳性鼠与Flp工具鼠交配去除阳性克隆筛选标记基因(该过程示意图见图5)后,再通过互相交配即可得到人源化IL15RA基因纯合子小鼠。示例性的F1代小鼠的鉴定结果见图6,其中,编号为F1-01至F1-13的小鼠均为阳性杂合小鼠,PCR测定引物如表3所示。
表3 PCR检测引物序列及目的片段长度
这表明使用本方法能构建出可稳定传代且无随机插入的IL15RA人源化基因工程小鼠。可通过常规检测方法确认阳性小鼠体内人源化IL15RA蛋白的表达情况,如流式细胞术检测(FACS)等。取骨髓细胞,用含10% FBS 的RPMI 1640完全培养液调整细胞浓度为106/ml,铺至24孔培养板内,每孔1 ml细胞,同时加入重组小鼠 GM-CSF(20 ng/ml)和IL-4(10ng/ml),37℃,5% CO2培养箱培养,此为培养的第0天。在第7天时,收集悬浮细胞及疏松贴壁生长的细胞即为树突状细胞(DC细胞)。分离DC细胞,按照如下染色方案进行流式检测:用抗鼠CD11c抗体Brilliant Violet 605™anti-mouse CD11cAntibody(mCD11c-BV605)和抗鼠IL15RA抗体Mouse IL-15R alpha APC-conjugated Antibody(mIL15RA-APC)、抗鼠CD45抗体Brilliant Violet 510™ anti-mouse CD45或抗人IL15RA抗体anti-human CD215 (IL-15Rα) Antibody,PE,Biolegend ™(hIL15RA-PE)识别染色后进行流式检测,流式分析结果表明,可在正常野生型小鼠骨髓细胞中经诱导后检测到鼠IL15RA蛋白(图7A),但未检测到人或人源化IL15RA蛋白(图7C);可在IL15RA人源化纯合子体内检测到人源化IL15RA蛋白(图7D),但未检测到鼠IL15RA蛋白(图7B)。这表明经人源化改造后小鼠体内IL15RA可正常表达。
提取野生型C57BL/6小鼠和人源化IL15RA小鼠骨髓细胞总RNA,利用逆转录试剂盒逆转录成cDNA,利用引物mIL15RA-RT-PCR-F:5’- GTGTGAACTCCAGGGAGAGG -3’(SEQ ID NO:27)和mIL15RA-RT-PCR-R:5’- GAGGACTTGTGACTGCCTGT -3’(SEQ ID NO:28)扩增大小为358bp的鼠IL15RA片段;
利用引物hIL15RA-RT-PCR-F:5’- TGACGGAGTGCGTGTTGAA -3’(SEQ ID NO:29),和hIL15RA-RT-PCR-R:5’- AGGCGGATGCTGTGAGTTC -3’(SEQ ID NO:30)扩增大小为354bp的人IL15RA片段;
PCR反应体系20μL,反应条件:95℃,5min;(95℃,30sec;60℃,30sec;72℃,30sec,35个循环);72℃,10min;4℃保温。使用GAPDH作为内参。
实验结果显示(见图8),野生型C57BL/6小鼠细胞中可检测到鼠IL15RA表达,人源化IL15RA纯合子小鼠细胞中可检测到人IL15RA的mRNA表达。
实施例2体内药效验证
利用本方法制得的IL15RA人源化小鼠可以用于评估靶向人IL15RA的调节剂的药效。例如,取IL15RA人源化小鼠纯合子皮下接种小鼠结肠癌细胞MC38,待肿瘤体积生长到约100mm3后根据肿瘤体积分为对照组或治疗组,治疗组随机选择靶向人IL15RA的药物X等,对照组注射等体积的生理盐水。定期测量肿瘤体积并称量小鼠的体重,可通过比较小鼠体重变化和肿瘤大小即可有效评估化合物的体内安全性和体内药效。
实施例3双基因或多基因人源化小鼠
利用本方法或制得的IL15RA基因人源化小鼠还可以制备双基因修饰或多基因修饰的小鼠模型。如前述实施例1中,囊胚显微注射使用的胚胎干细胞可选择来源于含有PD-1、PD-L1、CTLA4、OX40、LAG3、TIM3、CD73等其它基因修饰的小鼠,或者,也可在人源化IL15RA小鼠的基础上,利用分离小鼠ES胚胎干细胞和基因重组打靶技术,获得IL15RA与其它基因修饰的双基因或多基因修饰的小鼠模型。也可将本方法得到的IL15RA小鼠纯合子或杂合子与其它基因修饰的纯合或杂合小鼠交配,对其后代进行筛选,根据孟德尔遗传规律,可有一定机率得到IL15RA基因与其它基因修饰的双基因或多基因修饰的杂合小鼠,再将杂合子相互交配可以得到双基因或多基因修饰的纯合子,利用这些双基因或多基因修饰的小鼠可以进行靶向人IL15RA和其它基因调节剂的体内药效验证等。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
序列表
<110> 百奥赛图(北京)医药科技股份有限公司
<120> IL15RA基因人源化非人动物及其构建方法和应用
<130> 1
<160> 31
<170> SIPOSequenceListing 1.0
<210> 1
<211> 263
<212> PRT
<213> 小鼠(Mouse)
<400> 1
Met Ala Ser Pro Gln Leu Arg Gly Tyr Gly Val Gln Ala Ile Pro Val
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Leu Arg Val Thr Pro
20 25 30
Gly Thr Thr Cys Pro Pro Pro Val Ser Ile Glu His Ala Asp Ile Arg
35 40 45
Val Lys Asn Tyr Ser Val Asn Ser Arg Glu Arg Tyr Val Cys Asn Ser
50 55 60
Gly Phe Lys Arg Lys Ala Gly Thr Ser Thr Leu Ile Glu Cys Val Ile
65 70 75 80
Asn Lys Asn Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys
85 90 95
Ile Arg Asp Pro Ser Leu Ala His Tyr Ser Pro Val Pro Thr Val Val
100 105 110
Thr Pro Lys Val Thr Ser Gln Pro Glu Ser Pro Ser Pro Ser Ala Lys
115 120 125
Glu Pro Glu Ala Phe Ser Pro Lys Ser Asp Thr Ala Met Thr Thr Glu
130 135 140
Thr Ala Ile Met Pro Gly Ser Arg Leu Thr Pro Ser Gln Thr Thr Ser
145 150 155 160
Ala Gly Thr Thr Gly Thr Gly Ser His Lys Ser Ser Arg Ala Pro Ser
165 170 175
Leu Ala Ala Thr Met Thr Leu Glu Pro Thr Ala Ser Thr Ser Leu Arg
180 185 190
Ile Thr Glu Ile Ser Pro His Ser Ser Lys Met Thr Lys Val Ala Ile
195 200 205
Ser Thr Ser Val Leu Leu Val Gly Ala Gly Val Val Met Ala Phe Leu
210 215 220
Ala Trp Tyr Ile Lys Ser Arg Gln Pro Ser Gln Pro Cys Arg Val Glu
225 230 235 240
Val Glu Thr Met Glu Thr Val Pro Met Thr Val Arg Ala Ser Ser Lys
245 250 255
Glu Asp Glu Asp Thr Gly Ala
260
<210> 2
<211> 267
<212> PRT
<213> 人(human)
<400> 2
Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr
20 25 30
Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser
35 40 45
Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys
50 55 60
Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala
65 70 75 80
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp
85 90 95
Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr
100 105 110
Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu
115 120 125
Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala
130 135 140
Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr
145 150 155 160
Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser
165 170 175
Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln
180 185 190
Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile
195 200 205
Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu
210 215 220
Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu
225 230 235 240
Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg
245 250 255
Asp Glu Asp Leu Glu Asn Cys Ser His His Leu
260 265
<210> 3
<211> 5262
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctggcccaa gaaaagacaa taacaataac gttaagaagc aggcttggga caaacaagag 60
aaagggtagt tcttttagat gtcttcctct agccttttcc ctgaagagct cacaagtctc 120
tggagatact gatcgatagc tggaaaatgt ccagccacaa ataatgtaat agctcactca 180
caggtccaaa ggtccaggct ttctcagaca gagggaacat tttctaaaag gtcaggccta 240
gtatgtttca ctgaattctc cattctggtc tactgccaag tgtccttaag gtcatttgaa 300
ttggttttta tgacagttcc ttcccacccc acaccattaa tccatcccca agtatttccc 360
aagcaatgta acatacggct aagtacccca aatgaggatt taaagatttg gaaacacaga 420
gctaaacaca tgggactggc aagccacaca cacacacaca cacacacaca cacacacaca 480
cacacacaca cacaaataaa gaaataaata aatgtatttt ttaaagttaa atgaggcaga 540
accttcccct aatgaataaa gatttcaagg gaccaatcac tgggcaagta ggtgggaatt 600
tccagtcagt gagaggaata gggaagtcag gagaaagata cttccttttg gaccagaaag 660
agcatggaga cacaatgtgg gtagcgagag gcccacgatt taggtaggga tcctctggga 720
tgcgccactg gaggatttat aacttaaata gctaataaaa ttaggatgtg tcttagttag 780
agttttactg ctgtgaacag acaccatggc caaggcaagt cttataaaaa caacatttaa 840
ttgatgctgg cttgcaggtt cagaggttca gtccattacc atcaaggtgg gagcatggca 900
gcattcaggc aggcatggtg caggaggagc tgagagttct atgtcttcat ccaaaggctg 960
ctagtgaaag actgactttc aggcaactag gatgaggatc tcaagcccac acccacagtg 1020
acacacctac tccaaccagg tcacacctat tccaacaagg ccacacctcc aaatggtgcc 1080
actccctggt ccaagaatat acaaaccatc acaggatgct agtttccgca cccagcaatt 1140
gtgttacctg tagattctaa actaagattg tgtggtgttt tcctttactc ggtgactcaa 1200
ctgagttcca gagaaaagta cggtggcagg gcaccccagc cagccatggg aatttggaag 1260
tgtagggcat gtgtagcaac accccgccat gggaacttag cgagccgggt ggagagattt 1320
tgaagctctg aatcggagag tctgctgggc agagaacagg ctgggctgtc cactggtgcc 1380
ttgctggtgg tagtgtggat tgctttttta tatatttcac gcaacagtta aagccctaga 1440
gtagaagttt aggtgatctt ataaagacaa atgtgtaaaa gaaatctcca tttatggagt 1500
caatgccatg atctggaaga tggtgtggac aggctgctgt tcacactttt gtgtcataaa 1560
caaagcacca tttgctctgc tgcacttgta gacagcagca gtccccaaac cacagttcct 1620
ggacctgcag agcctgtacc atctggaact tggaaataca ggttcccggg ctctgcctca 1680
gatcctccag ctcagtagtt ctgggttgga gccagctagt tttaacaagc tctacagatg 1740
gatccacaca taactcctga gcattactga aaacatggac ctgaagtgac aggaggcagc 1800
agaggaagga tgccactgca gcagtgataa gctcgccctg ggagacaaag ctacggctaa 1860
gactccaaac acagaccaag tggaattaac agcacgggac cctggtcttg aaggaaggag 1920
ctctaaatgt tgtaaatagg tggttaataa gaagagggag tggtgccaag tcaggtatca 1980
aaagccacag ctagaggagt aactaaagga aactgtcttc tgcaactcac aatcttggct 2040
gaactgtcag tgcctcttag aggttccttc aagaaggatc ctggcagagc acttgtatag 2100
catatgcaag gccctgagtt tgatccatag cactgaagag gaggaggggg gaagagaaag 2160
aagaagagga ggaggaagaa gaggaagagg aggagaagga ggtctaagaa tagttaattt 2220
tctattgcaa taatagaata cctgagactc tgtggttcat agagagtcaa aatttattta 2280
gctcatgttg ctacaacctg ggaaatcctg gcatgggacc atctccacat attctaaatt 2340
atataatgca tttacagtta tacgtgggat agctatataa tgttaagtgg aattatgacc 2400
tggctattgt ttccatttgg aataataata agggaataat aataagggaa tgtgaactgt 2460
gtgtcaagtt gacaagattc ggacttgtga taagatactc taggttgtca acttgatgac 2520
atctgagatt aactaaaacc catgctgctg ggcacacctg tgagggagta tcttggttgg 2580
ctcttctgag ctgcacctct gggtggcagc ccacttgaaa gaacatgaag gaaggaagga 2640
aggaaggaag gaagtgtttg ctttttgtct gcttgccctc actctggctg gcaagttcat 2700
ccattctgtt tctcagttcg tttgctggta tcttcagggt tccgttgtag actgaggacc 2760
agcacctctc taggacttcc ctgggactct agttccacat gagtatgctg ataacgtttg 2820
gtctcgtgga ctgaacaact acctttctct caggaggtag ccattgttga ctatgttata 2880
attactgcta acacattctc cagtttacag cctctccaat gataaattct gaatgctttt 2940
atctggacgt atgtgatgag tgctcagagt tgaaagaatt ctggagaaaa cttcaatact 3000
tagaagatgt gaaacaactt gttgataaaa aaaaaaaaat ccctgagtct gaaggcaaaa 3060
gagaaagcag tggagatgaa aaggtggtcc tgaaatcatg ggtggattca cactcaaact 3120
atttcaaatt caaacctgtt gaacctaagt aaaaatgttc taaaaggaag tcaggcttgt 3180
tctgtgcgat tggagacaaa gagaaaagaa agcccaagat gggctggaga gatagcttag 3240
caggtaacag tacttgcctc tcttacagag aacccaggct cagttccttc catccacatg 3300
gtggctcaca gtcatttctg actctagatt ccaagttcta gagcatccaa caccctcttc 3360
tgacttactg ggcacagtgc gcacatatag cacacataca aacattcagg taaaacactc 3420
aaacatataa ggtaaaattt aaaaatccaa aaggaatgcc tgggtagaga aatggcttgg 3480
tggttaagaa cactggccgc ttgtccatag gacctggatt tgattcccag catccacaga 3540
gcagttttaa aaaatcatat gtagctccag ctctaggaga tctgacaacc tcttctggac 3600
tccctgggca ccaaggatac gtgtggtcca cacgtattca ggcaaggcac taatgcacat 3660
aaaggttttg gttttgtttg ttttgttgtt gttttgagac agggtctttc tgtgtagtcc 3720
tggctgtcct agagcttgct gtgtagacca ggctagcttc taactcacag aaatcctcct 3780
gcctctgcct ctggagttct ggggttaaaa agtcaaacac aactctggca tagaaatagc 3840
caacattttg gacccctcat ctatttctag aaccctcaga catcatgttc taaaactcct 3900
cacctctacc acacagaatt ctcccattga agccagtttt agctggagtt ttgcagtttg 3960
agagctgacc tatctaggca gttcatcttt gggacagttt gtctcgttcc tagtttcttc 4020
tgaaaaagtg aaataatcag agaaactgac tacgtatagg aaataaaaca aaccagacta 4080
aatcacagca gggagtcaaa ctccagcttg ttactgaaga aaagtgtcta tccatcatcc 4140
tttccattgt ttgttttaaa caaggcctca ctatgtagcc ctagctggtc tggaactcac 4200
taagtagacc aggctggcct caaactcaca gagttctgcc tgcctctctg cctctgcctc 4260
tgcctctgcc tcttcctctg cctctgcctc tgcctctgcc tctgcctctg cctctgcctc 4320
tgcctctgcc tctgcctctg cctctgcctc tgcctctgcc gagtgctgag attaaagctt 4380
gagcctccac accaagccta actataattc tttccataca tatctgcaca tctagtattt 4440
ccaggtattg tactaggatc aaaaatacaa tgttaagcaa attaggactg ggccaaatat 4500
agatgggaaa gatcatagaa ccttaggact caggttacca gtaaacaaat acaggttttc 4560
ttagaaggta aacatggcag tcagcaaaga aagttggtgg gaggtagtca gcggaaagtg 4620
tggtgggttg tgaaagagga tcggggagaa acaaaggtct tcgattatgg ggaaccaaag 4680
ggtaaagaca tctggaaggg cagattctga caaaaacctt gtaggcttct tcatgaattt 4740
gtggttgtat tttttttttt cctgtgagag tagcaggctc cgaaggaaga gtaaagtcaa 4800
gtacacctgg ctaacaatta tgctggtggc tgggtggggg gtggcacaca aggacacagg 4860
tctatagact cttgctattg atcaaaaaca acaatgtgac atccctcaaa aaagcttgat 4920
ccaaatgggg aaagatggat agcttcgaga gctatcttga gcttggaggt gggtgggcag 4980
tggagggtaa agggaatgtt tgctgatggc tcttgggtat ccagccgcac tgaatggatg 5040
gaatttaaag gaaactccta caagattttt gtatagtggt gtcctttctc accataccca 5100
tgtgtttttg cctggcttct acactcaggg gcccctggga tatgttcaac cctagctaca 5160
gagctgatac ttgggctccc ttgaggacac ccatgagtaa acccagtctt ctcgtctctc 5220
tcccctctca tgcactttgc aggcaccacg tgtccacctc cc 5262
<210> 4
<211> 3757
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgacacagc gcagctcact caccagttcc ctccacttcc tgacatttca gtttatatct 60
ttcatttttc tctgaagtac gtttatcatt ccttccttta aaaaaatatt cctcacgtga 120
catattggct ttgaccttgc acttttatca tttagcaatg aactgacata gttgttaact 180
actgacatag ttttttaact ctgtaccgta tacgtctcta gatcctgctt ccgggttctt 240
gcaggagtga ctattagcat tcttgaaatg actattagca tgcagtcact aggactaaag 300
tgttaactaa ctatccatgt aaaaggatag gacttcatct ggagatgcag tcagttgaca 360
aaacacaacc tggccctgga ttttaccacc agaacttata tatatataac tgtttgagcc 420
tggttttgtc tctgtaactc tctgaaacat agtgatgaat tctgaaagaa agctattaat 480
caaaattcac cataagaatt gacagctgtt aatgtaagat catttgatac aaatactgta 540
agccccagaa gtcattaata aatcactaaa ctaagattag tatatagtca agaattttct 600
ttctttcttt tttttttttt tttttttggt ttttcaagac agagtttctc tgtgtagtcc 660
tggctgtcct ggaactcact ctgaccaggc tggtcttaaa ctcagaaatc ctcctgcctc 720
ttcctcgcaa gtgctgggat taaaggcatg cgccaccacc gcccagctaa gaattttctt 780
aaaaggcata aaaaaaaaat ggtgctggag agatggctca atggttgaga gcactgactg 840
gggttcaatt cccagcaccc atgcggcagc tcacaactgt ctgtaactcc attcccagta 900
attccagttc cagaggatct gacaccctca cacagacata catgcaggct aaacaccaat 960
gcacatgaaa ttaaagttaa aaataaatct tttttttaaa aatcaaaaaa atgctacatg 1020
ctaacgaaca tagaaagtga ttttaaagca ttttcaaaat atctttataa cagagtggtt 1080
tttaatctaa tatcctgaat ttagagttat agccttgtga gataataatt acttaatata 1140
tccagaaatg tatgtgtcat ctttttaata aataatatat tatattctta aattaattgg 1200
taaatttggt atatacagcc aataaacatc acaatagaat aatattataa caatatttta 1260
aagtacatct cggtgcattt taaagcagta attgtaacat ctttgtcatc tgctgaatca 1320
attctttcca ttgactgatt tttttttttt taacaaattg gttgatgggt agatttgcca 1380
ttctacagac aggctttggt gtactcatcc atatttctga tttctggttt atccatattt 1440
ctgatttctg gtttctgtct cacacaaaac tctttactta atcatggctt gaaaatttat 1500
ttatccataa gaaaacatta tatgagccat ggtagatatc actgaccaag atacttaggg 1560
cctaaaaacc aagggaactc ccagacaggt agacacacag acacagccag acacacagac 1620
acagccagac acacagacac agaatacaga gcctccccct gctcttgcta tccgggagct 1680
tttcactgta tttaatgcta aatacaagaa tgttttttta aaaagccttg tgagtatcta 1740
gcaaccttca tccataccat caagcctggt ccaccatggt ccccgtctcc tctctccatc 1800
ccttgctacc catgctggtt tctagaggat gatcagcgga gttggaggga gggtggggtg 1860
gcatggtctg ttccagcaac cttgctaaag ccctggtgga tggatggcaa ggttgaagat 1920
ctctaaatag gattcaaact cttgctatgt gcacattcgg gaagcctagg ttttcctaat 1980
ctctaaacag cacagacctc tgagagtggg ggtggagggt gacatttgct aatgtaaccc 2040
actcagtacc gggcatacat gttcagtaag agatatgttg actgaagggt ggtcctgtcc 2100
tctttttaaa acctgtgaat gaactgcttt ataggcagcc ttctcagccg tgccgtgttg 2160
aggtggaaac catggaaaca gtaccaatga ctgtgagggc cagcagcaag gaggatgaag 2220
acacaggagc ctaaccacac gatttcagaa actcagggag acctgcccag ctgatcctga 2280
agagaagata caagggcagt tagtccagct gcgcctggac acaggagaaa gcccaatgtg 2340
ataatagagg tctctggtat gatctgtttt actgagcctg ggtgcctccc atattcaggc 2400
ctggatcccc ggagtccgga aggacaccac ctttcttcct ggttcatcac agaggccaac 2460
ttcccagagt acaagcagcc tgagctctcc tgggagtctc cattaaaagc ctcgagttcc 2520
actcagagat taactcaaag cagctgatct gctagactct ttttctattc cctactctga 2580
cttgcagttt acagagattg acaaggctcc cattgtcttc caaggctcct ctggcacagg 2640
agatgtctgt aaagaagaca gcaacatttg agctcctgaa gacttggccc tttgctgctt 2700
tgcacctatt ggaggagagc agagaacaga agaagagata ctgagccaat gaaccctttc 2760
gtataggatt catgacaaaa ccaaactcag tgactatata tgtatgtgtg tgtgtgtgtg 2820
tatgtaaaag tgtatattta catatacatt tatatttata ctttcttttc tattatatct 2880
acatattgta tatgatttat atttgaaagt gctttgtgta gacaaaataa aatatctatt 2940
ttcagtacaa aaagcattaa aattatcaac aaaaaaaaat gttcctcaaa ggaaaaaagg 3000
agaaagaggg gtgaaatggt tctaatgtga aagagatctc aggttctctt tcaaaaacag 3060
agtcacacca ccactgccct gcttttggtg ggatttttaa ccaaatttcc tagtgagtag 3120
aagcaacgtg aactaaacaa tggcaagtct gagcccagct agcattagac acataccttt 3180
ctctctctct ctccccactc cctccctttc tcctccctcc ctccttccca ccttcttcat 3240
ttctcccctt cctacccctt tcctgttatc acaaccattt ataaaatcat aagacaaatt 3300
ccacttcttt gttttattta tttatttatt attattattt gttggagagc cgtggataaa 3360
aagggatttg cccctagtag gcaagtattt caccactaaa tcatatccac atatctacct 3420
ttacagacaa aataatagta gcctggagaa gttgatctgc ccaaagtcac atagctcaac 3480
taatgctacc acttggacaa aactggggtg ggaagttggg taggagagga gctctggtca 3540
tgagtcagct gccaatgggc ctcctgcatc cctgggtaaa ggggaaccac cataagtatt 3600
tgggagatga gttaaacacc tggtgcacaa gctaccattc ctaactggct tctgccagac 3660
atcagtgtat gtggtttctt tcatcaaaac taaaggaagt ctttccgcat aggtcatggg 3720
cttcctcaga acatggtaga ctagttaggt tgtttaa 3757
<210> 5
<211> 9882
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgtccgtgg aacacgcaga catctgggtc aagagctaca gcttgtactc cagggagcgg 60
tacatttgta actctggttt caagcgtaaa gccggcacgt ccagcctgac ggagtgcgtg 120
ttgaacaagg ccacgaatgt cgcccactgg acaaccccca gtctcaaatg cattagtgag 180
tagcccttgc caccccacct tcctccctcc cccaccctgc caaggctgca cagagagaag 240
accccatctc aggggtcaag ggatctgtgc tgaggtctga ggctaaacac accagtgggg 300
gccgggcacg gtggctcaca cctgtaatcc cagcactttg ggaggccgag gcgggaggat 360
cacttgaagt caggagttcc agatcagccc ggccaacatg gtgaaacccc gtttctacta 420
aaaatacaaa aagtagccgg gcgtggtggc aggcgcctgt aatcccagct acttgggagg 480
ttgaggcagg agaattgctt gaacccggga ggtagaggtc acagtgagcc gcgatcacac 540
cactgcactc cagcctgggt gacacagcaa gactccatct caaaaaagaa agaaaaaaag 600
aaagacacca gtgtgatcct cagcaacagc tcctggcgtt ggaaacacac aggtgctggg 660
gtcacaggat tctgggttta aatcctggct ttgccgccta ccacccagga ggccttgggc 720
agctcttgtg atattctggc actctggctg gtcatctgga aaacggactt gatatccaca 780
ctgcgggctg tgggagggtg gcgtgaaccc tggtaaatat taactcttat tacatgtagt 840
gctttgattt gagacgatgc tggcaggaga atttggctgc agagctgggc gggcactcat 900
gagcacgcta gtgtgagaaa tgtttggatt tcccatgaga aggtggatgt tggaagccat 960
cactgctcct ctctcagtac ccagccccgg ggatgggcac aggccctgga gcagggctgc 1020
cctttgctca gtcacctgtg catggtgtcc agcaggggtg agggaaggtg gcaagaatca 1080
actttttttt ttaactgtct gcacctggat aaacgtccca gaagcagcca aaccatctcg 1140
ggtgggaacc acttggagct gtttctggaa cactcaggcc cacctctgca ggcttctctc 1200
cttccccagt tcatctctgg ggcagggatg agcgcgcccc tgcatggagc ggccccatag 1260
ctcacgccct ctgcagcagg ggacagaggg gagctccttg aggcgctgga cattgccact 1320
ggcttggtag tggtagctca gcctcctccc ccagaacaga gcgagtcaca caggcagcag 1380
agggaaggca gggaggaagc ctccgacttg ttccaagaaa tcactcgctg gggacacagc 1440
accccgtggc atcagtgagt gaatgggaaa gctgagcgtg gcggggtgag caggatctgt 1500
gggaaccacg gtgggtctta ctctgccagt cctgggactc cctggaacct gtctgcccga 1560
ggtaaaatga gggctttctc actgcagggc aagtcaggtc tcctcgacac tccatgtcct 1620
ttgggcaaat gaatccggga tggagaagag ctatgggctc cagggtcagg atgggagagg 1680
aaagaagaga acccggaatc aggattttag agtccaaaga ctggaggcct ctttaggttt 1740
cacctagccc aaaggatcct tccacgttct gatcactcac aactccctct gttatgcttc 1800
ccaaactctt gtgctttgaa tgactttttt cctgctcaac aagtgaaatg taagccaggt 1860
atggtggctt acacctgtaa tcccagcact ttgggaggcc gagatgggag ggatttcttg 1920
agaccaggag ttcgagacca gcctcagcaa catagcaaga ccctgtctct accaaaaaaa 1980
ttaaaagata aaaattacct gggcattgag gcacgtacat gtagtcccag ctactcagga 2040
gactgaggca ggaggatctc ttgagcccgg gaggtcaagg ctgcagtgag ttgtgatcac 2100
actactgcac tccaacctgg gcaacggagc aagaccctgt ctcaaacaaa caaaatagaa 2160
tctattgagt gatcatttat tctcccaaga ataaccagca ttataaaatt gaattgatat 2220
cattaatata atctcagcca aaataaattt gatatttaac ttgtgcagtc tttttggaaa 2280
tgcaagaaaa atagtttgca gtctatcttt gcattcgggt ggggaaacat tgtttcttat 2340
gcttttaacc tgtctgagga agccagaggc ctccactgta agttcatgtg ttgatatatt 2400
ttatttgtat cccgttctga agccagccca ggttccaggc tcaggatcat aagtgccatg 2460
tggttatcct ctctctagga gaccctgccc tggttcacca aaggccagcg ccaccctcca 2520
cagtaacgac ggcaggggtg accccacagc cagagagcct ctccccttct ggaaaaggta 2580
ggaaggtcag aaacttctcg aggacattca ttccctccca acacccagag gcccgctcac 2640
caatcctgca gactcacgga cctctgtggt gtgccactgc agacttaggc aatagcgcaa 2700
acgtcttatt caatcaagaa taagaacgtc caacttaatt ttctttttca aatcaaagtt 2760
gcttgatatc caggtgttat atgtgcagaa ttaatagtgg tttaaaactc actaatatgt 2820
catatcaaat ttagttgtag acacttatct ttgtcaagaa ctgaaagcag actgaggttt 2880
gaggttgcat gtgtgttagc cctcaacatt ttcatgggtg ctggcagaaa agagggcact 2940
ccaggatggg aggccagcag gcagcgggag cttcagcatg tttacatcaa ttccctcgtc 3000
ccctagccag caggggcagc acagttgggc ctgaacagat gcctgtccac tcagcgggct 3060
gcactgtggg agaggaagcc tgggcttccg agacccaagc gttttgtcat gggcagtaat 3120
cacacctgct ctctgctcca ggggagacca tgtccccgtc ttccaaggcg cttgctgtag 3180
aagcaccctt gagaggatgg tccagagaaa gggccatcag ctcctctgtc tgcaagatgt 3240
gcaaaaatgt gagagacctg tggagagctg cccccaccag acctaaacac cagcacatcc 3300
cagtgactgc tctattatag aggcgctgcc tgaagctgcc agtgtcctgt gtttcaacag 3360
agtctttggt gcttgtcagg cccaatttcc tcagcgcttt ctcagtgttc agaaaatttg 3420
aaatcttgga tgctttcttg ggatcctccg ccctgcatgc tatgatacct ttgtgctaca 3480
gttcactctt ggctttagtg ttgcatcgtc cagcctgtgg ctttccctgg tcctgtctac 3540
tcctttgccc ccggggggat gggcctgtct gttgtcattc atactacagg actcagccat 3600
cctttgcgga gtgtcagttg tcagccgtgg gtacacactg acatcacctg ggcactccag 3660
ttggcctggg tgcaatctgg gactcacgcc tctccagccc caggtggatc tttttttttt 3720
ttttttttga gatggggtct tgctctattg cccagtctgg agggcagagg tgaaatcttg 3780
gctcactgca acctctgcct cccaggttca agtgattttc ctgcctcagc ctcccaagta 3840
gctgggatca caggcgtgca ccaccacgcc tggctaattt ctgtattttt agtagagaca 3900
gggtttcacc atgttggtca gtctggtctc aaactcctga cctcaggtga tccgcaagcc 3960
ttagtctccc aaagtgctgg gattacaggt gtgagccact gcacccagcc cacaggtggt 4020
tcttaagtgg ccaaggctga ggcctgcatc ttaagggagg aagaagcagc taaacaaggt 4080
tccctcctag tgagtcacct gcacagggag gaaggggttg aggggctggc tccatttaaa 4140
cttgaagtaa ttctacaacc cctttgatct gagtcacacc tggttcaccc aaggaggaga 4200
atgatcaagt aggctctccc agccccacct tcgcatcccc aatacctggt ccgatggcca 4260
ttgtcgagac acaatggtgc agtcagaagc cccgatgtga cagcagcctt tgacccgagc 4320
tggcacgacc gtgggcactt ggctgaggat gccttggtgc gtcctgtgca gggagccctg 4380
gagcattggc agtgagcgta gggcaagtta tatgaaaggt ggtgacacag gattaagggg 4440
cgtgaggcct tctcatgacc tgtgtggctg ggagaccatg ggaacgcggc aacaacagtg 4500
cgtccagaga gccacagcgc ttggcctcac gtttcctaga ttccgtggct gtcacaatgg 4560
aagacacatt tttcatggag agggaacagc acagttacgc cacaccctta cagtgcaggg 4620
gcagccacct tccagggaag gacaaggaag acagggaaga cgctgaacac aaggcagcct 4680
ctgttcctga gagcaagctc atcaggacgc tttcctccca cacagcccgg agagttcagg 4740
ccagagccca ggcttcccgt gtttcacacg cagccgctcc gagcgtcctc ggccagcgca 4800
gcttcacctg ctctcagctg agcctccagc gttgggcctg ccttctctag taaacaagct 4860
gaacgactca gatctttgac accttttctt ttcttccccc cacctttttt tttaagagac 4920
aggggtctca ctctgtcccc caggctagag tgcaatggct caattatagg tcactgcagc 4980
ctcaaactcc taggctaagg tgattctctt gcctcagcct ctcagatagc tggaactata 5040
ggcacatgcc caccacacct ggataattta tcgttattct tatttttgta gaaacagggt 5100
cttgcggcag ggcacagtgg ctctcgcccg taatcccagc actttgggag gccaaggtgg 5160
gtggatcacc taaggtcagg agtttgagac cggcctggcc aacgtggtga aaccccatct 5220
ctattaaaaa tacaaaaatt agctgggcgt ggtggtggac acctgtaatt ccagctactt 5280
gggaggctga ggcagggaga attgcctgaa cccggaaggc agaggttgca gtgagctgag 5340
atcgcgccac tgcactccaa ccttggcaac agagcaagat tccatctcaa aaaaagaagg 5400
aaaagaaaca gggtcttgct gtgttgccca ggctggtctt aaactcctgg gctcaagtga 5460
tcctcacacc tcagcctccc aaagtgttgg gattataggc atgacccact gtgtctggct 5520
gatcttttct ttatacgtgc cgtgatagta gccatgttct ttagtgtttt cacaggtcct 5580
atggcccttc cagaggaacg gctatgtcac cctctcaagg agcagttggc tgtgagagag 5640
ggagggctgg tcagggtgac tcactatggc agctcccaca tcaccccgtg ctgcagctga 5700
gaggagggga gtctgcacac tgatgttgtc cctgccttgg actctcctac agagcccgca 5760
gcttcatctc ccagctcaaa caacacagcg gccacaacag cagctattgt cccgggctcc 5820
cagctgatgc cttcaaaatc accttccaca ggaaccacag agataagcag tcatgagtcc 5880
tcccacggca ccccctctca gacaacagcc aagaactggg aactcacagc atccgcctcc 5940
caccagccgc caggttagca ctcgctttgc cccaggagag gtcagttccc cattgctgct 6000
tctgagatcg gggctgagat ccggcacaag gcagctttat accatcaatc caaagtcagg 6060
attcactggg cagcctgctg gccccggcca ccagccacgg cgtcctggcc tgacctcttt 6120
ctccgtggct ttctctgtgg ctggggcagc tgcaaaagca agcccacacc cccccaagat 6180
gtgacagggc cagccttatc acccgcatgg cagagcctgt gcagctggga gtggcatctg 6240
tgccctcctg cagtaggagg tggtgaagct gcctacagtc cggggccagg atctgtggaa 6300
tggaggagct gagggtaatg gggaccttct gtaaggctta tctgattctt aagtgtggcc 6360
agcacctcag tgagctaccc tgcttgttct ttgctaaaat gagtctggtt acgccagcca 6420
cgagtctccc agggtgctaa gatgggagcc aagccatgca gattttctcc tgcttcatga 6480
ggaactagga cctctaggac tcgagccgtg atgctgtcct ctcttttccg caggtgtgta 6540
tccacagggc cacagcgaca ccactggtaa gtgtgtccct ttgtccggta tgtttatgat 6600
cagggtgacc gcagtcccag ggtgctgggt ggtcctggcc caggcccgct gccccagcac 6660
agccctgaca gcagcccctt ccacactcag aaggcctcca tcagataata agttaagtaa 6720
tagctgattt atggaattct ctgtttgatg tgcccatttt tctaaatatt ggtagcttct 6780
agctgtcagg ctactggggt tgaacccaga gagctggcac cccaaataag ggctcaaggt 6840
cagctcttca cagcagggtg cacctctttt gcatggagca tccaaaacta caggtgctga 6900
tgccagcact gacgttacag catctctgcc agaagctgcc aattcctctc cagggcgtgg 6960
caccgtgcac ttaacagaga agctgtccac attttcggta taaaatgcta ctcttggcca 7020
ggcatggtgg ctcacacctg taatcccagc agtttgggag gccgaaatgg gaggattgct 7080
tgaggccaga atttgaggcc agcctggtca acatagcaag accccatctc tattaaaaaa 7140
aaaaaaaaag aaaaagaaaa agaaaaaaag ttaacaaaac aaaataaaac caaaaatctg 7200
ctcctcttta cagaagtcac acgcaatgct aaggctagga gaatagaaaa gagaacgtgc 7260
aagtgatgat aaggaggaaa aggggaaagc gtttacaaga ctaaaagaaa aagctgaagg 7320
gaggggagga gagctgagag aggaggggag aacaggagga agaagggaag caggcaggga 7380
ggaagaacgc aggtaatgag aagaatgcaa gtcatgctcc aggctgacaa gcttccaaag 7440
taacaaaaaa cccaactgta atttgatatg gacaaaaatt attttaatct tctgttcata 7500
aatttttctg ctagagaatg actcatattt ttgtggtgtc atttttacct gcatcaaacg 7560
tcatctggga aaacatcaag tgactttgga cattttagca gttaattgat tagaatcgtt 7620
taaaaaacaa tcaggctggg cgaggtagct cacacctata atcccagcag tttgggaggc 7680
cgaggggggc tgatcactgg aggccaggag tcccagacca gcctggacaa catgacaaaa 7740
ccccatcttt actaaaaata caaaaaatta gccagacatg gtggcacatg cctgtagtcc 7800
cacctactcg ggatgctgag gtgggagaat tgctggaacc caggaggcag aggttgcagt 7860
gagctgtgat cacaccactg cactctggcc tgggcaacag gacaggaccc tgtctcaaaa 7920
aaaagacgaa aagcaaatcc catttctaac tcaaaaatcc ccttgcagga aggaagcaac 7980
acttgctacc agatttccag gcatccttcc agagagactg tataaataaa catatagagg 8040
ataaggaatg ctggacagtt gcttcttttc tccttttctg taccatgttt gtttccactt 8100
gctggtatac cttgtagatc ctcccatatc aggacacaaa aaccatctca tcgtttttaa 8160
cagctacaga atattccact gtatggaatt cctaccatta atttagccaa ccccctatct 8220
ggacatgtga ttgctacatt ctgttgttat cttacaaaca gccattaatt gaaaatccat 8280
aggtgcaaga acatctgtag gataaaatcc tacatataaa gttcaggggt cattttttct 8340
atatacattt tccaacaact atttgatgat gggctataat ctataatctg cttttgctag 8400
ttcatgactt ttattagatt aaaaaatcag ccgggcgctg tggctcatgc ctgtaattcc 8460
aacactttgg gaggccaagg tgggcagatc acttgaggcc aggagttcga gaccagcctg 8520
gccaacatag caaaacacca tctctaccaa aaatacaaaa attagccagg tgtggtggca 8580
ggcgcctgaa atcccagcta ctcaggaggc tgaggtagga gaatcgtttg aacctgggag 8640
ggggaggttg cagtgagcaa actcgagccg ctgcactcca gcctgggcaa cacagtgaga 8700
ttccgtcgca gaaaaaaaaa aaaaaagaag aagaaaagaa aaagttggcc aggcgtggtg 8760
gttcacgcct gtaatcccag cacttcggga ggctaaggca ggcggatcac gaggtcagga 8820
gttcaagacc agcctgccta acatagtgaa accctgtctc tactaaaaat acaaaaatta 8880
gccgggcatg gtggcacctg cctgtagtcc cagctactcg ggaggctgag gcaggagaat 8940
tgcttgaaca ctggagctgg aggttgtagt gagctgagat cacgccactg cactccagtt 9000
tgggcaacaa aatgacactt catctcaaaa agaaagaaag aaagaaaaag ttgtaggtag 9060
actggacgca gtggctcatg cctgtaaacc cagcactttg ggaggctgag gcaggtggat 9120
cacttgaggt caggagtttg agactagcct ggccaacatg gtgaaacccc gtctctactg 9180
aaaatacaaa aaattagcca ggcgtggtgg tgtgtgcctg taatcccagc tactcagaag 9240
gctgaggcag gagaatcact tgagttcagg aggcagaggt tgcaatgagc cgagatagtg 9300
cctctgcact ccagtctagg tgacagaggg agaccctgtc tcaaaaaaaa aaaaaaaaaa 9360
aattgcaagt gaaaaaaaaa tgaaattgga aggaaaatgg cactgtgtgc tattgtttct 9420
aaatctgtga agttgaagca aagtgggcgt tgtactgggg acagcagagg aggggtgtgt 9480
ccctgtctgc tttttcaacc ctggctttag cagcaggaat gctctctcct catcgcactc 9540
ttggctttgc ccttgaggct cttactgctc cctcctgact tgctccgtcc ctgccccgtc 9600
accccaggca ctctgcctga gaaggacaag gatgaggagc agaggtgtgg gacgactggg 9660
agctggcacc ctcagcagga aagagaatgt gtttttgcct agacatagaa tcgggggaat 9720
tgcatggacc ttaagaaatg tccttagcag gaggctggtt gggaggcact tggatccctc 9780
tgtaagaaca tccatggtaa agaatttgtg gttcgtagca atgaatgtgc ttctgggtat 9840
ctcagcgtgg tctcctccct ttcagtggct atctccacgt cc 9882
<210> 6
<211> 87
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
catgtcagcc ttgattctgt atttctaata gcagaacata tggaattccg aagttcctat 60
tctctagaaa gtataggaac ttcaggt 87
<210> 7
<211> 102
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tctctagaaa gtataggaac ttcatcagtc aggtacataa tggtggatcc atgacacagc 60
gcagctcact caccagttcc ctccacttcc tgacatttca gt 102
<210> 8
<211> 1673
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
tctctgcaat ccgggggcta aagcaaaagc gaaagcgaat actccttagc agcggtctgg 60
tcctggagct gagctcgcca cctcccaggg accccagcct tgcgtcccgt tgggtcactg 120
ctggggacaa ttggccatgg cctcgccgca gctccggggc tatggagtcc aggccattcc 180
tgtgttgctg ctgctgctgt tgctactgtt gctcccgctg agggtgacgc cgggcaccac 240
gtgtccacct cccatgtccg tggaacacgc agacatctgg gtcaagagct acagcttgta 300
ctccagggag cggtacattt gtaactctgg tttcaagcgt aaagccggca cgtccagcct 360
gacggagtgc gtgttgaaca aggccacgaa tgtcgcccac tggacaaccc ccagtctcaa 420
atgcattaga gaccctgccc tggttcacca aaggccagcg ccaccctcca cagtaacgac 480
ggcaggggtg accccacagc cagagagcct ctccccttct ggaaaagagc ccgcagcttc 540
atctcccagc tcaaacaaca cagcggccac aacagcagct attgtcccgg gctcccagct 600
gatgccttca aaatcacctt ccacaggaac cacagagata agcagtcatg agtcctccca 660
cggcaccccc tctcagacaa cagccaagaa ctgggaactc acagcatccg cctcccacca 720
gccgccaggt gtgtatccac agggccacag cgacaccact gtggctatct ccacgtccgt 780
cctcttggtt ggtgcagggg ttgtgatggc tttcctggcc tggtacatca aatcaaggca 840
gccttctcag ccgtgccgtg ttgaggtgga aaccatggaa acagtaccaa tgactgtgag 900
ggccagcagc aaggaggatg aagacacagg agcctaacca cacgatttca gaaactcagg 960
gagacctgcc cagctgatcc tgaagagaag atacaagggc agttagtcca gctgcgcctg 1020
gacacaggag aaagcccaat gtgataatag aggtctctgg tatgatctgt tttactgagc 1080
ctgggtgcct cccatattca ggcctggatc cccggagtcc ggaaggacac cacctttctt 1140
cctggttcat cacagaggcc aacttcccag agtacaagca gcctgagctc tcctgggagt 1200
ctccattaaa agcctcgagt tccactcaga gattaactca aagcagctga tctgctagac 1260
tctttttcta ttccctactc tgacttgcag tttacagaga ttgacaaggc tcccattgtc 1320
ttccaaggct cctctggcac aggagatgtc tgtaaagaag acagcaacat ttgagctcct 1380
gaagacttgg ccctttgctg ctttgcacct attggaggag agcagagaac agaagaagag 1440
atactgagcc aatgaaccct ttcgtatagg attcatgaca aaaccaaact cagtgactat 1500
atatgtatgt gtgtgtgtgt gtgtatgtaa aagtgtatat ttacatatac atttatattt 1560
atactttctt ttctattata tctacatatt gtatatgatt tatatttgaa agtgctttgt 1620
gtagacaaaa taaaatatct attttcagta caaaaagcat taaaattatc aac 1673
<210> 9
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
acagcagtgt cagccatcag gaaaa 25
<210> 10
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccctgagatg gggtcttctc tctgt 25
<210> 11
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
gctcgactag agcttgcgga 20
<210> 12
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
ctcgggaatg gagcaatctt aggca 25
<210> 13
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
tcctatcagg cagggttcac aaggt 25
<210> 14
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 14
aggagcctaa gagtcccttc ctcac 25
<210> 15
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 15
cagatcccca gccttttgca acatc 25
<210> 16
<211> 26
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 16
tcaagaaccc agaatgaatt tgcagt 26
<210> 17
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggatcggcca ttgaacaaga t 21
<210> 18
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 18
cagaagaact cgtcaagaag gc 22
<210> 19
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 19
gcatagggag tacgggggat agata 25
<210> 20
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 20
aaaggaagca ctatgccctg cagtt 25
<210> 21
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 21
ctgtgaagtt gaagcaaagt gggcg 25
<210> 22
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Met Ala Ser Pro Gln Leu Arg Gly Tyr Gly Val Gln Ala Ile Pro Val
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Leu Arg Val Thr Pro
20 25 30
Gly Thr Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp
35 40 45
Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
50 55 60
Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu
65 70 75 80
Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys
85 90 95
Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr
100 105 110
Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser
115 120 125
Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala
130 135 140
Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser
145 150 155 160
Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly
165 170 175
Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala
180 185 190
Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr
195 200 205
Val Ala Ile Ser Thr Ser Val Leu Leu Val Gly Ala Gly Val Val Met
210 215 220
Ala Phe Leu Ala Trp Tyr Ile Lys Ser Arg Gln Pro Ser Gln Pro Cys
225 230 235 240
Arg Val Glu Val Glu Thr Met Glu Thr Val Pro Met Thr Val Arg Ala
245 250 255
Ser Ser Lys Glu Asp Glu Asp Thr Gly Ala
260 265
<210> 23
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcaggcactg agccctatga tacag 25
<210> 24
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 24
cccggaagca ggatctagag acgta 25
<210> 25
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 25
gacaagcgtt agtaggcaca tatac 25
<210> 26
<211> 24
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 26
gctccaattt cccacaacat tagt 24
<210> 27
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 27
gtgtgaactc cagggagagg 20
<210> 28
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 28
gaggacttgt gactgcctgt 20
<210> 29
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgacggagtg cgtgttgaa 19
<210> 30
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 30
aggcggatgc tgtgagttc 19
<210> 31
<211> 12304
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gtatctattg agcatgctga catccgggtc aagaattaca gtgtgaactc cagggagagg 60
tatgtctgta actctggctt taagcggaaa gctggaacat ccaccctgat tgagtgtgtg 120
atcaacaaga acacaaatgt tgcccactgg acaactccca gcctcaagtg catcagtaag 180
tagccagtct ccacactgtc ccacacactc cagccaaggc tgaacaggga gctgtgggag 240
ccagagacat ctgttctgag atctggagct aactaagcca gtaggacatg aagcatcacg 300
ccctcccccc atgggaatac attggtagtg ggttctgtca cccaacattg gttcatctcc 360
tatctctgcc atttagccat tttgtgtcct tgggcaaatc ccttaggctc ttggaactct 420
ggttggtcac tcaaaatatg cacctgatgt caacagtggt ttgtagaatt gggtggaacc 480
tggtaaatgt taactattac agctgtcagt gtttgttttg gcatgacagt agcaggaggt 540
ttgtctatag atgatgctgg gtgtgcaaac tcaccaccct gagagctttt tggatcacta 600
acaacgcata agtctgtggg agaacataga ctgtttttgt ttgctggaac ccaggaagtt 660
gggtaaacct aacaaacagt atagctctcg agtacccact tctagtgtcc agcagatgct 720
caagtacaac aataataata aaataatcta ggacgtgatg gtgcatgcct tcaatcccag 780
cactcagaag gcaaatggat ctctaaggtc cagtcccagc ctgaacaata aagagcaaca 840
aagcaatgaa ggttatgaga atcaacttta aaaaaaaatc accatggttg gatgatgttg 900
gttggccaac atttttggaa tggaagccgc ttggtggtgt gcttccaagt aacactatcc 960
cccagtctgc ttcctagaaa aaataaaaag tggcccgagt cttctaggag agtaagtact 1020
gctcaagcaa gaatcccatg aatgtgatct atcttaggat gtcctcccct gtccttctct 1080
atcctgtttt ttgacacagg gcctctcact gaatccagca cttactgact gactgagtag 1140
tgagctgtgc agagctgcct gcctccactc cctgccaaca gcactaaggc tgcagacttg 1200
ggccaccatg ctcagagttt tacaagtgta ttgaggatca gaacccagat ccttatgtgt 1260
gtaccacagc tatcactcca gccttgtcct caggatctca taacccaaag actggaggga 1320
tctttaagtc agcctcatct aatccaaagg ttttgctcca cagtcatttt accaccagca 1380
gctctcttca ctgtttcccc ctagctcctg tgcactcaat aatttcccct aaataagtga 1440
actccattga gtgatatttt gtttttcccc atgaaaaact aataaaactg atgtttcgtg 1500
tgtgattccc atacaaaaat atttgatgac tgacccgggg gaagatacta gaaaaagcag 1560
tttacaggct gtacatttga gtcgggaaac cagtattctt tctagccttg tcccaggaag 1620
ccagaaatga atgtgcaccg attcattatt tttatttgta gctagttctg aaactgccca 1680
gatactgccc tcagaaatca taaatgccat gtgactattc tctctctagg agacccctcc 1740
ctagctcact acagtccagt gccaacagta gtgacaccaa aggtgacctc acagccagag 1800
agcccctccc cctctgcaaa aggtaggaac cctgggaaaa cgtgaagaac taatgcttac 1860
cccgccagct cccttatcat agatgccatt gctcactgct gatcactgca aacctaggca 1920
acagaacttt ataagattca gaacttgtag ctgggtggtg gtggcatgca cctttaatcc 1980
cagcactggg gaggcagaaa cagatggatc tctgagttca aggtcagcct gatctacaga 2040
gcaagtttca ggaaatacag cattatacag agaaacccct tcttccaaaa cccaaaacca 2100
aaccaaagta aataaataaa taaataaata aataaataaa taaataaaaa aaatcagaac 2160
ttatagtggc ttagggtttt tctagtcaaa gttatttgat atccagaatt cacatgtgca 2220
taatttataa tggcttagga tcggccatta acatgtcaga gcatagggag ctgtagatgc 2280
ccacctttat cgagcactga gacttgatct cattatgttg tcttcccaag ggaggctttc 2340
ccacatggca caggaaatct gaagcagatg tatgtgtgta ccaattcata tctctctgga 2400
ttgtgccagg gcagcctagg gtcatagagc ctgaatgtct tatacttggt ggcaaaggtg 2460
cctgtgtttg ttccagaggg aggtgtggtg tctgtcttcc aaagctacca tgcatgtcct 2520
gggcaagaca gaccacagaa agggtaatga ggatggagcc ctcttttggc aaaataaaga 2580
caatgtgagg gactcattga agagctttta tttatttatt cttaagtgga agggttgggg 2640
gtgggttgag gcagggtctc actatgtagc tctagctgtc ctagaactca ctgtgtagac 2700
caggctggcc tcaaaattac agagattcat ctgcctctgc ctcttgagcc ctgggattaa 2760
aggtgtgagc catcaagtcc agctccccaa tcttcttcat taacacatta atctgaatgc 2820
ttgttctagt gtctttttta atgataggct aaaatatccc aacagagagc aacttagggg 2880
agaaagggtt cattttatct tacgtttcca ggtgggaagc catcgctggg aaagtcacag 2940
aggcaggagc ttgaagaagt cagtcgcatc acacccacag tcaagagtgg aaagaaatga 3000
aagtgtgcat gttcatttgc ctaattgtgc cctgctttaa tttctccaca cgtttttttt 3060
ttaatagaat tatttattta ttttatgtgt gtgagtacac tgtagctgtc ttcagacaca 3120
ccagaagaga gcatcagatc ctgttaccga cggtagtgag ccaccatgtg gttgctggga 3180
attgaactca ggacctctgg aagagcagtc agtgagtgct cttaaccact gagccatcta 3240
tccagcctca gtttctccac tcttaagagc acaggagccc ttgcctatgg aatggagcca 3300
ctgacggggg agaggagggt cttcccacat caactaactt cgtcaagaca atccgccata 3360
gacatgctgg acagcccaaa ccagtataga caatttaaag tgtcaagtta aagccaacca 3420
tcacaaggct gtcatacaga aatactgcat gaccatcaca tctctctcag tccctgcctt 3480
ctttctttgc ttcctttctg atagggtttc aataggtatc ccaagctagc ttcaaactca 3540
tcctccagaa tgctcagatt atatgtgcca ctgaacccag ctcattgttt tgtccttcgc 3600
ttgagtgttg tgtaggctag ctcaatgtct ttagcatttg tctccatatt aacattggaa 3660
acctgtgcgc tctatcctgt gatactctcg tttcccactg agtgctttgt tctccctgtt 3720
acatgatccc atttgtgact tctccttgtt tcttccttct actcccaaat atggaaacct 3780
ctttttcaat caccctaact cgctcaactg gtcagactat cctgtatgcc ccatgtcagt 3840
cttcaataca caaggaactt actgtaatgc tcccgtttgt ttagggtaag atctgagaat 3900
cagcaacgct ggaagcttcc cgggtggttc agaggcgtcc cagcggagga agtttgttct 3960
aaggaggatg aggggtttct gaatttcatg ccagcgcaca gcatagaagg ctccagttca 4020
gaagttaata gtaataaaat cgggctggca ctataggtca ttgatagaat gtttccctag 4080
catgcatgag gctctcccag acatttggtc agacattcct ctctgagaca aacatgtaca 4140
atcataatct tggacactaa ctggtatagt cgcaggtgct tagatgtgag tgctctgcgg 4200
ggttggtagt tgagggaggg agagatacaa atgagtagtc ccagtggact gaagggtctg 4260
agcatctctt acatggagaa tggctaagag agtatgggaa caggacactc catgcctctg 4320
gagcactagg atgcctacca ggctttactc aggctgaatt tcgcaagtga gattttgcca 4380
cagtgcaaga cccattttca agagaaggga atagagaata gggaagcggt aggccccttt 4440
atagtttagg ggcatccgtt tgctaataac gaacaaggaa gacaggaacc taaacccagg 4500
catcctccat gccagcagga actaatgcag aacagtctcc tcagataccg cctagggcat 4560
ctggagaggc cggaataaag ctgcagcttc ttatttcata atgtcgtgcc aacgtgtaga 4620
tagtcggctg tgatggagca cacctttgat ctcagcactc aggaggcaga gccagctgtc 4680
agaaccttgt gagtttgaag acatcctggt ctacacaaca agttccgggt cagccaggga 4740
tacataaaag actctgtctc aaggaacaac aacagaactg gaagagaaca ccagcattcc 4800
tgctatcttt tgcaaatata gttccacttg tgtcttggca tcgagtcttt aagaatataa 4860
cataccatct ccagagaaca aggatatctg tctgtctctc tgtctctcct tctctctctc 4920
tgtctttctc tctctctctc tctcagtgat tatattattt agagttatca caagtactat 4980
tgtccagggg ctagctatgc catctactta ggggaccgtt tttttttttt tttgaaaggg 5040
tgctggccag aacaagctca ttaaagtaag tccttgggtc ccaggcacta gtctgaattt 5100
ttatgtctct gtcttggaat ttcctgcaga gccagaagct ttctctccca aatcagatac 5160
cgcaatgacc acagagacag ctattatgcc tggctccagg ctgacaccat cccaaacaac 5220
ttctgcagga actacaggga caggcagtca caagtcctcc cgagccccat ctcttgcagc 5280
aacaatgacc ttggagccta cagcctccac ctccctcagg ataacaggtt agcctgagct 5340
ttgcacaggg acaggccagt ttcccacata ctgcttctga tacctgggtt gggatcttac 5400
aagaagatgt ctacattagc tttggtccac ttatgctcaa tttgggactg actgagaggt 5460
ccagtcgcct gttgtcaatg gttagttggt tattgcaatc cccactgtaa ggatatcaga 5520
aaggaaaccc ctcctctcac atatgacatg gcttattgat gtgaatgcta gagagtggca 5580
aacgtatctg ccagtgtgga gctgtgctct gtggagtaga aaaaccacca tcaattgctc 5640
ctttctaagg catggccaat gcttagatgt gaccaaatac ctcagcaagt taaccagttt 5700
gttttgcaaa gaccaatttg gttctaagat agtagctaat ctctccagct tcatgagaca 5760
ctaatgatct tgggagtcca gtcctgattc atctctcctc tctcctctgc agagatttct 5820
ccccacagtt ccaaaatgac gaaaggtgag tatttctttt gttccatgtg tttataactg 5880
gggcactaca aagcagaccc tgcatgctca acagcctgat ccagtaccac tctaccacag 5940
taactagcac taacccgttg cttctctgaa atgtctccat tagagaataa tttgtagaat 6000
atcctagtta ttgaatttgt cattagacat gatcatttaa gatgagtagc ttctaactgt 6060
cacactagag gcatacaaag cagggagtta gtacaccaca aaggagcttg atggttcagc 6120
tactcacagt taagtcccac tgggattcac tcacattcag gcatagaagg aaatcacagg 6180
agcctgccac tgccactaag ctaactccat tcggccagaa gctgcaggga cctcacccag 6240
actggagcat tgtgcattgt atagaaaggt agcccagatg gctccattgt gaagaagaca 6300
tagtgcctta cagaagaaaa tgaaagccac atataagcca ggcaggagga aaacaaacag 6360
tgcttgtgaa aaggtagacg gagatctttg agaagaaaga gaagagcagg agagaaggga 6420
gggctggggt gcagatcagt gcagggagct tgctcaacaa gtccaaggct caaacttaga 6480
tcaggatact ttgacttctt ttcctattcc tgtccctttt tatttccatg tcttgtctta 6540
ttgttctggc tatggtttca aaaactcagt tgaacgggag tgaagagaat ggatcttgat 6600
cagagaattg tttttggcct ccaatgctgt aagaaaaaga ctggggtgta aagagtatga 6660
cgttaatact ttagtcagac aagctttaaa aggaacaaaa ggcactgcag agatggttca 6720
ggggtaaaga gtatctactg ctcttgcaga ggaccagcat tcagttccca ccagcccaga 6780
ggaataggtt acaaccactt ataactccag ctccagggca tacaatagcc ttttctgtgg 6840
gcacctgcac actcatgagt atgtgtacat agacacacac agacacacac acacacacac 6900
acacacacac acagagagag acacagacat acagagacac agacacacag acacacacac 6960
agacacacat acacacagac acacatacac acagagacac agacacacac acagacacac 7020
acagacacac acagacacac acacacacac acagagagac acagacacac acacacacag 7080
acacagacac acacacacac acacacacac acagacacac acagacacac acacacccca 7140
caaataaata tgtgtaaagg gataaaataa tatgattata atttaacatg gacaaaactc 7200
ttttcacttt ctattcataa gttttcctgc ctaagaatgg ctcgctggct tttgtggtcc 7260
tgtttttacc tgcactaagt aacatctgaa aatcaaatgt tgctttagat agattagcag 7320
ttttgttagt tttgtaaatg atcaaatgca aagccaggcc tggtggtgaa ggttcgtacc 7380
ctcagttact caggaggctg aggcaagagg attgcaagtg caagaccaac ctggacaact 7440
cagtgaaatc ctgtattaaa gttaaaatta caaaaaaggc tgtgaactgg ctcagtgata 7500
ggggagttgg tcagtcctca gcactggggg aaacaggaga gagacagaca ggcggggaat 7560
ggacagatag atgatagatg gatggatgga tggatagata gatagataga tagatagata 7620
gataagatgg atggataaaa gcaagacaat ttagaaagac tctttgaaga atattttcat 7680
ggaaaaattc acttactgac aaggacatgt aaataaaacc acattcatga tttaagatca 7740
ctacaagaat tagccaaact catttccccc tatgacacat agcacattac tcttcccatg 7800
tggttttaga attttttctt tgtttttcaa attcttaata atttgcacat ggtctgaaac 7860
cataaaaaca ttaagctaag catggaggca tatgtctgta atctcagaac tcagaaggcc 7920
aaggcaggag gattactgtc catttaagat ccacctagct acataaaatg aataaatctt 7980
aaaatggggt gggggggggc tggagagatg gcttagcagt taagagcact gactgctctt 8040
ccagaggacc tgggttcaat tcccagcaac cacatggtga ctcacaacca tctgtaatgg 8100
gatctgatgc cctcttctgg tacttatata cataaaataa ataaatcttt ttaaaaaaat 8160
ccacctggtt acatagtatc aggaccatcg tagctacaca gcaataccat acctcaaaaa 8220
aaaattaaaa taaaaacaat caaaagttgg cacaaattga acaataaaag ctgttgcctc 8280
ttactcaaag catcctggga gtcaggagca gacttttctg cctcctttta gataggctgt 8340
gcaaagccaa gcacacacag caagagtgta aggacagagt ttgtcgttca agcacacaca 8400
gcaagagtgt aaggacagag tttgtcgttt cacgtctctt cacttactgg ctgtcctgat 8460
ttcctttttg ttgctgtgat aaaaacaatc taaccaaact caacttgcgt aggaaagggt 8520
ttatctggcc tatgattcca gatcactgag catcactgag gcacgtcaga gcagaagctc 8580
gggtggagct tgaagatgaa gccagagagg actgctgctt gctcatttac cttctgcctc 8640
atgcttagat aactttctta gatttttaaa aaatgttttt tgaaagatta atttttatta 8700
tgttaatatt atattatttt atcactttat tgtttattat cttattattt ttattacttt 8760
attattgctc acacatgtga gcaagtgctc tggaagtcca gaggcataaa aacctccagg 8820
acctagagtt gcaggagata gtgatggtgg gatgcagccg atgtggatgc tgggaaccaa 8880
aatcagatcc tgtactagag cagtatgtgc tcttaatgat gaactattta tctccaatac 8940
agccaggacc acagccaggg aacagcgtgt aacacagtgg gctgggcctt ctacatcaat 9000
ccaatagtaa ttggagactt aggtgcaagt atattcagat ggaagtatat ttggatgaaa 9060
cagtctacac gtacaatttt tacagagagt ttcaagaaat taatgagcta acactcatct 9120
ttccagatca tgcaaagatc aaaaactatt ctcttctcct ctgaaatgca aatcgataaa 9180
cattatctaa acccctacaa aggacaaaga taattttgtg caagagactg tgggcaatac 9240
aaagattttt aaactttaaa catggcttca tttatgaata aggttttctt ccttaaatat 9300
agtcatgaga atgacttact ataagagaag ccaaattttt agcaaagaaa tgaaaatgtg 9360
atgttgagtt tttattgtct gctaacaacc catatgttga acttaagatc agtctagttt 9420
tgtaaaagca tttgttacag tgcatttaaa gtttgccatt tacacgcctt ccatcccagc 9480
cagccaagat ctgcgagtac cctgtcatcg gttacacaca gcagatgctc attggctcct 9540
tagacagaag gtgaggatct ttttctccat gtataagcaa tcatctgagc acatctctgt 9600
aaggcaatag caacaaggtt taaccgtagt tgttgcttaa tcgtttaact gataaccaat 9660
atcctggtat tttgttttat tgtttttgtt gtttttcgct aaagactgac ccaaaggcat 9720
gctagacaag tgctccacac tgaggtatgt ccccagccta tagtgtttaa aatgggtcct 9780
cactgttctt ttaaattttt tatttttatg ttatgtatat gtacctgcat gaatgcatgg 9840
tagtgcacct tgtgtgtaca ggagcctgca gaggtcagaa gagagcatca catagatctc 9900
ttggaactgg agttacaggc agttgtgaac tactgtgggt gctaagaact gaacctggag 9960
ccgggcgtgg tggcacacac ctttaatgcc agcactcggg aggcagaggc aggcggattt 10020
ctgagttcga gtctagcctg gtctacagag agagttccag gatagccaga gctatacaga 10080
gaaaccctgt ttttaaaaaa caaaacaaaa aacaaacaaa caaaaaacct gaacctgggt 10140
ccttggcagg agcagtgtgt gctcctacaa acggaaccat ctctccagcc ccagttatca 10200
ctattttaaa aagttgtttc ttttaatcta acagaaaaca gccaatagct tatgttcaat 10260
aagtactgaa tgaattagca aatgaatacc agaacttaaa agacttcagt aatttaatac 10320
atagtaagaa aatgtggtta atattttcat tgtcttaact atcaactggt tcacctgaca 10380
gcctttcttg tcaaaaactt tattcagtta tcccagtatc tctgagcttc acctatgctg 10440
ctattggaga ctcctgactt tatttctaaa atctttagct cagaagacag tttgcgggag 10500
ttggttctca ccttccacct tgtagttccc aggtactgaa ctcaggatgt tggtgacaga 10560
tgcctttact cattgagcta actctgagcc caagacttga ttacaaaatg caagcccaac 10620
acttccactg ctgtcctcag ctcactaggc atgagaaggt aattcttcag atatgagttt 10680
gctgtggtca gtgcggtctg catttgatgg tcactgcacc acacacacac acacacacac 10740
acacacacac acacacacac catcttaaat tcatgaaaaa ttttaaggct gagctaaaac 10800
aatccatttc tgagtgtcca atttgaaccc aacatctctt taatttattt gtttttcaga 10860
acctagggtc tcattgcatg gtaggcaaat actctaacta catcctcaag tcctgagctc 10920
aactcttctt agcaatgtag gacaataagg gagaaaaaac cctcagccca tgactaacaa 10980
gtggtcgctc tcccttcagc accttcctct gatgcagact cacagcccct gcaaaactgg 11040
cctggtttgc ccgggtctgc taagaaatct gctccctctg attcacacac agtgatttca 11100
tcttcctcct ggaacactgg ccctggctgt ctctgctgcc ttcgccatct taactccaac 11160
ctatattgtg ggctgatgtg gaggtgggac aatggctctg tggccccctg caccatagtc 11220
atctgttctc tatcatctcc tccttctcct ttcttatatc aaacttctgt ttcttccttt 11280
tcccttcaga aacacaaaag ggtggagaac ttgacatatg atctgttaat tcattgtctc 11340
cagaagtctg gtttcaattt tttgtccaac actgcgtgga ttaattgaaa cgaggctggg 11400
gttaagtctc ggaattcaaa tggttttgtg atatcatcag cccaggagac ttctttcctg 11460
acgatgactc tgcacatgtc aactcttatt ccaaaagcca ttgtgaaagt acagccactc 11520
ctcactgcca gtaactcctc ttttccaata agtcactccc cagcagtcaa tgggtgctaa 11580
gccagccagt gtggagacag tctacatgca acaaatttga aagaaaaaaa aagtcaatct 11640
gagaccttga aacatctttc attaatgtat tgtttcctaa tatttttgta acaactttgt 11700
gctgctgagt ttattaactt tctgaaacaa tgtcttcctc ctgtgatttt actgcttcta 11760
atacagtatc attacaaaac tctgagggca aatcctctta atgaaagtaa agagtatcta 11820
cagtctcatc atgacttgat attttgtaac caacccccat tagactcaag aattttctgt 11880
acatatggtt gtgacttgtt ggaaatttct taagtgtgtt ttcaaccagc tttcaatttt 11940
tggtctatga cttctagatt tatttttatt aaatagggat tgcataggga gtacggggga 12000
tagatatgtg tgtgtgagag tagatagcca cagaagccag aagagggtat ccgattccct 12060
ggagctggga ttacagatgg ttgtaatctg tgtgtgtggc gggaaacagt gtgggctctt 12120
aattgctgcg cttctttcca gacccaagaa atgttaataa tgacaagctg gctagaagaa 12180
catccgtcct gggcaagaat ggaaattacc aagaatttac taattcttgg ccacgaatgc 12240
cttcatggca gtcaagcggt tctagcttta gttctgttct ttttcagtgg ccatctctac 12300
atcg 12304
Claims (5)
1.一种IL15RA基因人源化改造的非人动物的构建方法,其特征在于,所述的非人动物的基因组中包含人IL15RA基因或者编码人IL15RA蛋白的核苷酸序列,所述的人IL15RA基因如SEQ ID NO:5所示,所述人IL15RA蛋白如SEQ ID NO:2第37至211位氨基酸所示,所述的构建方法包括用包含SEQ ID NO:5所示核苷酸序列替换至非人动物IL15RA基因座上,所述的核苷酸序列在非人动物体内通过内源调控元件进行调控,所述的替换至非人动物IL15RA基因座为替换非人动物IL15RA基因中编码SEQ ID NO:1第40至211位的核苷酸序列,或者替换SEQ ID NO:31所示序列相同的核苷酸序列,所述的非人动物内源IL15RA蛋白的表达降低或缺失,所述的非人动物为小鼠。
2.根据权利要求1所述的构建方法,其特征在于,所述的非人动物体内表达人源化IL15RA蛋白,所述的人源化IL15RA蛋白包括人IL15RA蛋白的胞外区和跨膜区,所述的人源化IL15RA蛋白如SEQ ID NO:22所示的氨基酸序列。
3.根据权利要求1或2所述的构建方法,其特征在于,使用靶向载体进行非人动物的构建,其中,所述的靶向载体包含编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列或包含SEQ ID NO:5所示核苷酸序列。
4.一种靶向载体,其特征在于,所述的靶向载体包含来源于人IL15RA基因的部分,所述来源于人IL15RA基因的部分如编码SEQ ID NO:2第37至211位氨基酸的核苷酸序列或如SEQID NO:5所示核苷酸序列,所述的靶向载体还包含5’臂和3’臂,所述的5’臂的核苷酸序列如SEQ ID NO:3所示,所述的3’臂的核苷酸序列如SEQ ID NO:4所示。
5.权利要求1-3任一构建方法构建的非人动物在IL15RA基因或蛋白相关研究中的应用,所述应用不是疾病的诊断和治疗方法,所述的应用包括:
A)涉及人类细胞的免疫过程的产品开发,制造或筛选人类抗体中的应用;
B)作为药理学、免疫学、微生物学和医学研究的模型系统中的应用;
C)涉及人类细胞的免疫过程的生产和利用动物实验疾病模型,用于病原学研究中的应用;
D)在体内研究人IL15RA信号通路调节剂的筛选、药效检测、评估疗效、验证或评价;或者,
E)研究IL15RA基因功能,研究人IL15RA抗体,研究针对人IL15RA靶位点的药物、药效,研究免疫相关疾病药物以及抗肿瘤或炎症药物方面的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110173729.9A CN112481303B (zh) | 2021-02-09 | 2021-02-09 | Il15ra基因人源化非人动物及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110173729.9A CN112481303B (zh) | 2021-02-09 | 2021-02-09 | Il15ra基因人源化非人动物及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112481303A CN112481303A (zh) | 2021-03-12 |
CN112481303B true CN112481303B (zh) | 2021-05-25 |
Family
ID=74912456
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110173729.9A Active CN112481303B (zh) | 2021-02-09 | 2021-02-09 | Il15ra基因人源化非人动物及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112481303B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101250519A (zh) * | 2007-11-27 | 2008-08-27 | 江苏省原子医学研究所 | 一种可溶性白细胞介素15受体α亚单位的原核表达及纯化方法 |
WO2010071836A1 (en) * | 2008-12-19 | 2010-06-24 | Inserm | Il-15 mediated nk and t cell maturation |
CN111304248A (zh) * | 2018-12-25 | 2020-06-19 | 百奥赛图江苏基因生物技术有限公司 | 人源化细胞因子il15基因改造非人动物的构建方法及应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102376041B1 (ko) * | 2013-10-15 | 2022-03-18 | 리제너론 파마슈티칼스 인코포레이티드 | 인간화된 il-15 동물 |
-
2021
- 2021-02-09 CN CN202110173729.9A patent/CN112481303B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101250519A (zh) * | 2007-11-27 | 2008-08-27 | 江苏省原子医学研究所 | 一种可溶性白细胞介素15受体α亚单位的原核表达及纯化方法 |
WO2010071836A1 (en) * | 2008-12-19 | 2010-06-24 | Inserm | Il-15 mediated nk and t cell maturation |
CN111304248A (zh) * | 2018-12-25 | 2020-06-19 | 百奥赛图江苏基因生物技术有限公司 | 人源化细胞因子il15基因改造非人动物的构建方法及应用 |
Non-Patent Citations (4)
Title |
---|
Anderson,D.M. et al..NP_002180.1.《GenBank》.1999, * |
Expression of IL-15RA or an IL-15/IL-15RA fusion on CD8+ T cells modifies adoptively transferred T cell function in cis;Jesse Rowley et al.;《Eur J Immunol.》;20090228;第491-506页 * |
NP_002180.1;Anderson,D.M. et al.;《GenBank》;19990319 * |
NP_032384;Giri,J.G. et al.;《GenBank》;20000104 * |
Also Published As
Publication number | Publication date |
---|---|
CN112481303A (zh) | 2021-03-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109136274B (zh) | 人源化cd40基因改造动物模型的制备方法及应用 | |
CN111304246B (zh) | 一种人源化细胞因子动物模型、制备方法及应用 | |
CN111057721B (zh) | 人源化IL-4和/或IL-4Rα改造动物模型的制备方法及应用 | |
CN108424928B (zh) | 人源化基因改造动物模型的制备方法及应用 | |
CN109913493B (zh) | 人源化cd3基因改造动物模型的制备方法及应用 | |
CN112779285B (zh) | 人源化il-10和il-10ra基因改造动物的构建方法和应用 | |
CN112430621B (zh) | Il2ra基因人源化的非人动物的构建方法及应用 | |
CN111793646B (zh) | Il1r1基因人源化改造的非人动物的构建方法及其应用 | |
CN111218425B (zh) | 人源化转基因动物 | |
CN112300265B (zh) | Il33基因人源化的非人动物的构建方法和应用 | |
CN112501205B (zh) | Ceacam1基因人源化非人动物的构建方法及应用 | |
CN114751973B (zh) | Siglec15基因人源化非人动物的构建方法和应用 | |
CN112553213B (zh) | Cx3cr1基因人源化的非人动物及其构建方法和应用 | |
CN113881681B (zh) | Ccr8基因人源化非人动物及其构建方法和应用 | |
CN112481303B (zh) | Il15ra基因人源化非人动物及其构建方法和应用 | |
CN112553252B (zh) | Tnfr2基因人源化的非人动物的构建方法和应用 | |
CN112501204B (zh) | Il21r基因人源化非人动物及其构建方法和应用 | |
CN112501203B (zh) | Il17rb基因人源化的非人动物的构建方法及应用 | |
CN114853871B (zh) | Csf1和/或csf1r基因人源化的非人动物及其构建方法和应用 | |
CN113388640B (zh) | Ccr4基因人源化的非人动物及其构建方法和应用 | |
CN112501202B (zh) | Cxcr4基因人源化的非人动物及其构建方法和应用 | |
CN113774086A (zh) | A2ar基因人源化非人动物及其构建方法和应用 | |
CN113234139A (zh) | Tnfsf9基因人源化的非人动物及其构建方法和应用 | |
CN113831403A (zh) | Sting基因人源化非人动物的构建方法及应用 | |
CN115010800A (zh) | Pvrig基因人源化非人动物的构建方法及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |