CN115260303A - Construction method and application of CD70 gene humanized non-human animal - Google Patents
Construction method and application of CD70 gene humanized non-human animal Download PDFInfo
- Publication number
- CN115260303A CN115260303A CN202210609446.9A CN202210609446A CN115260303A CN 115260303 A CN115260303 A CN 115260303A CN 202210609446 A CN202210609446 A CN 202210609446A CN 115260303 A CN115260303 A CN 115260303A
- Authority
- CN
- China
- Prior art keywords
- gene
- humanized
- human
- protein
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 257
- 101150088890 CD70 gene Proteins 0.000 title claims abstract description 134
- 238000010276 construction Methods 0.000 title claims abstract description 33
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 claims abstract description 209
- 239000002773 nucleotide Substances 0.000 claims abstract description 180
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 180
- 102100025221 CD70 antigen Human genes 0.000 claims abstract description 139
- 102000053826 human CD70 Human genes 0.000 claims abstract description 68
- 230000008685 targeting Effects 0.000 claims abstract description 33
- 239000013598 vector Substances 0.000 claims abstract description 32
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 27
- 239000003814 drug Substances 0.000 claims abstract description 25
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 19
- 238000012216 screening Methods 0.000 claims abstract description 18
- 201000010099 disease Diseases 0.000 claims abstract description 17
- 241001465754 Metazoa Species 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 55
- 101100273745 Homo sapiens CD70 gene Proteins 0.000 claims description 50
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 43
- 210000004027 cell Anatomy 0.000 claims description 32
- 150000001413 amino acids Chemical class 0.000 claims description 25
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 18
- 238000003780 insertion Methods 0.000 claims description 17
- 230000037431 insertion Effects 0.000 claims description 17
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 16
- 230000001086 cytosolic effect Effects 0.000 claims description 15
- 229920001184 polypeptide Polymers 0.000 claims description 15
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 15
- 230000014509 gene expression Effects 0.000 claims description 14
- 230000001105 regulatory effect Effects 0.000 claims description 14
- 102100027207 CD27 antigen Human genes 0.000 claims description 13
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 13
- 238000006467 substitution reaction Methods 0.000 claims description 13
- 230000001225 therapeutic effect Effects 0.000 claims description 13
- 210000001519 tissue Anatomy 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 10
- 238000011161 development Methods 0.000 claims description 10
- 230000000857 drug effect Effects 0.000 claims description 10
- 238000011160 research Methods 0.000 claims description 9
- 238000012217 deletion Methods 0.000 claims description 8
- 230000037430 deletion Effects 0.000 claims description 8
- 238000011156 evaluation Methods 0.000 claims description 8
- 238000012795 verification Methods 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 6
- 238000001727 in vivo Methods 0.000 claims description 6
- 210000000056 organ Anatomy 0.000 claims description 6
- 102000008096 B7-H1 Antigen Human genes 0.000 claims description 5
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 5
- 101150013553 CD40 gene Proteins 0.000 claims description 5
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 5
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 5
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 5
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims description 5
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 5
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 5
- 238000010362 genome editing Methods 0.000 claims description 5
- 108020004999 messenger RNA Proteins 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 102100023990 60S ribosomal protein L17 Human genes 0.000 claims description 4
- 101100510617 Caenorhabditis elegans sel-8 gene Proteins 0.000 claims description 4
- 108091092195 Intron Proteins 0.000 claims description 4
- 125000000539 amino acid group Chemical group 0.000 claims description 4
- 108091026890 Coding region Proteins 0.000 claims description 3
- 230000004720 fertilization Effects 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000013011 mating Effects 0.000 claims description 3
- 230000000259 anti-tumor effect Effects 0.000 claims description 2
- 210000005260 human cell Anatomy 0.000 claims description 2
- 230000001900 immune effect Effects 0.000 claims description 2
- 230000002906 microbiologic effect Effects 0.000 claims description 2
- 230000000144 pharmacologic effect Effects 0.000 claims description 2
- 238000010171 animal model Methods 0.000 abstract description 23
- 229940079593 drug Drugs 0.000 abstract description 20
- 239000002547 new drug Substances 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 3
- 230000006801 homologous recombination Effects 0.000 abstract 1
- 238000002744 homologous recombination Methods 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 51
- 108020004414 DNA Proteins 0.000 description 41
- 108700024394 Exon Proteins 0.000 description 36
- 241000699670 Mus sp. Species 0.000 description 32
- 241000283984 Rodentia Species 0.000 description 23
- 241000700159 Rattus Species 0.000 description 19
- 206010061218 Inflammation Diseases 0.000 description 12
- 239000003550 marker Substances 0.000 description 12
- 239000012634 fragment Substances 0.000 description 11
- 230000004054 inflammatory process Effects 0.000 description 11
- 238000011577 humanized mouse model Methods 0.000 description 10
- 238000005215 recombination Methods 0.000 description 10
- 230000006798 recombination Effects 0.000 description 10
- 210000004881 tumor cell Anatomy 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- 210000001744 T-lymphocyte Anatomy 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 7
- 238000012239 gene modification Methods 0.000 description 7
- 241000282693 Cercopithecidae Species 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 241001529936 Murinae Species 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 210000004443 dendritic cell Anatomy 0.000 description 5
- 210000001671 embryonic stem cell Anatomy 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 208000032839 leukemia Diseases 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 210000001541 thymus gland Anatomy 0.000 description 5
- 208000023275 Autoimmune disease Diseases 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 4
- 241000699800 Cricetinae Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 238000002105 Southern blotting Methods 0.000 description 4
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 4
- BVOVIGCHYNFJBZ-JXUBOQSCSA-N Thr-Leu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O BVOVIGCHYNFJBZ-JXUBOQSCSA-N 0.000 description 4
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000005017 genetic modification Effects 0.000 description 4
- 235000013617 genetically modified food Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 206010003571 Astrocytoma Diseases 0.000 description 3
- 241000252212 Danio rerio Species 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 208000017604 Hodgkin disease Diseases 0.000 description 3
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 3
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 3
- 102100022297 Integrin alpha-X Human genes 0.000 description 3
- 208000008839 Kidney Neoplasms Diseases 0.000 description 3
- BQHLZUMZOXUWNU-DCAQKATOSA-N Met-Pro-Glu Chemical compound CSCC[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N BQHLZUMZOXUWNU-DCAQKATOSA-N 0.000 description 3
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 3
- 206010038389 Renal cancer Diseases 0.000 description 3
- 238000011579 SCID mouse model Methods 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 235000013330 chicken meat Nutrition 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 208000005017 glioblastoma Diseases 0.000 description 3
- 108010078144 glutaminyl-glycine Proteins 0.000 description 3
- 108010092114 histidylphenylalanine Proteins 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 201000010982 kidney cancer Diseases 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- 108010084525 phenylalanyl-phenylalanyl-glycine Proteins 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 3
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- VJVQKGYHIZPSNS-FXQIFTODSA-N Ala-Ser-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N VJVQKGYHIZPSNS-FXQIFTODSA-N 0.000 description 2
- FHETWELNCBMRMG-HJGDQZAQSA-N Asn-Leu-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FHETWELNCBMRMG-HJGDQZAQSA-N 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- ABLQPNMKLMFDQU-BIIVOSGPSA-N Cys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CS)N)C(=O)O ABLQPNMKLMFDQU-BIIVOSGPSA-N 0.000 description 2
- JIVJQYNNAYFXDG-LKXGYXEUSA-N Cys-Thr-Asn Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O JIVJQYNNAYFXDG-LKXGYXEUSA-N 0.000 description 2
- NMPSRDYYNIYOSJ-IHPCNDPISA-N Cys-Trp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CS)N NMPSRDYYNIYOSJ-IHPCNDPISA-N 0.000 description 2
- 102000016607 Diphtheria Toxin Human genes 0.000 description 2
- 108010053187 Diphtheria Toxin Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000699694 Gerbillinae Species 0.000 description 2
- PRBLYKYHAJEABA-SRVKXCTJSA-N Gln-Arg-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O PRBLYKYHAJEABA-SRVKXCTJSA-N 0.000 description 2
- PXXGVUVQWQGGIG-YUMQZZPRSA-N Glu-Gly-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N PXXGVUVQWQGGIG-YUMQZZPRSA-N 0.000 description 2
- DNPCBMNFQVTHMA-DCAQKATOSA-N Glu-Leu-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O DNPCBMNFQVTHMA-DCAQKATOSA-N 0.000 description 2
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 2
- GWCRIHNSVMOBEQ-BQBZGAKWSA-N Gly-Arg-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O GWCRIHNSVMOBEQ-BQBZGAKWSA-N 0.000 description 2
- UHPAZODVFFYEEL-QWRGUYRKSA-N Gly-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN UHPAZODVFFYEEL-QWRGUYRKSA-N 0.000 description 2
- JJGBXTYGTKWGAT-YUMQZZPRSA-N Gly-Pro-Glu Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O JJGBXTYGTKWGAT-YUMQZZPRSA-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
- LBCAQRFTWMMWRR-CIUDSAMLSA-N His-Cys-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O LBCAQRFTWMMWRR-CIUDSAMLSA-N 0.000 description 2
- WJGSTIMGSIWHJX-HVTMNAMFSA-N His-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N WJGSTIMGSIWHJX-HVTMNAMFSA-N 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- DURWCDDDAWVPOP-JBDRJPRFSA-N Ile-Cys-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N DURWCDDDAWVPOP-JBDRJPRFSA-N 0.000 description 2
- 108010025815 Kanamycin Kinase Proteins 0.000 description 2
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- 108091092724 Noncoding DNA Proteins 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
- TUYWCHPXKQTISF-LPEHRKFASA-N Pro-Cys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N2CCC[C@@H]2C(=O)O TUYWCHPXKQTISF-LPEHRKFASA-N 0.000 description 2
- FCRMLGJMPXCAHD-FXQIFTODSA-N Ser-Arg-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O FCRMLGJMPXCAHD-FXQIFTODSA-N 0.000 description 2
- BYCVMHKULKRVPV-GUBZILKMSA-N Ser-Lys-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O BYCVMHKULKRVPV-GUBZILKMSA-N 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- VEIKMWOMUYMMMK-FCLVOEFKSA-N Thr-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 VEIKMWOMUYMMMK-FCLVOEFKSA-N 0.000 description 2
- UJGDFQRPYGJBEH-AAEUAGOBSA-N Trp-Ser-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N UJGDFQRPYGJBEH-AAEUAGOBSA-N 0.000 description 2
- MBLJBGZWLHTJBH-SZMVWBNQSA-N Trp-Val-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 MBLJBGZWLHTJBH-SZMVWBNQSA-N 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- PMDOQZFYGWZSTK-LSJOCFKGSA-N Val-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C PMDOQZFYGWZSTK-LSJOCFKGSA-N 0.000 description 2
- YQMILNREHKTFBS-IHRRRGAJSA-N Val-Phe-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N YQMILNREHKTFBS-IHRRRGAJSA-N 0.000 description 2
- 208000008383 Wilms tumor Diseases 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 102000025171 antigen binding proteins Human genes 0.000 description 2
- 108091000831 antigen binding proteins Proteins 0.000 description 2
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 2
- 229960002756 azacitidine Drugs 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 210000001728 clone cell Anatomy 0.000 description 2
- 238000013377 clone selection method Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000000139 costimulatory effect Effects 0.000 description 2
- 229940085936 cusatuzumab Drugs 0.000 description 2
- 108010004073 cysteinylcysteine Proteins 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010036413 histidylglycine Proteins 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 108010027338 isoleucylcysteine Proteins 0.000 description 2
- 208000022013 kidney Wilms tumor Diseases 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 201000008026 nephroblastoma Diseases 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000003234 polygenic effect Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229960001603 tamoxifen Drugs 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 230000005760 tumorsuppression Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- LGFCAXJBAZESCF-ACZMJKKPSA-N Ala-Gln-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O LGFCAXJBAZESCF-ACZMJKKPSA-N 0.000 description 1
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 1
- FOHXUHGZZKETFI-JBDRJPRFSA-N Ala-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C)N FOHXUHGZZKETFI-JBDRJPRFSA-N 0.000 description 1
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- 201000003076 Angiosarcoma Diseases 0.000 description 1
- 208000031873 Animal Disease Models Diseases 0.000 description 1
- OVVUNXXROOFSIM-SDDRHHMPSA-N Arg-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O OVVUNXXROOFSIM-SDDRHHMPSA-N 0.000 description 1
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 1
- MMGCRPZQZWTZTA-IHRRRGAJSA-N Arg-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N MMGCRPZQZWTZTA-IHRRRGAJSA-N 0.000 description 1
- FLYANDHDFRGGTM-PYJNHQTQSA-N Arg-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FLYANDHDFRGGTM-PYJNHQTQSA-N 0.000 description 1
- COXMUHNBYCVVRG-DCAQKATOSA-N Arg-Leu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O COXMUHNBYCVVRG-DCAQKATOSA-N 0.000 description 1
- JEOCWTUOMKEEMF-RHYQMDGZSA-N Arg-Leu-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JEOCWTUOMKEEMF-RHYQMDGZSA-N 0.000 description 1
- URAUIUGLHBRPMF-NAKRPEOUSA-N Arg-Ser-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O URAUIUGLHBRPMF-NAKRPEOUSA-N 0.000 description 1
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 1
- SUEIIIFUBHDCCS-PBCZWWQYSA-N Asn-His-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SUEIIIFUBHDCCS-PBCZWWQYSA-N 0.000 description 1
- HPASIOLTWSNMFB-OLHMAJIHSA-N Asn-Thr-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O HPASIOLTWSNMFB-OLHMAJIHSA-N 0.000 description 1
- PSLSTUMPZILTAH-BYULHYEWSA-N Asp-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PSLSTUMPZILTAH-BYULHYEWSA-N 0.000 description 1
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 1
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 1
- XWKBWZXGNXTDKY-ZKWXMUAHSA-N Asp-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O XWKBWZXGNXTDKY-ZKWXMUAHSA-N 0.000 description 1
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 1
- 208000036170 B-Cell Marginal Zone Lymphoma 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
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 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
- 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
- 108020004705 Codon Proteins 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- OHLLDUNVMPPUMD-DCAQKATOSA-N Cys-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N OHLLDUNVMPPUMD-DCAQKATOSA-N 0.000 description 1
- JAHCWGSVNZXHRR-SVSWQMSJSA-N Cys-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CS)N JAHCWGSVNZXHRR-SVSWQMSJSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 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
- 206010015548 Euthanasia 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
- ZFADFBPRMSBPOT-KKUMJFAQSA-N Gln-Arg-Phe Chemical compound N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O ZFADFBPRMSBPOT-KKUMJFAQSA-N 0.000 description 1
- JFSNBQJNDMXMQF-XHNCKOQMSA-N Gln-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)C(=O)O JFSNBQJNDMXMQF-XHNCKOQMSA-N 0.000 description 1
- QYKBTDOAMKORGL-FXQIFTODSA-N Gln-Gln-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N QYKBTDOAMKORGL-FXQIFTODSA-N 0.000 description 1
- PKVWNYGXMNWJSI-CIUDSAMLSA-N Gln-Gln-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O PKVWNYGXMNWJSI-CIUDSAMLSA-N 0.000 description 1
- DWDBJWAXPXXYLP-SRVKXCTJSA-N Gln-His-Arg Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N DWDBJWAXPXXYLP-SRVKXCTJSA-N 0.000 description 1
- HWEINOMSWQSJDC-SRVKXCTJSA-N Gln-Leu-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O HWEINOMSWQSJDC-SRVKXCTJSA-N 0.000 description 1
- VZRAXPGTUNDIDK-GUBZILKMSA-N Gln-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N VZRAXPGTUNDIDK-GUBZILKMSA-N 0.000 description 1
- ZBKUIQNCRIYVGH-SDDRHHMPSA-N Gln-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZBKUIQNCRIYVGH-SDDRHHMPSA-N 0.000 description 1
- NVHJGTGTUGEWCG-ZVZYQTTQSA-N Gln-Trp-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O NVHJGTGTUGEWCG-ZVZYQTTQSA-N 0.000 description 1
- HNAUFGBKJLTWQE-IFFSRLJSSA-N Gln-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)N)N)O HNAUFGBKJLTWQE-IFFSRLJSSA-N 0.000 description 1
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 1
- PJBVXVBTTFZPHJ-GUBZILKMSA-N Glu-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N PJBVXVBTTFZPHJ-GUBZILKMSA-N 0.000 description 1
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 1
- PMNHJLASAAWELO-FOHZUACHSA-N Gly-Asp-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PMNHJLASAAWELO-FOHZUACHSA-N 0.000 description 1
- QCTLGOYODITHPQ-WHFBIAKZSA-N Gly-Cys-Ser Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O QCTLGOYODITHPQ-WHFBIAKZSA-N 0.000 description 1
- VNBNZUAPOYGRDB-ZDLURKLDSA-N Gly-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)CN)O VNBNZUAPOYGRDB-ZDLURKLDSA-N 0.000 description 1
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 1
- HAXARWKYFIIHKD-ZKWXMUAHSA-N Gly-Ile-Ser Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HAXARWKYFIIHKD-ZKWXMUAHSA-N 0.000 description 1
- HJARVELKOSZUEW-YUMQZZPRSA-N Gly-Pro-Gln Chemical compound [H]NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O HJARVELKOSZUEW-YUMQZZPRSA-N 0.000 description 1
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-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
- HVCRQRQPIIRNLY-IUCAKERBSA-N His-Gln-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)NCC(=O)O)N HVCRQRQPIIRNLY-IUCAKERBSA-N 0.000 description 1
- ZUPVLBAXUUGKKN-VHSXEESVSA-N His-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CC2=CN=CN2)N)C(=O)O ZUPVLBAXUUGKKN-VHSXEESVSA-N 0.000 description 1
- JBSLJUPMTYLLFH-MELADBBJSA-N His-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC3=CN=CN3)N)C(=O)O JBSLJUPMTYLLFH-MELADBBJSA-N 0.000 description 1
- SKYULSWNBYAQMG-IHRRRGAJSA-N His-Leu-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SKYULSWNBYAQMG-IHRRRGAJSA-N 0.000 description 1
- BRQKGRLDDDQWQJ-MBLNEYKQSA-N His-Thr-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O BRQKGRLDDDQWQJ-MBLNEYKQSA-N 0.000 description 1
- 101000914673 Homo sapiens Fanconi anemia group A protein Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein 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
- HDOYNXLPTRQLAD-JBDRJPRFSA-N Ile-Ala-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)O)N HDOYNXLPTRQLAD-JBDRJPRFSA-N 0.000 description 1
- WEWCEPOYKANMGZ-MMWGEVLESA-N Ile-Cys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N WEWCEPOYKANMGZ-MMWGEVLESA-N 0.000 description 1
- AREBLHSMLMRICD-PYJNHQTQSA-N Ile-His-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N AREBLHSMLMRICD-PYJNHQTQSA-N 0.000 description 1
- HYLIOBDWPQNLKI-HVTMNAMFSA-N Ile-His-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N HYLIOBDWPQNLKI-HVTMNAMFSA-N 0.000 description 1
- HODVZHLJUUWPKY-STECZYCISA-N Ile-Tyr-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)CC)CC1=CC=C(O)C=C1 HODVZHLJUUWPKY-STECZYCISA-N 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- 102220470475 L-seryl-tRNA(Sec) kinase_C57L_mutation Human genes 0.000 description 1
- 102000017578 LAG3 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
- 241000880493 Leptailurus serval Species 0.000 description 1
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 1
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 1
- FOEHRHOBWFQSNW-KATARQTJSA-N Leu-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)N)O FOEHRHOBWFQSNW-KATARQTJSA-N 0.000 description 1
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 1
- IWTBYNQNAPECCS-AVGNSLFASA-N Leu-Glu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IWTBYNQNAPECCS-AVGNSLFASA-N 0.000 description 1
- WQWSMEOYXJTFRU-GUBZILKMSA-N Leu-Glu-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O WQWSMEOYXJTFRU-GUBZILKMSA-N 0.000 description 1
- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 1
- JRJLGNFWYFSJHB-HOCLYGCPSA-N Leu-Gly-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JRJLGNFWYFSJHB-HOCLYGCPSA-N 0.000 description 1
- IWMJFLJQHIDZQW-KKUMJFAQSA-N Leu-Ser-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IWMJFLJQHIDZQW-KKUMJFAQSA-N 0.000 description 1
- VDIARPPNADFEAV-WEDXCCLWSA-N Leu-Thr-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O VDIARPPNADFEAV-WEDXCCLWSA-N 0.000 description 1
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 1
- SEOXPEFQEOYURL-PMVMPFDFSA-N Leu-Tyr-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O SEOXPEFQEOYURL-PMVMPFDFSA-N 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- YEIYAQQKADPIBJ-GARJFASQSA-N Lys-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)N)C(=O)O YEIYAQQKADPIBJ-GARJFASQSA-N 0.000 description 1
- XNKDCYABMBBEKN-IUCAKERBSA-N Lys-Gly-Gln Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O XNKDCYABMBBEKN-IUCAKERBSA-N 0.000 description 1
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241000699729 Muridae Species 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
- 208000009525 Myocarditis Diseases 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- HNFUGJUZJRYUHN-JSGCOSHPSA-N Phe-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 HNFUGJUZJRYUHN-JSGCOSHPSA-N 0.000 description 1
- PPHFTNABKQRAJV-JYJNAYRXSA-N Phe-His-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PPHFTNABKQRAJV-JYJNAYRXSA-N 0.000 description 1
- METZZBCMDXHFMK-BZSNNMDCSA-N Phe-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N METZZBCMDXHFMK-BZSNNMDCSA-N 0.000 description 1
- CXMSESHALPOLRE-MEYUZBJRSA-N Phe-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)O CXMSESHALPOLRE-MEYUZBJRSA-N 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 1
- VCYJKOLZYPYGJV-AVGNSLFASA-N Pro-Arg-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O VCYJKOLZYPYGJV-AVGNSLFASA-N 0.000 description 1
- LGSANCBHSMDFDY-GARJFASQSA-N Pro-Glu-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O LGSANCBHSMDFDY-GARJFASQSA-N 0.000 description 1
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 1
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 1
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 241000605112 Scapanulus oweni Species 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 1
- RHAPJNVNWDBFQI-BQBZGAKWSA-N Ser-Pro-Gly Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O RHAPJNVNWDBFQI-BQBZGAKWSA-N 0.000 description 1
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 1
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 description 1
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-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
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 208000031673 T-Cell Cutaneous Lymphoma 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
- 239000004098 Tetracycline Substances 0.000 description 1
- 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 1
- YBXMGKCLOPDEKA-NUMRIWBASA-N Thr-Asp-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YBXMGKCLOPDEKA-NUMRIWBASA-N 0.000 description 1
- MSIYNSBKKVMGFO-BHNWBGBOSA-N Thr-Gly-Pro Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N)O MSIYNSBKKVMGFO-BHNWBGBOSA-N 0.000 description 1
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 1
- DEGCBBCMYWNJNA-RHYQMDGZSA-N Thr-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O DEGCBBCMYWNJNA-RHYQMDGZSA-N 0.000 description 1
- JAWUQFCGNVEDRN-MEYUZBJRSA-N Thr-Tyr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O JAWUQFCGNVEDRN-MEYUZBJRSA-N 0.000 description 1
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 1
- 208000000728 Thymus Neoplasms Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- RERIQEJUYCLJQI-QRTARXTBSA-N Trp-Asp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N RERIQEJUYCLJQI-QRTARXTBSA-N 0.000 description 1
- FEZASNVQLJQBHW-CABZTGNLSA-N Trp-Gly-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O)=CNC2=C1 FEZASNVQLJQBHW-CABZTGNLSA-N 0.000 description 1
- 108091005956 Type II transmembrane proteins Proteins 0.000 description 1
- ADBDQGBDNUTRDB-ULQDDVLXSA-N Tyr-Arg-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O ADBDQGBDNUTRDB-ULQDDVLXSA-N 0.000 description 1
- KHUVIWRRFMPVHD-JYJNAYRXSA-N Tyr-Met-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(O)=O KHUVIWRRFMPVHD-JYJNAYRXSA-N 0.000 description 1
- HMPMGPISLMLHSI-JBACZVJFSA-N Tyr-Trp-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N HMPMGPISLMLHSI-JBACZVJFSA-N 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 206010073328 Undifferentiated nasopharyngeal carcinoma Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- FBVUOEYVGNMRMD-NAKRPEOUSA-N Val-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N FBVUOEYVGNMRMD-NAKRPEOUSA-N 0.000 description 1
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 1
- DLMNFMXSNGTSNJ-PYJNHQTQSA-N Val-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N DLMNFMXSNGTSNJ-PYJNHQTQSA-N 0.000 description 1
- YTNGABPUXFEOGU-SRVKXCTJSA-N Val-Pro-Arg Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O YTNGABPUXFEOGU-SRVKXCTJSA-N 0.000 description 1
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 1
- 230000009471 action 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
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 238000003556 assay Methods 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
- 239000011425 bamboo Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000026900 bile duct neoplasm Diseases 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000007969 cellular immunity 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
- 230000009194 climbing Effects 0.000 description 1
- 238000013373 clone screening Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 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
- 201000007241 cutaneous T cell lymphoma Diseases 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
- 238000003255 drug test Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 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
- 201000003444 follicular lymphoma Diseases 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 201000003115 germ cell cancer Diseases 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 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
- 108010085325 histidylproline Proteins 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 108010077158 leucinyl-arginyl-tryptophan Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 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
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 201000000564 macroglobulinemia Diseases 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
- 238000005259 measurement Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 230000002503 metabolic effect 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
- 238000010172 mouse model Methods 0.000 description 1
- 201000005962 mycosis fungoides Diseases 0.000 description 1
- 230000002956 necrotizing effect Effects 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 230000000324 neuroprotective effect Effects 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
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 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
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 230000001737 promoting 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
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- -1 scRNA Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 206010042863 synovial sarcoma Diseases 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000010809 targeting technique Methods 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 208000008732 thymoma 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
- 201000008827 tuberculosis Diseases 0.000 description 1
- 239000000439 tumor marker Substances 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
- 238000012800 visualization Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
-
- 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
- A01K67/0276—Knock-out vertebrates
-
- 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
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
-
- 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
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
-
- 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
- A01K2267/0368—Animal model for inflammation
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Microbiology (AREA)
- Animal Husbandry (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Diabetes (AREA)
- Rheumatology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Mycology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a construction method of a CD70 gene humanized non-human animal, which is characterized in that a nucleotide sequence for coding a human CD70 protein is introduced into the genome of the non-human animal in a homologous recombination mode, the animal body can normally express the human or humanized CD70 protein, can be used as an animal model for researching the human CD70 signal mechanism and screening drugs for tumors and immune related diseases, and has important application value for the research and development of new drugs for immune targets. The invention also provides a humanized CD70 protein, a humanized CD70 gene, a targeting vector of the CD70 gene and application of the constructed non-human animal in the field of biomedicine.
Description
Technical Field
The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a CD70 gene modified non-human animal model and application thereof in the field of biomedicine.
Background
CD70 is one of the Tumor Necrosis Factor (TNF) superfamily members, is a type II transmembrane protein, and is mainly expressed in activated T cells, B cells, natural Killer (NK) cells and dendritic cells. The interaction between CD70 and its receptor, CD27, promotes the activation, proliferation and differentiation of T cells, B cells and NK cells, which are essential conditions for the generation of immune responses. In cellular immunity, the main biological effect of CD70/CD27 is to promote the activation of T cells and the proliferation of T cells, so that the T cells become effector T cells and memory T cells; in humoral immunity, the primary role of CD70/CD27 is to promote B cell differentiation and proliferation of plasma cells; for NK cells, the direct action of CD27 and CD70 on the surface of NK cells can increase the killing activity of NK cells, promote the differentiation of NK cells and release IFN-gamma.
Under physiological conditions, CD70 is highly precisely regulated by antibodies, antigens, cytokines, etc., and thus is expressed on the surface of activated T cells and B cells for a short period of time. In addition to expression on normal lymphocytes, CD70 is also highly expressed on the surface of a variety of tumor cells, especially blood-borne tumors such as hodgkin's lymphoma, non-hodgkin's lymphoma, leukemia, and fahrenheit macroglobulinemia (Waldenstroms macroglobulinemia), and is a potential target for partial tumor targeting and immunotherapy; among non-blood-derived tumors, the high expression of CD70 is found in undifferentiated nasopharyngeal carcinoma and germ cell cancer at the earliest, and subsequent studies find that CD70 is also highly expressed in tumors such as thymoma, nephroblastoma, glioblastoma, astrocytoma and ovarian cancer, particularly kidney cancer, which is currently considered as a novel specific tumor marker for kidney cancer. In addition, the research finds that the costimulatory signal provided by the CD27/CD70 interaction plays a promoting role in the occurrence and development of various autoimmune diseases, and selective intervention of the costimulatory pathway can alleviate or reduce the development of certain autoimmune diseases. Such as Systemic Lupus Erythematosus (SLE), rheumatoid Arthritis (RA), etc., may be a specific therapeutic target for autoimmune diseases.
At present, monoclonal antibodies targeting CD70 have entered human clinics and show better safety and therapeutic effects. For example, humanized monoclonal antibody Cusatuzumab obtained by Janssen early-paid in 3 hundred million dollars and invested in 2 hundred million dollars shows good safety and tolerance, also shows a certain anti-cancer effect and shows the potential for treating the disease in a clinical test of stage I/II of CD70 positive advanced cutaneous T cell lymphoma; in another phase I/II clinical trial of acute myelogenous leukemia, cusatuzumab in combination with Azacitidine had a higher overall remission rate than the historical data for Azacitidine alone.
The experimental animal disease model is an indispensable research tool for researching etiology and pathogenesis of human diseases, developing prevention and treatment technologies and developing medicines. However, due to the differences between the physiological structures and metabolic systems of animals and humans, the traditional animal models cannot reflect the real conditions of human bodies well, and the establishment of disease models closer to the physiological characteristics of human bodies in animal bodies is an urgent need of the biomedical industry. Therefore, because of the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, it remains the greatest challenge to construct "efficient" humanized animal models for new drug development.
In view of the huge application potential of the CD27/CD70 signal pathway in the treatment field of tumors, autoimmune diseases and the like, in order to further explore the relevant biological characteristics, improve the effectiveness of the preclinical pharmacodynamic test, improve the success rate of research and development, make the preclinical test more effective and minimize the research and development failure, the development of a non-human animal model of the CD27/CD70 signal pathway is urgently needed in the field. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research.
Disclosure of Invention
The application uses human normal or mutant genes to replace homologous genes of animal genomes, and establishes a normal or mutant gene animal model which is closer to the human physiological or disease characteristics. The humanized animal model for cell or tissue transplantation is improved, and more importantly, the humanized animal model can express or partially express human protein in an animal body due to the insertion of human gene segments, can be used as a target of a drug only capable of recognizing human protein sequences, and provides possibility for screening anti-human antibodies and other drugs at the animal level.
In a first aspect of the invention, a humanized CD70 protein is provided, wherein the humanized CD70 protein comprises all or part of a human CD70 protein.
Preferably, the humanized CD70 protein comprises an extracellular domain, a transmembrane domain and/or a cytoplasmic domain of the human CD70 protein.
Preferably, the humanized CD70 protein comprises all or part of the extracellular domain of a human CD70 protein. Further preferred are those comprising at least 50 to at least 154, e.g. 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD 70. Further preferably, the extracellular domain comprises an extracellular domain of human CD70 in which 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids are deleted from the C-terminus and/or N-terminus of the extracellular domain.
Preferably, the humanized CD70 protein further comprises a portion of a non-human animal CD70 protein, and further preferably, the portion of the non-human animal CD70 protein comprises a transmembrane region and/or a cytoplasmic region of the non-human animal CD70 protein; still more preferably, the polypeptide further comprises a part of extracellular domain of the non-human animal CD70 protein, and further preferably comprises 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) consecutive amino acid sequences of C-terminal and/or N-terminal of the extracellular domain of the non-human animal.
Preferably, the portion of the human CD70 protein and the portion of the non-human animal CD70 protein are directly linked or indirectly linked (e.g., a peptide linker).
In one embodiment of the present invention, the humanized CD70 protein comprises an extracellular domain of human CD70 protein, a transmembrane domain and a cytoplasmic domain of a non-human animal, and preferably further comprises a partial extracellular domain of a non-human animal.
Preferably, the portion of human CD70 protein comprises SEQ ID NO:2, amino acid sequence as shown in positions 48-193 or 39-193; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 48-193 or 39-193, is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 48-193 or 39-193, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 48-193 or 39-193, including substitution, deletion and/or insertion of one or more amino acid residues.
Preferably, the portion of the non-human animal CD70 protein comprises SEQ ID NO:1, positions 1-44 or 1-49; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99% identical to the amino acid sequence shown in positions 1-44 or 1-49; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-44 or 1-49, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 1-44 or 1-49, including substitution, deletion and/or insertion of one or more amino acid residues.
In one embodiment of the present invention, the humanized CD70 protein comprises SEQ ID NO:2, amino acid sequence shown in positions 48-193 or 39-193 and SEQ ID NO:1, 1-44 or 1-49.
Preferably, said part of the human CD70 protein comprises all or part of the amino acid sequence encoded by the human CD70 gene.
Preferably, the part of the human CD70 protein comprises all or part of an amino acid sequence encoded by exon 1, exon 2 and/or exon 3 of the human CD70 gene; preferably, the humanized CD70 protein comprises all or part of an amino acid sequence encoded by exons 1 to 3 of the human CD70 gene. Further preferably, the humanized CD70 protein comprises all or part of an amino acid sequence encoded by one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of the human CD70 gene. Still more preferably, the humanized CD70 protein comprises part of exon 1 and the amino acid sequence encoded by exons 2 to 3 of human CD70 gene, wherein the part of exon 1 is preferably a nucleotide sequence encoding a transmembrane region and/or an extracellular region.
Preferably, the portion of the non-human animal CD70 protein comprises a partial amino acid sequence encoded by the non-human animal CD70 gene. Preferably a partial amino acid sequence encoded by exon 1 of the non-human animal CD70 gene.
In a specific embodiment of the present invention, the humanized CD70 protein comprises a part of the amino acid sequence encoded by exon 1 of the non-human animal CD70 gene, and all of exons 2 to 3 of the human CD70 gene, and preferably further comprises a part of the amino acid sequence encoded by exon 1 of the human CD70 gene.
Preferably, the non-human animal can be selected from any non-human animal such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc., which can be genetically engineered to become genetically humanized.
Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or a mouse.
Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdcscid IL-2rγnullMouse, NOD-Rag 1-/--IL2rg-/-(NRG) mouse, rag 2-/--IL2rg-/-(RG) mice, NOD/SCID mice or nude mice.
Preferably, the amino acid sequence of the humanized CD70 protein derived from human CD70 protein is identical to the amino acid sequence of SEQ ID NO:2 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%.
Preferably, the amino acid sequence of the humanized CD70 protein derived from the non-human animal CD70 protein has a sequence similar to that of SEQ ID NO:1 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%.
Preferably, the humanized CD70 protein comprises at least 50 amino acid sequences identical to the corresponding amino acid sequence of human CD70, and further, the 50 amino acid sequences are the amino acid sequences from the first amino acid of the C-terminus.
In one embodiment of the present invention, the amino acid sequence of the humanized CD70 protein comprises one of the following groups:
a) Is SEQ ID NO:11, all or part of an amino acid sequence;
b) And SEQ ID NO:11 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical in amino acid sequence;
c) And SEQ ID NO:11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or
d) And SEQ ID NO:11, comprising substitution, deletion and/or insertion of one or more amino acid residues.
In a second aspect of the invention, there is provided a nucleic acid encoding the humanized CD70 protein.
In a third aspect of the invention, a humanized CD70 gene is provided, said humanized CD70 gene comprising a portion of a human CD70 gene.
Preferably, the humanized CD70 gene encodes the humanized CD70 protein.
Preferably, the humanized CD70 gene comprises all or part of exons 1 to 3 of human CD70 gene, further preferably comprises any one, two or three or two consecutive exons of exons 1 to 3 of human CD70 gene, further preferably comprises part of exon 1, all of exon 2 and part of exon 3 of human CD70 gene, wherein part of exon 1 comprises at least 10bp to at least 310bp of exon 1, such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp contiguous nucleotide sequence, and part of exon 3 comprises at least 100bp to at least 567bp of exon 3, such as 100, 200, 300, 350, 380, 381, 382, 383, 384, 385, 386, 387, 388, 385, 389, 390, 400, 500, 550, 7bp contiguous nucleotide sequence, preferably also comprises introns 1-2 bp and/or 2bp contiguous nucleotide sequence.
Preferably, the humanized CD70 gene comprises the genomic DNA sequence, cDNA sequence or CDs sequence of all or part of the human CD70 gene.
In one embodiment of the present invention, the humanized CD70 gene comprises a partial nucleotide sequence of the human CD70 gene comprising one of the following groups:
(A) SEQ ID NO:5, all or part of a nucleotide sequence set forth in seq id no;
(B) And SEQ ID NO:5 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;
(C) And SEQ ID NO:5 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or
(D) Has the sequence shown in SEQ ID NO:5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
Preferably, the humanized CD70 gene further comprises a portion of a non-human animal CD70 gene, and further preferably comprises all or part of exon 1 and/or all or part of exon 3 of a non-human animal CD70 gene.
In one embodiment of the present invention, the humanized CD70 gene comprises a portion of exon 1 of non-human animal CD70, a portion of exon 1 of human CD70 gene, all of exon 2, a portion of exon 3, and a portion of exon 3 of non-human animal CD70 gene. Preferably, the gene further comprises intron 1-2 and/or intron 2-3 of the human CD70 gene.
Preferably, the humanized CD70 gene comprises all or part of the nucleotide sequence encoding the transmembrane, cytoplasmic, and/or extracellular regions of the human CD70 protein. Further preferably, the humanized CD70 gene comprises all or part of the nucleotide sequence encoding the extracellular domain of human CD70 protein.
Preferably, the humanized CD70 gene comprises a nucleotide sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193, is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, comprising a polypeptide having the sequence encoding SEQ ID NO:2, the nucleotide sequence at the 39 th to 193 th positions or the 48 th to 193 th positions comprises a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions.
Preferably, the humanized CD70 gene further comprises a partial nucleotide sequence encoding a non-human animal CD70 protein. Further preferably comprises all or part of the nucleotide sequence encoding the cytoplasmic region and/or the transmembrane region of the non-human animal CD70 protein, and still further preferably comprises part of the nucleotide sequence encoding the extracellular region of the non-human animal CD70 protein.
In one embodiment of the invention, the humanized CD70 gene comprises a nucleotide sequence identical to a nucleotide sequence encoding SEQ ID NO:1, amino acid sequence 1-44 or 1-49, or a nucleotide sequence that has at least 90% identity to a nucleotide sequence encoding SEQ ID NO:1, 1-44 or 1-49 amino acid sequence.
Preferably, the humanized CD70 gene further comprises 5'UTR and/or 3' UTR of the non-human animal CD70 gene.
Preferably, the non-human animal can be selected from any non-human animal such as rodent, zebrafish, pig, chicken, rabbit, monkey, etc. which can be genetically modified to make a gene humanized.
Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or a mouse.
Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdcscid IL-2rγnullMouse, NOD-Rag 1-/--IL2rg-/-(NRG) mice, rag 2-/--IL2rg-/-(RG) mice, NOD/SCID mice or nude mice.
Preferably, the nucleotide sequence of the humanized CD70 gene comprises a nucleotide sequence identical to SEQ ID NO:6 and/or SEQ ID NO:7 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:6 and/or SEQ ID NO: 7.
Preferably, the nucleotide sequence of the humanized CD70 gene comprises a nucleotide sequence identical to SEQ ID NO:8 and/or SEQ ID NO:9 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:8 and/or SEQ ID NO:9, or a nucleotide sequence shown in the specification.
In one embodiment of the present invention, the mRNA transcribed from the humanized CD70 gene comprises any one of the following groups:
(a) The amino acid sequence of SEQ ID NO:10, or a portion or all of a nucleotide sequence set forth in seq id no;
(b) And SEQ ID NO:10 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;
(c) And SEQ ID NO:10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; or
(d) And SEQ ID NO:10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
Preferably, the humanized CD70 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a substance that is conventionally inducible or repressible.
In one embodiment of the invention, the specific inducer is selected from the tetracycline System (Tet-Off System/Tet-On System) or Tamoxifen System (Tamoxifen System).
In a fourth aspect of the invention, there is provided a targeting vector comprising a donor nucleotide sequence, said donor nucleotide sequence comprising one of the following group:
a) All or part of a nucleotide sequence encoding a human or humanized CD70 protein;
b) A nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic region of the human CD70 protein, preferably encoding all or part of the extracellular region of the human CD70 protein, further preferably a nucleotide sequence encoding at least 50 to at least 154, e.g. 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154, consecutive amino acids of the extracellular region of human CD70, further preferably a nucleotide sequence encoding an extracellular region of human CD70 with 0-20 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and/or the N-terminus of the extracellular region, more preferably a nucleotide sequence encoding an extracellular region comprising SEQ ID NO:2 from position 39-193 or from position 48-193; alternatively, the code comprises a nucleotide sequence identical to SEQ ID NO:2, positions 39-193 or 48-193, or at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, a polypeptide having the sequence encoding SEQ ID NO:2, the nucleotide sequence from 39 th to 193 th or from 48 th to 193 th, including nucleotide sequences with one or more nucleotide substitutions, deletions and/or insertions;
c) All or part of a human or humanized CD70 gene; or,
d) All or part of exons 1 to 3 of the human CD70 gene, preferably any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, further preferably part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein part of exon 1 comprises at least 10bp to at least 310bp of exon 1, such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp of a consecutive nucleotide sequence, and part of exon 3 comprises at least 100bp to at least 7bp of exon 3, such as 100, 200, 300, 350, 380, 400, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567bp of a consecutive nucleotide sequence, preferably also comprising exons 1-2 and/or 3-introns; even more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO:5 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO:5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
Preferably, the targeting vector further comprises a 5 'arm and/or a 3' arm.
The 5' arm (or 5' homologous arm) and the transformation region 5' end homologous DNA fragment, which is selected from the non-human animal CD70 gene genome DNA 100-10000 nucleotides in length. Preferably, the 5' arm has at least 90% homology with NCBI accession No. NC — 000083.7. Further preferably, the 5' arm sequence is identical to SEQ ID NO:3 or as shown in SEQ ID NO:3, respectively.
The 3' arm (or 3' homologous arm) and the 3' end of the switching region to be changed are homologous DNA fragments selected from 100-10000 nucleotides in length of non-human animal CD70 gene genome DNA. Preferably, the 3' arm has at least 90% homology with NCBI accession No. NC — 000083.7. Further preferably, the 3' arm sequence is identical to SEQ ID NO:4 or as shown in SEQ ID NO:4, respectively.
Preferably, the targeting vector further comprises a non-human animal 3' UTR.
Preferably, the targeting vector further comprises a non-human animal 5' UTR.
Preferably, the transition region to be altered is located at the non-human animal CD70 locus. Further preferably, the transition region to be altered is located on exons 1 to 3 of the non-human animal CD70 gene. Further preferably, the gene is located in exon 1 and/or exon 3 of the non-human animal CD70 gene.
Preferably, the targeting vector comprises a sequence identical to SEQ ID NO:6 and/or SEQ ID NO:7 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:6 and/or SEQ ID NO: 7.
Preferably, the targeting vector comprises a sequence identical to SEQ ID NO:8 and/or SEQ ID NO:9 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:8 and/or SEQ ID NO:9, or a nucleotide sequence shown in the specification.
Preferably, the non-human animal can be selected from any non-human animal such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc., which can be genetically engineered to become genetically humanized.
Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or a mouse.
Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdcscid IL-2rγnullMouse, NOD-Rag 1-/--IL2rg-/-(NRG) mouse, rag 2-/--IL2rg-/-(RG) mice, NOD/SCID mice or nude mice.
Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Still more preferably, the gene encoding the negative selection marker is a gene encoding diphtheria toxin subunit a (DTA).
In one embodiment of the present invention, the targeting vector further comprises a resistance gene for positive clone selection. Further preferably, the resistance gene selected by the positive clone is neomycin phosphotransferase coding sequence Neo.
In one embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is a Frt recombination site (a conventional LoxP recombination system can also be selected). The specific recombination system is provided with two Frt recombination sites which are respectively connected to two sides of the resistance gene.
In a fifth aspect of the invention, there is provided a cell comprising the targeting vector described above.
In a sixth aspect of the invention, the targeting vector and the cell containing the targeting vector are provided for use in CD70 gene modification. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.
In the seventh aspect of the invention, a method for constructing a non-human animal humanized with a CD70 gene is provided, wherein the non-human animal expresses a human or humanized CD70 protein, and/or the genome of the non-human animal comprises the human or humanized CD70 gene.
Preferably, the humanized CD70 protein is the humanized CD70 protein described above.
Preferably, the humanized CD70 gene is the humanized CD70 gene described above.
Preferably, the non-human animal has reduced or absent expression of endogenous CD70 protein.
Preferably, the genome of the non-human animal comprises a part of the human CD70 gene, further preferably comprises all or part of exons 1 to 3 of the human CD70 gene, further preferably comprises any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, further preferably comprises part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein the part of exon 1 at least comprises at least 10bp to at least 310bp of exon 1, e.g., 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp contiguous nucleotide sequence, a portion of exon 3 comprising at least 100bp to at least 567bp of exon 3, e.g., 100, 200, 300, 350, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567bp contiguous nucleotide sequence, preferably further comprising intron 1-2 and/or intron 2-3; even more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO:5 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO:5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
Preferably, the genome of the non-human animal comprises all or part of a nucleotide sequence encoding a human CD70 protein, further preferably comprises all or part of a nucleotide sequence encoding an extracellular region, a cytoplasmic region and/or a transmembrane region of a human CD70 protein, further preferably comprises all or part of a nucleotide sequence encoding an extracellular region of a human CD70 protein, wherein the part of the extracellular region of a human CD70 protein comprises at least 50 to at least 154, such as 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD 70. Further preferably, the extracellular domain comprises an extracellular domain of human CD70 in which 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids are deleted from the C-terminus and/or N-terminus of the extracellular domain.
In one embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; or, comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO:2, positions 39-193 or 48-193, or at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39-193 or from position 48-193 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, a polypeptide having the sequence encoding SEQ ID NO:2, the nucleotide sequence at the 39 th to 193 th positions or the 48 th to 193 th positions comprises a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions.
Preferably, the construction method comprises introducing any one of the following nucleotide sequences into the CD70 locus of the non-human animal:
a) All or part of a nucleotide sequence encoding a human or humanized CD70 protein;
b) A nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic region of the human CD70 protein, preferably encoding all or part of the extracellular region of the human CD70 protein, further preferably a nucleotide sequence encoding at least 50 to at least 154, e.g. 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154, consecutive amino acids of the extracellular region of human CD70, further preferably a nucleotide sequence encoding an extracellular region of human CD70 with 0-20 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and/or the N-terminus of the extracellular region, more preferably a nucleotide sequence encoding an extracellular region comprising SEQ ID NO:2 from position 39-193 or from position 48-193; alternatively, the code comprises a nucleotide sequence identical to SEQ ID NO:2, positions 39-193 or 48-193, or at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, a polypeptide having the sequence encoding SEQ ID NO:2, the nucleotide sequence from 39 th to 193 th or from 48 th to 193 th, and comprises a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions;
c) All or part of a human or humanized CD70 gene; or,
d) All or part of exons No. 1 to 3 of human CD70 gene, preferably any one, two or three or two consecutive exons of exons No. 1 to 3 of human CD70 gene, further preferably part of exons No. 1, all of exons No. 2 and part of exons No. 3 of human CD70 gene, wherein part of exons No. 1 comprises at least 10bp to at least 310bp of exons No. 1, such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp of consecutive nucleotide sequence, and part of exons No. 3 comprises at least 100bp to at least 7bp of exons No. 3, such as 100, 200, 300, 350, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567bp of consecutive nucleotide sequence, preferably also comprises exons No. 1-2 and/or 2-3; even more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO:5 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO:5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
Preferably, the human or humanized CD70 gene, nucleotide sequence encoding the human or humanized CD70 protein is regulated in the non-human animal by regulatory elements. The regulatory element is endogenous or exogenous.
Preferably, the regulatory element comprises an endogenous promoter.
Optionally, the endogenous regulatory element is a non-human animal CD70 gene regulatory element, and the exogenous regulatory element is a human CD70 gene regulatory element.
Preferably, the introduction is insertion or substitution.
Preferably, the CD70 locus is introduced into the non-human animal to replace a corresponding region of the non-human animal, preferably to replace all or part of a nucleotide sequence encoding a human CD70 protein in the genome of the non-human animal, more preferably to replace a nucleotide sequence encoding part of an extracellular domain of an endogenous CD70 protein in the genome of the non-human animal, and even more preferably to replace all or part of exons 1 to 3 of the CD70 gene in the genome of the non-human animal.
In a particular embodiment of the invention, the CD70 locus introduced into the non-human animal is a replacement for the nucleotide sequence encoding SEQ ID NO:1 from amino acid 50 to amino acid 195.
In a specific embodiment of the present invention, part of exon 1, all of exon 2, and part of exon 3 of the non-human animal CD70 gene are replaced; it is further preferred that the intron 1-2 and/or the intron 2-3 of the non-human animal are also replaced.
Preferably, the construction method comprises inserting or replacing at the non-human animal CD70 locus all or part of the nucleotide sequence encoding the non-human animal CD70 protein with a cDNA sequence comprising the protein encoding human CD70 or all or part of the nucleotide sequence encoding the human or humanized CD70 protein.
Preferably, the constructing method comprises inserting or replacing at the non-human animal CD70 locus all or part of the nucleotide sequence encoding the transmembrane region, cytoplasmic region and/or extracellular region of the non-human animal CD70 protein with all or part of the nucleotide sequence comprising the transmembrane region, cytoplasmic region and/or extracellular region encoding the human CD70 protein. Further preferably, all or part of the nucleotide sequence encoding the extracellular domain of the non-human animal CD70 protein at the CD70 locus is inserted or substituted with all or part of the nucleotide sequence comprising the extracellular domain encoding the human CD70 protein. In one embodiment, the polypeptide is encoded by a polypeptide comprising the sequence encoding SEQ ID NO:2, amino acids 48-193, or a substitution to a nucleotide sequence encoding SEQ ID NO:1, amino acids 50-195. In one embodiment, the polypeptide is encoded by a polypeptide comprising the sequence encoding SEQ ID NO:2, amino acids 39-193, or a substitution to a nucleotide sequence encoding SEQ ID NO:1, amino acids 45-195.
Preferably, the construction method comprises inserting or replacing a nucleotide sequence comprising the humanized CD70 gene at the non-human animal CD70 locus. Preferably, the construction method comprises inserting or replacing a portion of the endogenous CD70 gene at the CD70 locus of the non-human animal with a portion comprising the human CD70 gene. Preferably, the construction method comprises inserting or replacing all or part of exons 1 to 3 of the non-human animal CD70 gene with a recombinant vector comprising all or part of exons 1 to 3 of the human CD70 gene. Preferably, the construction method comprises inserting or replacing part of exon 1, all of exon 2, and part of exon 3 of the non-human animal CD70 gene with a gene comprising part of exon 1, all of exon 2, and part of exon 3 of the human CD70 gene, preferably intron 1-2 and/or intron 2-3 are replaced.
Preferably, the construction method comprises modifying the coding frame of the non-human animal CD70 gene, and inserting any nucleotide sequence comprising the above into the endogenous regulatory element of the non-human animal CD70 gene, wherein the coding frame of the modified non-human animal CD70 gene can adopt a functional region for knocking out the non-human animal CD70 gene or adopt a sequence for inserting a segment so that the non-human animal CD70 protein is not expressed or the protein with reduced expression or expressed is not functional, and further preferably, the coding frame of the modified non-human animal CD70 gene can be all or part of the nucleotide sequence from exon 1 to exon 3 of the non-human animal CD70 gene. Preferably, the insertion is performed by first disrupting the coding frame of the endogenous CD70 gene in the non-human animal and then performing the insertion operation. Or the insertion step can cause frame shift mutation to the endogenous CD70 gene and realize the step of inserting the human sequence.
Preferably, the genome of the non-human animal comprises a humanized CD70 gene on at least one chromosome.
Preferably, at least one cell in the non-human animal expresses a human or humanized CD70 protein.
Preferably, the non-human animal is constructed using gene editing techniques including gene targeting using embryonic stem cells, CRISPR/Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases or other molecular biology techniques.
Preferably, the targeting vector of the present invention is used for construction of a non-human animal.
Preferably, the construction method comprises introducing the targeting vector into non-human animal cells, culturing the cells, transplanting the cultured cells into oviduct of female non-human animals, allowing the female non-human animals to develop, and identifying and screening the non-human animals with humanized CD70 genes.
Preferably, the construction method further comprises mating the non-human animal humanized with the CD70 gene with another genetically modified non-human animal, performing in vitro fertilization or directly performing gene editing, and screening to obtain the polygenetically modified non-human animal.
Preferably, the other gene is at least one selected from the group consisting of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, and LAG 3. Preferably, the human or humanized CD70 gene or other gene is homozygous for the endogenously modified locus; preferably, the human or humanized CD70 gene or other gene is heterozygous for the endogenous modified locus.
In an eighth aspect of the invention, there is provided a humanized non-human animal, wherein the non-human animal expresses a human or humanized CD70 protein, and/or wherein the genome of the non-human animal comprises a human or humanized CD70 gene.
Preferably, the humanized CD70 protein is the humanized CD70 protein.
Preferably, the humanized CD70 gene is the humanized CD70 gene described above.
Preferably, the nucleotide sequence encoding the human or humanized CD70 protein or the nucleotide sequence of the human or humanized CD70 gene is operably linked to endogenous regulatory elements at the endogenous CD70 locus in at least one chromosome.
Preferably, the non-human animal has reduced or absent expression of endogenous CD70 protein.
Preferably, the non-human animal further comprises other genetic modifications, the other genes selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, LAG 3.
Preferably, the human or humanized CD70 gene and/or said further gene is homozygous for the endogenous modified locus.
Preferably, the human or humanized CD70 gene and/or said other gene is heterozygous for the endogenous modified locus.
In a ninth aspect of the invention, a CD70 gene-deleted non-human animal is provided.
Preferably, the non-human animal lacks all or part of the nucleotide sequence of the CD70 gene.
Preferably, the non-human animal lacks all or part of exons 1 to 3 of the CD70 gene. Further preferably, all or part of the nucleotide sequence of one, two, three or a combination of two consecutive exons from exons 1 to 3 is deleted. Still more preferably, part of the nucleotide sequence of exon 1, all of exon 2 and part of exon 3 are deleted. Most preferably, the nucleotide sequence of part of exon 1, intron 1-2, all of exon 2, intron 2-3 and part of exon 3 are deleted.
Preferably, the non-human animal has a deleted portion of exon 1 of the CD70 gene that does not include at least a non-coding region, and more preferably, does not include a portion encoding a cytoplasmic region and/or a transmembrane region. Still more preferably, the non-human animal further lacks a nucleotide sequence encoding a portion of the extracellular domain of exon 1 of the CD70 gene.
In one embodiment of the present invention, the nucleotide sequence of the exon 3 of the endogenous CD70 gene deleted in the non-human animal does not include at least a non-coding region.
Preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the cytoplasmic, transmembrane and/or extracellular domain. Further preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular domain and/or the transmembrane domain. In one embodiment of the invention, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular region.
The tenth aspect of the invention provides a method for constructing the non-human animal with the CD70 gene deleted, wherein the method for constructing the non-human animal comprises the step of constructing the non-human animal by using the targeting vector.
In the eleventh aspect of the present invention, there is provided a method for constructing a polygene-modified non-human animal, comprising the steps of:
i) Providing the non-human animal or the non-human animal obtained by the construction method;
II) mating the non-human animal provided in the step I) with other genetically modified non-human animals, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-genetically modified non-human animal.
Preferably, the other genetically modified non-human animal comprises a non-human animal modified with the genes PD-1, PD-L1, CD27, CD40, OX40, 4-1BB and/or LAG 3.
Preferably, the polygenic modified non-human animal is a two-gene humanized non-human animal, a three-gene humanized non-human animal, a four-gene humanized non-human animal, a five-gene humanized non-human animal, a six-gene humanized non-human animal, a seven-gene humanized non-human animal, an eight-gene humanized non-human animal, or a nine-gene modified non-human animal.
Preferably, each of the plurality of genes modified in the genome of the polygenic modified non-human animal may be homozygous for the endogenous modified locus.
Preferably, each of the plurality of genes modified in the genome of the polygenetically modified non-human animal may be heterozygous for the endogenous modified locus.
In a twelfth aspect of the present invention, there is provided a non-human animal or its progeny obtained by the above construction method.
In a thirteenth aspect of the present invention, an animal model is provided, wherein the animal model is derived from the above non-human animal, the non-human animal obtained by the above construction method, or the above non-human animal or its progeny. Preferably, the animal model is a tumor-bearing or inflammatory animal model.
In a fourteenth aspect of the present invention, there is provided a method for producing an animal model, which comprises using the above-constructed humanized non-human animal of CD70 gene, a non-human animal lacking CD70 gene, or a multi-gene-modified non-human animal or its progeny. Preferably, the method further comprises the step of implanting the tumor cells.
In a fifteenth aspect of the present invention, there is provided a use of the above-mentioned humanized non-human animal of CD70 gene, a non-human animal deficient in CD70 gene, a multi-gene-modified non-human animal or progeny thereof, or a humanized non-human animal of CD70 gene, a non-human animal deficient in CD70 gene, a multi-gene-modified non-human animal or progeny thereof obtained by the above-mentioned construction method, for preparing an animal model.
In a sixteenth aspect, the invention provides the cell, the tissue or the organ, wherein the cell, the tissue or the organ expresses the humanized CD70 protein, or the genome of the cell, the tissue or the organ comprises the humanized CD70 gene, or the cell, the tissue or the organ is derived from the non-human animal or the non-human animal obtained by the construction method.
In a seventeenth aspect of the present invention, there is provided a tumor tissue after tumor loading, wherein the tumor tissue expresses the humanized CD70 protein, or the genome of the tumor tissue contains the humanized CD70 gene, or the tumor tissue is derived from the above non-human animal or its progeny, the non-human animal obtained by the above construction method, or the above animal model.
In the eighteenth aspect of the invention, a CD70 gene humanized cell is provided, wherein the cell expresses human or humanized CD70 protein, or the genome of the cell contains humanized CD70 gene.
Preferably, the humanized CD70 protein is the humanized CD70 protein according to the first aspect of the present invention.
Preferably, the expression of endogenous CD70 protein in said cell is reduced or absent.
Preferably, the genome of said cell comprises all or part of the human CD70 gene. Further preferably, the cell comprises the humanized CD70 gene described above.
In a nineteenth aspect, the present invention provides an application derived from the above-mentioned humanized CD70 protein, the above-mentioned humanized CD70 gene, the above-mentioned non-human animal or its progeny, the non-human animal obtained by the above-mentioned construction method, the above-mentioned animal model, the above-mentioned cell, tissue or organ, or the above-mentioned tumor-bearing tissue, the application comprising:
(A) Use in the development of products involving CD 70-related immune processes in human cells;
(B) Use as a model system in association with CD70 for pharmacological, immunological, microbiological and medical research;
(C) To the production and use of animal experimental disease models for the study of CD 70-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;
(D) The application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human CD70 signal channel regulator is studied in vivo; or,
(E) The functions of the CD70 gene are researched, the medicine and the drug effect aiming at the target site of the human CD70 are researched, and the application in the aspects of CD 70-related immune-related disease medicines and anti-tumor medicines is researched;
(F) The application of the CD27/CD70 signal channel in the treatment fields of tumors, immune-related diseases and the like is researched.
Preferably, the use may be for therapeutic purposes.
Preferably, the use may be for non-therapeutic purposes.
In a twentieth aspect of the present invention, there is provided a method of screening for a human CD 70-specific modulator, said screening method comprising administering the modulator to an individual implanted with tumor cells, and detecting tumor suppression; wherein the individual is selected from the non-human animal or its offspring, the non-human animal obtained by the construction method, or the animal model.
Preferably, the modulator is selected from CAR-T, a drug. Further preferably, the drug is an antibody.
Preferably, the modulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.
Preferably, the detection comprises determining the size and/or proliferation rate of the tumor cells.
Preferably, the detection method comprises vernier caliper measurement, flow cytometry detection and/or animal in vivo imaging detection.
Preferably, the detecting comprises assessing the subject's body weight, fat mass, activation pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.
Preferably, the tumor cell is derived from a human or non-human animal.
Preferably, the method of screening for a human CD 70-specific modulator may be for therapeutic purposes.
Preferably, the method of screening for a human CD 70-specific modulator may be for non-therapeutic purposes. The screening method is used for screening or evaluating drugs, and detecting and comparing the drug effects of candidate drugs to determine which candidate drugs can be used as drugs and which can not be used as drugs, or comparing the drug effect sensitivity degrees of different drugs, namely, the treatment effect is not necessary but is only a possibility.
According to a twenty-first aspect of the present invention, there is provided an evaluation method of an intervention program, the evaluation method comprising implanting tumor cells into an individual, applying the intervention program to the individual in which the tumor cells are implanted, and detecting and evaluating a tumor suppression effect of the individual after applying the intervention program; wherein the individual is selected from the group consisting of the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or a progeny thereof, and the above animal model.
Preferably, the intervention regimen is selected from CAR-T, drug therapy. Further preferably, the drug is an antigen binding protein. The antigen binding protein is an antibody.
Preferably, the tumor cell is derived from a human or non-human animal.
Preferably, the method of assessing the intervention regimen may be for therapeutic purposes.
Preferably, the method of assessing the intervention regimen may be for non-therapeutic purposes. The evaluation method detects and evaluates the effect of the intervention program to determine whether the intervention program has a therapeutic effect, i.e. the therapeutic effect is not necessarily but only a possibility.
In a twenty-second aspect of the present invention, there is provided a use of the non-human animal obtained by the above construction method or a progeny thereof, or the above animal model, in the preparation of a human CD 70-specific modulator.
In a twenty-third aspect of the present invention, there is provided a use of the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model, in the preparation of a medicament for treating tumor, inflammation or immune-related diseases, derived from the above-mentioned non-human animal or its progeny.
The "immune-related diseases" described in the present invention include, but are not limited to, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver diseases, diabetes, pain, or neurological disorders, etc.
The term "inflammation" as used herein includes acute inflammation as well as chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, cellulolytic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).
"tumors" as referred to herein include, but are not limited to, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.
The CD70 gene humanized non-human animal can normally express human or humanized CD70 protein in vivo, can be used for drug screening, drug effect evaluation, immune disease and tumor treatment aiming at human CD70 target sites, can accelerate the development process of new drugs, and can save time and cost. Provides effective guarantee for researching CD70 protein function and screening related disease drugs.
The "cell" of the present invention may be a fertilized egg cell, an embryonic stem cell, an immune cell (e.g., a T cell, a B cell, a Natural Killer (NK) cell, and a dendritic cell), a tumor cell, or the like. The tumor cell may be blood-borne tumor such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia and Waldenstrom's macroglobulinemia, and may also be thymus tumor, nephroblastoma, glioblastoma, astrocytoma, ovarian cancer, etc. Thus, depending on the source of the cells, a portion of the cells described herein may develop into individual animals and a portion may not.
The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that include the stated specified components or steps, as well as any other specified components or steps, which are not materially affected. However, when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described herein.
The whole or part of the invention, the whole is a whole, and the part is a part of the whole or an individual forming the whole.
The "humanized CD70 protein" of the present invention comprises a part derived from a human CD70 protein and a part of a non-human CD70 protein. Wherein, the "human CD70 protein" is identical to the whole human CD70 protein, namely the amino acid sequence of the "human CD70 protein" is identical to the full-length amino acid sequence of the human CD70 protein. The "part of human CD70 protein" is a continuous or alternate 5-193 amino acid sequence consistent with the amino acid sequence of human CD70 protein. Preferably, the amino acid sequence of the human CD70 protein is identical to the amino acid sequence of the human CD70 protein in a sequence or at intervals of 10-146, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 146, 150, 160, 170, 180, 190, 193.
The "whole transmembrane region of human CD70 protein", "whole cytoplasmic region of human CD70 protein" or "whole extracellular region of human CD70 protein" according to the present invention means that the amino acid sequence thereof is identical to the full-length amino acid sequence of the transmembrane region, cytoplasmic region or extracellular region of human CD70 protein, respectively.
The "part of the extracellular region of the human CD70 protein" of the present invention is a sequence of 5 to 155 (preferably 10 to 146) consecutive or spaced amino acids identical to the amino acid sequence of the extracellular region of the human CD70 protein, for example, a sequence of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 146, 150, 155 consecutive amino acids identical to the amino acid sequence of the extracellular region of the human CD70 protein.
The "part of the non-human animal CD70 protein" according to the present invention refers to a sequence of contiguous or separated 5 to 195 (preferably 10 to 50) amino acids identical to the amino acid sequence of the non-human animal CD70 protein, for example, contiguous 5, 10, 20, 30, 40, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 195 amino acids identical to the amino acid sequence of the non-human animal CD70 protein.
The "humanized CD70 gene" of the present invention includes a portion derived from a human CD70 gene and a portion derived from a non-human CD70 gene. Wherein, the "human CD70 gene" is identical to the whole human CD70 gene, namely the nucleotide sequence is consistent with the full-length nucleotide sequence of the human CD70 gene. The "part of the human CD70 gene" is a nucleotide sequence of 20-9503bp (preferably 20-4842bp or 20-911 or 20-441) which is continuous or spaced and is consistent with the nucleotide sequence of the human CD70 gene, for example, 20, 50, 100, 200, 300, 400, 441, 500, 600, 700, 800, 900, 911, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 4000, 4842, 5000, 6000, 7000, 8000, 9000 or 9503bp is consistent with the nucleotide sequence of the human CD70 gene.
"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals. For example, the portion of exon 1 of the human CD70 gene, which comprises consecutive or spaced nucleotide sequences of 5-310bp (preferably 10-21 bp) identical to the exon 1 nucleotide sequence of the human CD70 gene. For example, the portion of exon 3 of the human CD70 gene, which comprises consecutive or spaced nucleotide sequences of 5-567bp (preferably 10-386 bp) identical to the nucleotide sequence of exon 3 of the human CD70 gene. In a specific embodiment of the present invention, the "part of exon 1" contained in the "human CD70 gene" at least includes the nucleotide sequence from the 48 th amino acid of the coding human extracellular region to the last nucleotide sequence of exon 1. In a specific embodiment of the present invention, the "part of exon 3" contained in the "humanized CD70 gene" at least includes a nucleotide sequence from the first nucleotide sequence of exon 3 to a stop codon.
The "xx to xxx exon" or "all of the xx to xxx exons" of the present invention comprise nucleotide sequences of exons and introns therebetween, for example, the "1 to 3 exons" comprise all nucleotide sequences of exon 1, intron 1-2, exon 2, intron 2-3 and exon 3.
The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 1-2" means an intron between exon 1 and exon 2.
The "part of the non-human animal CD70 gene" of the present invention is a nucleotide sequence of 20-3781bp (20-764 bp or 20-441 bp) continuously or intermittently, which is identical to the nucleotide sequence of the non-human animal CD70 gene, for example, a nucleotide sequence of 20, 50, 100, 200, 300, 400, 441, 500, 600, 700, 764, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3781 nucleotides is identical to the nucleotide sequence of the non-human animal CD70 gene.
The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "CD70 locus" refers to a DNA fragment of an optional stretch of exons 1 to 3 of the CD70 gene.
The "nucleotide sequence" of the present invention includes a natural or modified ribonucleotide sequence and a deoxyribonucleotide sequence. Preferably DNA, cDNA, pre-mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.
"treating" as referred to herein means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.
"homology" in the context of the present invention refers to the fact that, in the context of using amino acid sequences or nucleotide sequences, one skilled in the art can adjust the sequences to have (including but not limited to) 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% identity.
One skilled in the art can determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual,2nd Ed., 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. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, alanr.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, methods In ENZYMOLOGY (J.Abelson and M.Simon, eds. Inchief, academic Press, inc., new York), specific, vols.154and 155 (Wuetal. Eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); gene Transfer Vectors For mammarian Cells (J.H.Miller and M.P.Caloseds, 1987, cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., academic Press, london, 1987); handbook Of Experimental Immunology, volumes V (d.m.weir and c.c.blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1986).
In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment of the method of the present invention, the genetically modified animal is from a member selected from the group consisting of the family Pomaceae (e.g., hamster-like hamster), the family Kangalidae (e.g., hamster, new world rat and mouse, hamster), the superfamily muridae (true mouse and rat, gerbil, spiny rat, crowned rat) the families of marmotacaceae (climbing mice, rock mice, tailed rats, madagascar rats and mice), acanthomyidae (e.g. thorny sleep rats) and spacidomyidae (e.g. mole rats, bamboo rats and zokors). In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.
In a specific embodiment, the non-human animal is a rodent that is a C57BL, C58, CBA/Br, CBA/Ca, A/J, CBA/H/st, CBA/H, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola strain C57 BL/BL, C58, CBA/Br, CBA/Ca, A/J, CBA/ST, CBA/H, and CBA/H strainsMouse and NOD, NOD/SCID, NOD-Prkdcscid IL-2rgnullBackground mice.
The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.
All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention.
The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: schematic comparison of mouse CD70 gene and human CD70 locus (not to scale);
FIG. 2: schematic representation of humanization of mouse CD70 gene (not to scale);
FIG. 3: CD70 gene targeting strategies and targeting vector design schematic (not to scale);
FIG. 4: southern Blot assay results, where WT is wild type control;
FIG. 5: schematic representation (not to scale) of FRT recombination process for humanized mouse of CD70 gene;
FIG. 6: f1 generation mouse genotype identification result, wherein M is Marker, WT is wild type control, PC is positive control, H2O is water control;
FIG. 7: the mouse thymus tissue RT-PCR detection result is shown, wherein M is Marker, +/-H + is wild type C57BL/6 mouse, H/H is CD70 gene humanized homozygote mouse, H2O is water control.
Detailed Description
The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
In each of the following examples, the equipment and materials were obtained from several companies as indicated below:
BglII, draIII, aseI enzymes were purchased from NEB under the respective accession numbers R0144M, R0510L, R0526M;
c57BL/6 mice were purchased from national rodent laboratory animal seed center of the Chinese food and drug testing institute;
Brilliant Violet 510TManti-mouse CD45 Antibody was purchased from Biolegend, cat No. 103138;
PerCP/Cy5.5 anti-mouse TCR β chain from Biolegend, cat # 109228;
Brilliant Violet 605TManti-mouse CD11c Antibody, available from Biolegend under cat # 117334;
APC anti-mouse CD70 Antibody, available from Biolegend under cat No. 2204066;
PE anti-human CD70 Antibody from Biolegend, cat # 355103;
Zombie NIRTMfixable visualization Kit available from Biolegend, cat # 423106;
purified anti-mouse CD16/32 was purchased from Biolegend, cat # 101302.
Example 1 preparation of CD70 Gene-humanized mouse
A comparison scheme of mouse CD70 genes (NCBI Gene ID:21948, primary source.
To achieve the object of the present invention, a nucleotide sequence encoding a human CD70 protein may be introduced at the endogenous CD70 locus of a mouse, so that the mouse expresses the human or humanized CD70 protein. Specifically, the gene editing technology is used to replace the corresponding sequence of the mouse with the nucleotide sequence coding the human CD70 protein under the control of the regulatory element of the mouse CD70 gene, and the schematic diagram of the humanized CD70 locus is shown in FIG. 2, so as to realize the humanized transformation of the mouse CD70 gene.
Further design as shown in figure 3 of the targeting strategy diagram, which shows the targeting vector containing the mouse CD70 gene upstream and downstream homology arm sequences, and contains encoding human CD70 protein nucleotide sequence of A fragment. Wherein, the sequence of the upstream homology arm (5 'homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence from 57456455 to 57460839 of NCBI accession No. NC-000083.7, and the sequence of the downstream homology arm (3' homology arm, SEQ ID NO: 4) is the same as the nucleotide sequence from 57448179 to 57452246 of NCBI accession No. NC-000083.7; the human CD70 gene sequence (SEQ ID NO: 5) contained in fragment A is identical to the nucleotide sequence 6586020 to 6590861 of NCBI accession No. NC-000019.10; the connection between the upstream of the human CD70 sequence in the A fragment and the mouse is designed as Wherein the sequence "aacag"g" in "is the last nucleotide, sequence of the mouseThe first "c" in (a) is the first nucleotide in humans; the connection of the downstream of the human CD70 sequence and the mouse is designed asWherein the sequence "cctga"A" in "is the last nucleotide, sequence, of humanThe first "c" in (a) is the first nucleotide in the mouse.
The targeting vector also comprisesThe resistance gene selected by positive clone, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein the connection between the 5' end of the Neo-box and the mouse gene is designed as Wherein the sequence "cgcagThe last "g" of "is the last nucleotide, sequence, of the mouseThe first "C" in (a) is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo box and the mouse gene is designed asWherein the sequence "GCCGCThe last "C" in "is the last nucleotide of the Neo cassette, sequenceThe first "T" in (a) is the first nucleotide in the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin A subunit coding gene (DTA)) is constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse CD70 after being transformed is shown as SEQ ID NO:10, and the expressed protein sequence is shown as SEQ ID NO: shown at 11.
The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The method comprises the steps of transfecting a targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse by electroporation, screening the obtained cells by using a positive clone screening marker gene, detecting by using PCR and Southern Blot technology to confirm the integration condition of an exogenous gene, screening correct positive clone cells, detecting clones identified as positive by PCR by using Southern Blot (cell DNA is digested by BglII or DraIII or AseI respectively and hybridized by using 3 probes, the lengths of the probes and target fragments are shown in Table 1), detecting exemplary results are shown in FIG. 4, and detecting by further verification through sequencing, wherein 4 clones with numbers of 1-A11, 1-G02, 1-G12 and 3-E06 are positive clones without random insertion.
Table 1: specific probes and target fragment lengths
Restriction enzyme | Probe needle | Wild type fragment size | Recombinant sequence fragment size |
BglII | 5’Probe | 8.4kb | 5.8kb |
DraIII | 3’Probe | 20.7kb | 12.1kb |
AseI | Neo Probe | - | 10.4kb |
Wherein the PCR assay comprises the following primers:
PCR-F1:5’-CACTGCCACGGAATTCTTGAGGTGA-3’(SEQ ID NO:12),
PCR-R1:5’-TCATTCTGTCTTTTCGGTCACGCG-3’(SEQ ID NO:13);
PCR-F2:5’-GCTCGACTAGAGCTTGCGGA-3’(SEQ ID NO:14),
PCR-R2:5’-ACAGTCTGGACCTCCGAGGCA-3’(SEQ ID NO:15);
the Southern Blot detection comprises the following probe primers:
5’Probe:
5’Probe-F:5’-AGAAGCAACTTGCAGAAGGGACAGG-3’(SEQ ID NO:16),
5’Probe-R:5’-TTTAGAGGCCCTCGGAGATTGGTCT-3’(SEQ ID NO:17);
3’Probe:
3’Probe-F:5’-TCTCCTCTCTCTTTGTTGGCTCTGG-3’(SEQ ID NO:18),
3’Probe-R:5’-AAGGCCTTCTAGCACAGGATCTCCA-3’(SEQ ID NO:19);
Neo Probe:
Neo Probe-F:5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO:20),
Neo Probe-R:5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO:21)。
the selected correctly positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F0 generation chimeric mice and the wild mice are backcrossed to obtain F1 generation mice, and the F1 generation heterozygous mice are mutually mated to obtain F2 generation homozygous mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 5 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous CD70 gene mice. The somatic genotypes of the progeny mice can be identified by PCR (primers shown in Table 2), and the identification results of exemplary F1 mice (from which the Neo marker gene has been removed) are shown in FIG. 6, wherein 8 mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, and F1-08 are all positive heterozygous mice. This indicates that using this method, humanized mice of the CD70 gene can be constructed that can be stably passaged without random insertions.
Table 2: primer name and specific sequence
The expression of humanized CD70mRNA in positive mice can be confirmed by conventional detection methods, such as RT-PCR and the like. Specifically, 1 mouse of 9-week-old wild-type C57BL/6 mouse and 10-week-old humanized homozygote CD70 mouse prepared by the method were selected, and after cervical dislocation, thymus tissue was taken, and the primer sequences shown in Table 3 were designed to detect the mRNA expression of the thymus tissue of the C57BL/6 mouse and the humanized homozygote CD70 mouse. The results showed that only murine CD70mRNA expression was detected in C57BL/6 mouse thymus tissue (FIG. 7A); only expression of humanized CD70mRNA was detected in the thymus tissue of humanized homozygote mouse of CD70 gene (FIG. 7B), and no expression of murine CD70mRNA was detected (FIG. 7A).
Table 3: RT-PCR primer sequences
The expression of humanized CD70 protein in positive mice can be confirmed by flow cytometry. Specifically, 1 mouse of 8-week-old wild-type C57BL/6 female mice and 1 mouse of 9-week-old humanized homozygote of CD70 gene prepared in this example were selected, bone marrow cells were harvested after cervical-denuded euthanasia, and after incubation for 24 hours with 1ug/ml Lipopolysaccharide (LPS), anti-mouse CD45 antibody Brilliant Violet 510 was usedTManti-mouse CD45 Antibody (mCD 45), murine specific T cell surface Antibody PerCP/Cy5.5 anti-mouse TCR beta chain (mTCR beta), anti-mouse CD11c Antibody Brilliant Violet 605TMFlow-through CD70 Antibody (hCD 70) recognition staining was performed after anti-mouse CD11c Antibody (mCD 11 c), anti-mouse CD70 Antibody APC anti-mouse CD70 Antibody (mCD 70) or anti-human CD70 Antibody PE anti-human CD70 Antibody (hCD 70)The detection result shows that the proportion of mCD70 marker cells (characterized by mCD45+ mTCR beta-mCD 11C + mCD70 +) in Dendritic Cells (DC) (characterized by mCD45+ mTCR beta-mCD 11C +) in wild type C57BL/6 mouse bone marrow is 14.1 percent, and the proportion of hCD70 (characterized by mCD45+ mTCR beta-mCD 11C + hCD70 +) marker cells is 0.95 percent; in contrast, the percentage of hCD 70-labeled cells in CD 70-gene-humanized mouse bone marrow DC cells was 20.1%, and the percentage of mCD 70-labeled cells was 0.94%. The results show that the humanized CD70 protein can be successfully expressed in the bone marrow cells of the humanized mouse with the CD70 gene.
Example 2 preparation of double-humanized or multiple double-humanized mice
The method or the prepared CD70 gene humanized mouse can also be used for preparing a double-humanized or multi-humanized mouse model. For example, in example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other genetic modifications such as PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, LAG3, etc., or can be humanized CD70 mice and then used to obtain a mouse model with CD70 and other genetic modifications by using isolated mouse ES embryonic stem cells and gene recombination targeting technology. The homozygote or heterozygote of the CD70 mouse obtained by the method can also be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, the homozygote or heterozygote of humanized CD70 and double-gene or multi-gene modified heterozygote of other gene modification can be obtained with a certain probability according to Mendel genetic rules, then the heterozygote is mated with each other to obtain double-gene or multi-gene modified homozygote, and the in vivo efficacy verification of targeted human CD70 and other gene regulators can be carried out by utilizing the double-gene or multi-gene modified mice.
Example 3 drug efficacy verification
The humanized mouse with CD70 gene or the mouse modified by multiple genes prepared by the method can be used for evaluating the drug effect of the targeted human CD70 antibody. For example, a tumor animal model is constructed by taking a humanized homozygote mouse of the CD70 gene, the mouse is divided into a control group or a treatment group, the treatment group is injected with an antibody drug targeting human CD70, and the control group is injected with an equal volume of physiological saline. Then, the body weight, the tumor volume and the tumor related indexes of each group of mice are monitored, and the safety and the in-vivo drug effect of the antibody drug in the humanized CD70 mice can be effectively evaluated.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention. It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Sequence listing
<110> Baiosai Diagram (Beijing) pharmaceutical science and technology Co., ltd
Construction method and application of <120> CD70 gene humanized non-human animal
<130> BHCD70.002CN1-P0102021030231Z
<150> 2021106044751
<151> 2021-05-31
<160> 35
<170> SIPOSequenceListing 1.0
<210> 1
<211> 195
<212> PRT
<213> Mus musculus
<400> 1
Met Pro Glu Glu Gly Arg Pro Cys Pro Trp Val Arg Trp Ser Gly Thr
1 5 10 15
Ala Phe Gln Arg Gln Trp Pro Trp Leu Leu Leu Val Val Phe Ile Thr
20 25 30
Val Phe Cys Cys Trp Phe His Cys Ser Gly Leu Leu Ser Lys Gln Gln
35 40 45
Gln Arg Leu Leu Glu His Pro Glu Pro His Thr Ala Glu Leu Gln Leu
50 55 60
Asn Leu Thr Val Pro Arg Lys Asp Pro Thr Leu Arg Trp Gly Ala Gly
65 70 75 80
Pro Ala Leu Gly Arg Ser Phe Thr His Gly Pro Glu Leu Glu Glu Gly
85 90 95
His Leu Arg Ile His Gln Asp Gly Leu Tyr Arg Leu His Ile Gln Val
100 105 110
Thr Leu Ala Asn Cys Ser Ser Pro Gly Ser Thr Leu Gln His Arg Ala
115 120 125
Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ala His Gly Ile Ser Leu
130 135 140
Leu Arg Gly Arg Phe Gly Gln Asp Cys Thr Val Ala Leu Gln Arg Leu
145 150 155 160
Thr Tyr Leu Val His Gly Asp Val Leu Cys Thr Asn Leu Thr Leu Pro
165 170 175
Leu Leu Pro Ser Arg Asn Ala Asp Glu Thr Phe Phe Gly Val Gln Trp
180 185 190
Ile Cys Pro
195
<210> 2
<211> 193
<212> PRT
<213> Homo sapiens
<400> 2
Met Pro Glu Glu Gly Ser Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly
1 5 10 15
Cys Val Leu Arg Ala Ala Leu Val Pro Leu Val Ala Gly Leu Val Ile
20 25 30
Cys Leu Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu
35 40 45
Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His
50 55 60
Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala
65 70 75 80
Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu
85 90 95
Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu
100 105 110
Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu
115 120 125
Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg
130 135 140
Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro
145 150 155 160
Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu
165 170 175
Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
180 185 190
Pro
<210> 3
<211> 4385
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agactgctgg gctagagagg aaagcccagc gtattgtagt aggagtgtta ggttaggact 60
tagcaccaac ctttatatcc aacatggccc cagcccctca ttgaggaatt ctagcaggca 120
ggaagacttg gggggatgag ggggacagga caggaccaat tcacccctga ccacaccagc 180
atccctcccc cagagaaatc taagcaggga ctccacccct gagccacgcc cccaaggcca 240
gtggatgagt gtggatgaat gggcaacact tgccaactct atactgctct agcttccatc 300
ctcaccactg caaataataa taataataat aataataata aacatgatgt aaatcattgg 360
tacatggatt ctttataaag ttcaaagtcc tccagcagtt aatttcctag tgtactgcat 420
agttttatgt caatacaaat agggttggac aaggcatgac tgcatttcct tgattaatgt 480
tcaatgtagg agggcccagc acacaacatt tccatggaga tggtcctaag ccctacaaga 540
atgtaggctg agcaagtcag taagccagag atccccctag gctcctgctt cagtcctgcc 600
ttctctcgat gatgaggaac tataaagagg aacaaatcct tccctctcca aggtcacggt 660
gttcatcaac aataaaacct ctagcatgaa ccacatacat tagatgcctc aaagccccca 720
aaatggagct gaagaaatga gttagttatg agcaccggct gggactctcg ctgactctgg 780
agggctccat ttcagctagg ctgatcgatg agctgcggca atcctcctgt ctctgcttct 840
cagcaggcta ccattacaga caggcactga tgaaccccac tttcaggtat caaactcaga 900
gttcccattc ttgcttgatg gggcctgtac cagtaacttt aatttttttt cagaccttat 960
tttcaatgat gattcttaaa cactttaacc ttccttatag cccaccaccc accagaggta 1020
gtgggaaaga aaggatacag gagaagtgga cctgtttaga aaggttcttt ggagcaactc 1080
ctgcctgtgt tgtcaggaaa gaagttcaca ggttagcagc cgcagcttga tccactcaca 1140
aacacttcac ggacacacca gcagtccagt ttggtagagt caagatagca aacaggaatg 1200
aatccgaagt agtggctcta cctagcacta ggagaagctc tctctcagct gtgcctcttt 1260
cagggaagtt aagaacaatt catcagaccc acaggcgttg tacagctagc tccttaagcc 1320
tagctccgcc tctgtcactg tccatcaagc actttttttt tttttcattt ttcgaaacag 1380
ggtttctctg tgtagccctg gctgtcctgg aactcatttt atagaccagg ctggcctcga 1440
actcagaaat ccgcctgcct ctgcctccca agtgctgggt gtaaaggcat gggccaccac 1500
gactggctca tcaagtcctt tttataccct ccaaacctca catgttctcc aagggtcttg 1560
cctcaacact ccgccaatca gcatgagtcc gaggaagctg caagaaactg cagcacacca 1620
ccagaagact tttggtgctc tttttctctc tatagagtcc ctgtgagacc ccgacttaga 1680
gcgtttctcc ctgggaggta aagcccctga atgttccccc aagcctgttt tccctgatct 1740
ctaaaaatac agccagaaag aagctctttg tcctctacag ccagcaaaaa gcctttttgt 1800
ttctatacct tataaccaga gatacgataa ttgtaatttt accaaggaca cctggcagag 1860
aagagctgta gccaactctc ctcccaactc ctcccaactc ctcccaattc ctcagctagc 1920
tgttaaaagc cccctacggt taccactcag ggtggaactc ccctgccctg cgcagcgaag 1980
aggagttcgt cctcagctag ctgataataa aacctcttgc agtttgcatc aggtgtggtt 2040
ttctcaagtc attggggtgc tggccagctc atcccgggac ttgagcggag gcccagcttc 2100
aggggtctta catcccgaca aaggcagcat gttgttagca aagatccttc atcacgtgtc 2160
ctttcacgtg cttgctttag tagaacatgc cctctgctgt gtctgcttca gctaaacgtt 2220
ccttcaggag tctgccttag tctatcacct gtgtccactt caagaaaaca ctccttcact 2280
tgtctgccct agcaaaacac caccaacaca actgactttc caaagaaccc ctaagtttcc 2340
acttcagggc ctgtatccac tgagccatat ccccagccta cttcatattt tttaaagggt 2400
ttgttttgtt ttaatgtatg tgtggggggg cgatatgtgc acaggtgccc atggaaacca 2460
gagttgccaa atactcctag aactggaatt tcagatcgtt ttaaaccatt tgacatgggt 2520
gctgggaagt gagccctctc tctgggatag acacactcaa cctctgagat gtctctccaa 2580
ccccatgtta atctcactat acctgaattg caggagatta gatccagggg ccactgacca 2640
acatgttctc cccaaaacag aatctgaagg tggtggtggc ctctagctgg gccagctcaa 2700
acccatttca gaggcaacct ctccctggta ccctctcctg tagtaggcat ctcaacttcg 2760
gaaaaacttg agcttgtaga tgtgttccaa catttcatca aacagtctgg atcctgggta 2820
aaatagactt ccgtgaagaa cacacagcac tcttggggtg cccagagaag atccagaaga 2880
gaggacagca gagacaggag agatacaagc cccaggcctg ccaggctggg gcagctgctg 2940
cccagctcgg cacctcctgc ctcaagtgtt ttctcgcctc tgccagttct aagtaaactc 3000
tcagctgctc actctgctac cgtatctcat gtggcaaaga ggctagaaca aagtggaggc 3060
cacctgaaag caccaagcct ggttgtctgc ctgttcattc tttattgtct gacagcttcg 3120
tatatttgca aaaatgaatt gtagtcatga ccaccccctt tcccctctcc agtccactcc 3180
cctccttgtc ctgctgaaca ttttcttcca agcaagttct gctccccacc cctgtaatcg 3240
ccactgcctt acaggctttc gagtctcttt ctgtgtgggc tgcactgaat catgggcagg 3300
aggctggttt tctggggcaa tggcaactta ctggtggcca caacactgaa gaaagtgacc 3360
ttcctttccc agtggccaga actgcccaga aagcctcagg gaggggcaga atctcatggc 3420
ccagccccta tcaaggccaa accattcggg gacatcttgt gccaattttc acagctgagt 3480
taagtgtagg agtgcaatgg ctgtgtccac tttcaggtag gatcattgtc tcagtctccg 3540
ggtgacatgg atggggatgg gggtggggat cagcagtagc taatcaatat gctagctatc 3600
atattgagac caagccaagt gtggtggctc acaccagcaa tctagtagtc ctgcagtcag 3660
aaggatgtga gaggccagcc tgggctacat gacaagtttg acacctgcct gagctacttg 3720
aggagctgaa ggtcaccatg gacaccgtgg caaatactaa gccagcctgg gctctacggt 3780
gagttccaat gtgagctgtt tataatggcc ccccatggaa gagcctacaa tcgtgtccaa 3840
atattttggg aagcacaagg gattcctgga tgcggatgct tcctgatttc ctagcattca 3900
gcccaatact gaacctctaa tagatgctca gtgaatggtt tgtcgaatga atgaatgaat 3960
gaatgggcac cgagcagcag gagcaggggc tgaatgccca atcggaagct taaatattct 4020
gaagcaggga caggcctgct tcagtttgtc tgtgggatgg cagaaggtgc caaaagctcc 4080
aggggatttc cctgccctcc gagaagaggc ccagttcttc ccctgcatcg gacatccccg 4140
aggttctaag ggcaggtcaa ggcaggcaga agcttcaaaa gctcggctga ggaggctaca 4200
gcttcccgct gccttcaggc cgctgcttcc gtgcagggat gccggaggaa ggtcgccctt 4260
gcccctgggt tcgctggagc gggaccgcgt tccagcgcca atggccatgg ctgctgctgg 4320
tggtgtttat tactgtgttt tgctgttggt ttcattgtag cggactactc agtaagcagc 4380
aacag 4385
<210> 4
<211> 4068
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tactatggag gaggcctgtg cagaagctat aggcctctag gtgggaagtg agcactctaa 60
gcttgacctt caaagctccc tgtgataggt cacagctagg ccccacctcc aaagggtcca 120
catggaccct agattcaaac ctgaacattc caatcctggt cccaataggc ccatgggcat 180
ctcacagtac aaatgtattt aattcaaatt caacaccctc caccccttaa caccttcaat 240
gtctttcaaa agactgagtt cagaggcagc agaaacagct cagcagttag agcaccagct 300
gctcttgcag aggacctggg tttggttcca gcacctatag ggcagctcac agccagctct 360
aactccagct ccagggatcc cacactccgt tctggcctct gtgggcacag tgcacacatg 420
ttgcacagac agacatgaag gcaaaacacc catgcacaga aaaaatttta aaaacagaga 480
ggcctgtcta acagtctaaa ccaactaatc cttaactcgg gggggggggg ggagggcact 540
atgaaactag aatccatcaa ggttttcccg tggtagaaaa taaatcatcc aactgtcaag 600
tggggaggat ggaataggga ggcagatgag gccgaggcaa ggtgaaaccc cagcattgca 660
agttttgctc cctctactcc aaacctggtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 720
tgtgtgtgtg tgtttaaatg tcaaagggtt aaattgacag acaaaagttc attaaacaga 780
cagctaacct agcagacatc ctacctccct gacagcttct ttttgtgctc cagagaacag 840
aacacagcac ttacagtttg cacgagggat gaagctgggt gttgttcaga gccaggatct 900
gtccaggagc cagaggctta ctcttcagtc tagactggct ggctagtgag ctgccagggt 960
ccctcctgtg tccacctccc cagcatggga cacagacagg catgccatca gacacaaagt 1020
ttacatggct gccggagctc tgaacgcagg tttcccctga ctgtcttgtt cctatccaac 1080
ggtctgtcca attccaatgt gtgaactctg ggaacacgtt catttaggct gtagcaaata 1140
ttcacaggca gaaacggtga gtggactata aggaaactct gttctctgct atgcaaacca 1200
taaaaggatg tgttcttaag taaaataaaa atgacctcag tggaaatata ttgtgacggg 1260
aaaggactga ttttcaaggt ttgagcacgg ggggtattat aaagctgcta gggggaatct 1320
ccaccatcca cataggatga tgtcaacagg tgcagccttg aaacttcccc ttcaatgaca 1380
ctcaactgtg tagtaacttg atttatggta ctaagtgaca actgagagga gacctttaat 1440
cccagcactt gggaggcaga ggcaggcaga tctgaattca aagccagcct gatctacaga 1500
gtgattttca gggctataca gtgaaacctt gtgagagctg gggaagcgat ggctactctt 1560
ccagaggacc tgggtttgag tccccgtgct ttatggactg aaccatgcct ggacctttaa 1620
ggttcatttc ccctcaggat gtaggctcca gctctattct gtttctgaag gtctgtgcgt 1680
ataaagttgg gcatcatggc acacacctgt catcccaaca ttcccaggaa gctgaggcag 1740
gaggtttgca agtttgaggc caatcttagc tacatagtgt gctcttatct ggaaaaacaa 1800
aacaaacagt gggaatggga gggagaggaa gaaagggggt gtaaggggag agagagatag 1860
ggaagatggg ggcgggcatg gcagggaaga gagagagaag gaagaatggc gaaggaaatg 1920
caccacctct ctgttcgact ctattgtttg ttccgggtct catttacaaa acagaatagt 1980
tacacatatg tgcttttgaa actcgggatc tggacacaca cactgcacaa tggtcacttc 2040
tattcaacaa gttgaggtct cccgtctcct cagttgaccg tttctcctgc ctgcatgttt 2100
ctctttctgt ggaaatgcta tcaacagtcg atattattac cattgttact gctgaaagtt 2160
cctacagtct cggtcagggc ctccaactga ctcagataca caaccacaac ttctgagtaa 2220
ggtctcacac atttcacagg ggtctaggga gttcagcctt cttatttccc acgtctaagt 2280
aacaatatgc agtagcatcg ttcagggctg aataacactt cattgggtct ggatacccca 2340
ttagttgatt tggctagagc tgaaaataga ggatttcatt atgacatgtt cctacatggg 2400
gctcacattc acccccacaa tattctcttg tccccccttc ctcctacccc cttccccttt 2460
cctctaggcc tcatcaaatt gccccttctt gtatatttct ctctccaaat gcctaatatt 2520
agagaaaagg atggctttgt ctgagtctgt ctatgtctgt cttgttttaa taaaatggcc 2580
tctgtttcta tccataatag aaacatgatt taattccttt ttaaaaggtt ttaaagaatg 2640
tgatttattt attattgatg tgtatgtgat tctcacagtg catgtgcagg tccagggata 2700
gaacctagag tctgccttta cccactggat cagctcaatg cccccccccc accttttctt 2760
taacaggttt tgcacagtcc agtctggcct cacttcactc ttttttacag ttcaataaga 2820
cagcttcatt ttgtgcaatg tgtatctaga ccaaattttc tttttttgaa gacttattta 2880
tgtgtgaaag cccatagaag ccagaaatgg atatagtaac ctctggggag ggagttacag 2940
atggttgtga gtgccagtga gtgcagaatc gaattcagtc ctctgcaaga gctgcccagt 3000
gcgctaacca ctgggccact gctccagcct ctcagccttt tttaagagtc atcctcacag 3060
atatgaggtt tgttgggact ctggttagca ctttctgcca ttgctgcagt tggactcctt 3120
ttgcatactc tctgcccatt tctagtgctt acgaaacaga ttgaacttca ccaagaaagg 3180
aattttgctt ctactgaaaa gacaagccag ggcagcctct ctggggggtc ccattcttca 3240
gagcccaaaa ttaggacggt ggtgtgacct cagcatggac ccaaagaatg ttcaactgac 3300
tcaggacaga cacctgctca tggtactcac tgctgctccc ttcgcagtag ccaagaagtg 3360
tgccagcctg tgtgcctaac agcagattcg tgatgaggaa aacgtgacag cctggtggga 3420
atttctctat ttttgcaggc aatggagaca ggtaatgatg taagctgttg ctaggtccat 3480
gagaagagac tagtcacaat tctattccat tgatatatgt gtctatgcat atatccacgc 3540
ctcagtgttt attttgtctt tgagatagat gagagacaca cagagaatga agtatctatg 3600
gatggataaa tgaatgatag acagacagac aaacagatag atacctagat acatacatag 3660
acacagatat atagatatat agatagatct tagatagata catagatcaa aagacaggtg 3720
atagatgatg atagatatat agacagataa taatacatat agatgcatac atacacatat 3780
atagagagat acatacatac atacatacat atacatacat atgtgcatag ttacatacat 3840
ggaaacatag ataataattg tcctataaga cagcctgggc tcacctccac catcctgttt 3900
tcataagtga acttccagct ttcaactgtt ttctgatgga tttatcttga ttttgtttgt 3960
ttatttgacc aattggttgg ttgagcaact tttgagacaa ggtctcatgt agtctaagcc 4020
tcaaactatc tgtaaagctg aatacatcct tcaactcctg cttcttcc 4068
<210> 5
<211> 4842
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctgccgctcg agtcacttgg ggtgagttga gatggaaaag ttgggaagaa aacatagaga 60
ggcgcgtgac cgaaaagaca gaatgagatg ggtacaaaga ggccagagag gaagatctgg 120
tagggcagag acagagacca gaacagggag gcgaggcggg gaccaggctg cccggtgtag 180
gggctacgag acaggcagcc ctgccaggag gtacagggag atcccgggat gggaaaggta 240
ggcacacatg gaaatggaag atgactcggc tctggtgttc ccccggcagg ctgactcaga 300
ggctgctggg ggcttcacaa ggctgggcgt gggggcttcc tggggcctcc taggacggga 360
tggccccagc cactcgctcc gggtggggga ggggtccctt tggggaccgc gccgggcgcc 420
tttgcagcgt agagagtccg ctgcgcgcgg tgctctcgcg cccagtgaca tccaggaaaa 480
cgattcggga aacgaagaag ttcttttgaa ggtctcgact tcacgttccc cgctggttca 540
gacctgcttc ctctttaaga agtcttaaga gtaaaaaaaa ataaaatgaa ataaaatcac 600
cagtgcgcgc cgtgggatga gaggtggaaa ggaggatgga cagagaaaag agagctcctg 660
gcacagggga cacatagaac ctctctgctt acgtccgtgc cctgttttct ggtcttttct 720
tccagtggga cgtagctgag ctgcagctga atcacacagg taacacgggg gacgtggagg 780
gacggggaga agaagaggca cagagagaga aggaaggaga ggtagaaaga caagtgggga 840
gagacagaga gaaagagaca cagacagaga cggagggaga gagggaggga gagataggga 900
gggaaacgga gagggggaga cagagagaag acagagaggg agggagaggc ccagagaaag 960
ggagggagag acagaaaaaa gaacagagag agggacagag gggacagacg ggacaggaga 1020
agaaggggaa agaaagaaac agggagagac gcagggagag tggaagaggg agggagggag 1080
acaggcgggg gagacagaac gagagagagg gagagagacg gagaaagaga aaaaagacag 1140
atagaggaag agagaataaa aacgagagag gcgacctggc gtggttgctg gagcctgtaa 1200
tcccagcgct ttcggagggc gcggcgggtg gatcacttga gcccaggagt ttgagaccag 1260
cctggccaac atggtgaaac cccgtctcta ctaaaattac aaaaattagc cgggtgtggt 1320
ggcaggcacc tgtaatccca gctactcagg aggccgaggc aggagaatcg cctgaacctg 1380
ggaggcagaa gttgcagtga gccaagataa tggcactgct ctccagcctg ggtgacagag 1440
ggagattctg tcaaataaat aaataaaaag aagaaaggaa aagaaaaaga aagaaaggaa 1500
ggaaggaaaa gaaagagaag aaggaaggaa ggagagaggg agggaggtga gagagagaga 1560
aaggaaaaag aaagaagaaa gagaaaaaaa gaaataagag aaagaaaaga aagaaagcca 1620
gagagaggaa aagagaagga gaataacaga gaggaagaga cagagacaga gagggggaag 1680
aagaaaaaca gggcggggga gaaagagatc attcccaaga gaaaaaggaa aagggagaga 1740
acgagagaga gagagaggag agagagagcg agcaggtttg tgtttctggg catccactac 1800
ctttctctcc ctttccctct cagtccttca cattcgatct cctcccttgg aagaatcaaa 1860
ggggttttgt tagtaaacga aaacgctcac aggaaaggct acccttcctc cattcatgct 1920
acaagcacta attatacatc tgcagtgggc aggcactgtt ccggtactgc agtcgcgcac 1980
aaaacagaca gaaatccctg cctggtgagc ttcccttcta ctatcactcc taccgaggag 2040
gagatgacca aaaaaataaa aacgaacatg tgaaaaacag agcaatgcca cgtagagtaa 2100
aacaggccaa gagcaggttt tagagaaaaa atcaggcaag gaggtgggtg gggaatccgg 2160
gcatgccagg gttggggatt ttcatttaaa gtggagtcgc ggcggtcggg tgggggggtg 2220
gtatctgagt gggtggcatt ggcaagaagc cttgagggag aagaaatgaa cagttctgct 2280
ctaggtggaa gtctctacct cctcccagcc tctctcgaac gcttcttcct cttctgtttt 2340
tccacggttc atccccagag actaaaatta ctgcacgctt gcgtgttttg ttccttccat 2400
tttgtaatgg aagggacctt tgttgctctc tcttctcctt gggtctctga cccctgatat 2460
atcttccccc cctccccggc ctttgccata tttttcctgt aatttggtct tgaggcccct 2520
ttctggagag tggctgtcca gccccacatc gatggggcac caggggtggt ggccacaggt 2580
gttgacagtg tctttcacag gacagctctt tcttctggtg gatgatatca tgcctaagtg 2640
tgtgacaggg gaccaggtgt cactcttaca ggaaatttgc aaagcctggt aaacaccctt 2700
ggggctcctg tttgaccccg ttcagttcct tcctaacaag atagccagtc tccaggagag 2760
ccttgagtga ggaggagagt gaggctcagg tgtgtcagtc agataagttg cggagacagg 2820
agacgctgag atgaaacacg tgaaatatag ccgaggacct tggctcccgg ctgtgatccc 2880
agcgctttgg gaggctgagg tgggaggatc gcttgagccc aggagttcta gactatgttt 2940
cttgggaaac atagcaagac caccatctct agaacaaaat tttgaaaatt agctggtcat 3000
ggtggcgcat gccacctccc agtgactcgg gaggctgagg tgggagatag cttgagcccc 3060
agaggctgag gctgcagtga gctatgattg caccactgca ctccagcctg ggtgacagga 3120
ccagaccctg tctctaaaga aaataaagat cctagagaga agagggccaa cgggaaggta 3180
ggggagctct ctgagacaca cactcaacca gcgggtggaa agcaagaaaa aaaaagggtc 3240
ctgggagcca aagcttttat tggcgttcag ggtgtgaccc gagcaggcgt cccttgggga 3300
attctctcgt gcactctctt gcgcgctgtc tcgtgcgcac gctgtctctc tcgtgctgtc 3360
tgtctcactc tctcaggcac tctctcgctc actcccgtgc actctcgagc tctgtagctc 3420
tcatcctctc tcacacacac actctcccat gcactctctc gagctcactc tcacactctg 3480
tctcgtgcac tctctctcaa gctctctcgt cctctctcac acacacactc tctctcttgc 3540
tctctctctc atgctgtctc tcgtactctc tcctgctctc tcgtgctctc tctcactctc 3600
tcgtgcactc tccctcaagc tctctcgtcc tctctctcac actctctcgc tgtctcatgc 3660
tctctctccc tcatgatctc tctctctctc atgctgtctc ttgcactctc tcctgctctc 3720
tcgtgctctc tcgcactctc tcgtgcactc tccctcaagc tctctcgtcc tctcacacac 3780
tctctcgctc tctcatgctc tctctctcat gctctctgtc gcactctctc gtgcactctc 3840
gtcctctctc acacatacac tctctctctt gctctctctc tgcattctct ctctatatat 3900
ctctccctcc ccctctatct ttacccttgt ctgtttctgt tttttttttt tttttttttg 3960
tctctctctc tcctggtgtc tctctctccc tgtctctacc cctttcccct tacccctctc 4020
tccttccctt tagatctcta accttctatc cccctccctt ttttttgaga cagggtttct 4080
gtgaccaagg ctggagtgca gtggtgtgat catagctcac tgcagtctca gcctcccagg 4140
ctcaagcgat cctccagcct cagcctccca agagtagcca ggaccagagg cgtgcaccac 4200
cacacctggc tgacttttta ttttttgtag agatggggtc tcgccctgtt gtccaggctg 4260
gtcttgaact gctgacctca ggtgatccac ccgccttggc ctcctaaact gttaggattt 4320
caggcatgag ccaccacgtc caacctattc cttgatctct aactctcgat ctctatatct 4380
gactttctct atgaccccct gtgcctcagt ttccctaaac ctccatcctc tcatccctct 4440
aaccctgtgc ccccaggacc tcagcaggac cccaggctat actggcaggg gggcccagca 4500
ctgggccgct ccttcctgca tggaccagag ctggacaagg ggcagctacg tatccatcgt 4560
gatggcatct acatggtaca catccaggtg acgctggcca tctgctcctc cacgacggcc 4620
tccaggcacc accccaccac cctggccgtg ggaatctgct ctcccgcctc ccgtagcatc 4680
agcctgctgc gtctcagctt ccaccaaggt tgtaccattg cctcccagcg cctgacgccc 4740
ctggcccgag gggacacact ctgcaccaac ctcactggga cacttttgcc ttcccgaaac 4800
actgatgaga ccttctttgg agtgcagtgg gtgcgcccct ga 4842
<210> 6
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttggtttcat tgtagcggac tactcagtaa gcagcaacag ctgccgctcg agtcacttgg 60
ggtgagttga gatggaaaag 80
<210> 7
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgatgagacc ttctttggag tgcagtgggt gcgcccctga ccacaactcc aggatgactt 60
gtgaatattt tttttctttt 80
<210> 8
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
taaaatttta attctgttct tagtaacact cctcacgcag cacccagtgg atatcgaatt 60
ccgaagttcc tattctctag 80
<210> 9
<211> 121
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gaagttccta ttctctagaa agtataggaa cttcatcagt caggtacata atggtggatc 60
cactagttct agagcggccg ctactatgga ggaggcctgt gcagaagcta taggcctcta 120
g 121
<210> 10
<211> 842
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gaaggtgcca aaagctccag gggatttccc tgccctccga gaagaggccc agttcttccc 60
ctgcatcgga catccccgag gttctaaggg caggtcaagg caggcagaag cttcaaaagc 120
tcggctgagg aggctacagc ttcccgctgc cttcaggccg ctgcttccgt gcagggatgc 180
cggaggaagg tcgcccttgc ccctgggttc gctggagcgg gaccgcgttc cagcgccaat 240
ggccatggct gctgctggtg gtgtttatta ctgtgttttg ctgttggttt cattgtagcg 300
gactactcag taagcagcaa cagctgccgc tcgagtcact tgggtgggac gtagctgagc 360
tgcagctgaa tcacacagga cctcagcagg accccaggct atactggcag gggggcccag 420
cactgggccg ctccttcctg catggaccag agctggacaa ggggcagcta cgtatccatc 480
gtgatggcat ctacatggta cacatccagg tgacgctggc catctgctcc tccacgacgg 540
cctccaggca ccaccccacc accctggccg tgggaatctg ctctcccgcc tcccgtagca 600
tcagcctgct gcgtctcagc ttccaccaag gttgtaccat tgcctcccag cgcctgacgc 660
ccctggcccg aggggacaca ctctgcacca acctcactgg gacacttttg ccttcccgaa 720
acactgatga gaccttcttt ggagtgcagt gggtgcgccc ctgaccacaa ctccaggatg 780
acttgtgaat attttttttc ttttcaagtt ctacgtattt ataaatgtat atagtacaca 840
ta 842
<210> 11
<211> 195
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Pro Glu Glu Gly Arg Pro Cys Pro Trp Val Arg Trp Ser Gly Thr
1 5 10 15
Ala Phe Gln Arg Gln Trp Pro Trp Leu Leu Leu Val Val Phe Ile Thr
20 25 30
Val Phe Cys Cys Trp Phe His Cys Ser Gly Leu Leu Ser Lys Gln Gln
35 40 45
Gln Leu Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu
50 55 60
Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
65 70 75 80
Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly
85 90 95
Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val
100 105 110
Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
115 120 125
Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu
130 135 140
Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu
145 150 155 160
Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr
165 170 175
Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp
180 185 190
Val Arg Pro
195
<210> 12
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cactgccacg gaattcttga ggtga 25
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tcattctgtc ttttcggtca cgcg 24
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gctcgactag agcttgcgga 20
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
acagtctgga cctccgaggc a 21
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
agaagcaact tgcagaaggg acagg 25
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tttagaggcc ctcggagatt ggtct 25
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tctcctctct ctttgttggc tctgg 25
<210> 19
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aaggccttct agcacaggat ctcca 25
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggatcggcca ttgaacaaga t 21
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cagaagaact cgtcaagaag gc 22
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gtctgtggga tggcagaagg tgcca 25
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctcatgccac ccttcccact acttc 25
<210> 24
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ccacaggtgt tgacagtgtc tttcac 26
<210> 25
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tctagagatg gtggtcttgc tatgttt 27
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tacaacccag ctccctctca gcac 24
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gggaccagga ttggaatgtt caggt 25
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gacaagcgtt agtaggcaca tatac 25
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gctccaattt cccacaacat tagt 24
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgctggtggt gtttattact gtg 23
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cagggtgagg ttggtacaga g 21
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tagcggacta ctcagtaagc a 21
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cagtgtttcg ggaaggcaaa 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
accacagtcc atgccatcac 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tacagcaaca gggtggtgga 20
Claims (23)
1. A humanized CD70 protein, wherein the humanized CD70 protein comprises all or a portion of a human CD70 protein.
2. The humanized CD70 protein of claim 1, wherein the portion of the human CD70 protein comprises all or part of an extracellular region of the human CD70 protein, preferably comprises the amino acid sequence of SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; or, comprising a nucleotide sequence identical to SEQ ID NO:2 at positions 39-193 or 48-193 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.
3. The humanized CD70 protein of any one of claims 1 to 2, wherein the humanized CD70 protein further comprises a portion of a non-human animal CD70 protein, wherein the portion of the non-human animal CD70 protein comprises a cytoplasmic region and a transmembrane region of the non-human animal CD70 protein, and further preferably comprises a partial extracellular region; more preferably comprises SEQ ID NO:1, 1-49 or 1-44; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at positions 1-49 or 1-44 of at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%.
4. The humanized CD70 protein of any one of claims 1 to 3, wherein the amino acid sequence of the humanized CD70 protein comprises any one of the following groups:
a) The amino acid sequence of SEQ ID NO:11, and (b) the amino acid sequence shown in the figure;
b) And SEQ ID NO:11 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;
c) And SEQ ID NO:11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or
D) And SEQ ID NO:11, comprising the amino acid sequence of substitution, deletion and/or insertion of one or more amino acid residues.
5. A nucleic acid encoding the humanized CD70 protein of any one of claims 1 to 4.
6. A humanized CD70 gene comprising a portion of a human CD70 gene.
7. The humanized CD70 gene of claim 6, wherein the portion of the human CD70 gene comprises a portion of exon 1, a portion of exon 2, and a portion of exon 3 of the human CD70 gene, wherein the portion of exon 1 comprises at least 10bp of nucleotide sequence and the portion of exon 3 comprises at least 100bp of nucleotide sequence; preferably, the gene also comprises an intron 1-2 and/or an intron 2-3;
further preferably, said portion of the human CD70 gene comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO:5 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.
8. The humanized CD70 gene according to any of claims 6 to 7, further comprising a portion of a non-human animal CD70 gene, preferably comprising all or part of exon 1 and/or all or part of exon 3 of a non-human animal CD70 gene; further preferred comprises a nucleic acid sequence encoding SEQ ID NO:1, or 1-44, or a nucleotide sequence that hybridizes with a nucleic acid sequence encoding SEQ ID NO:1 or 1-44 of at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;
even more preferably, said humanized CD70 gene encodes a humanized CD70 protein according to any of claims 1 to 4.
9. The humanized CD70 gene of any one of claims 6 to 8 wherein the mRNA transcribed from the humanized CD70 gene comprises any one of the group consisting of:
a) SEQ ID NO: 10;
b) And SEQ ID NO:10 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;
c) And SEQ ID NO:10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; or,
d) Has the sequence shown in SEQ ID NO:10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
10. A targeting vector, said targeting vector comprising a donor nucleotide sequence, said donor nucleotide sequence comprising one of the following groups:
a) A nucleotide sequence encoding the humanized CD70 protein of any one of claims 1 to 4;
b) A nucleotide sequence encoding all or part of the extracellular region of the human CD70 protein, preferably encoding a polypeptide comprising SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; alternatively, the coding sequence comprises a nucleotide sequence identical to SEQ ID NO:2, positions 39-193 or 48-193, or at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% of the amino acid sequence identity;
c) The humanized CD70 gene of any one of claims 6-9; or,
d) Part of the No. 1 exon, the whole No. 2 exon and part of the No. 3 exon of the human CD70 gene, wherein the part of the No. 1 exon at least comprises 10bp nucleotide sequence, and the part of the No. 3 exon at least comprises 100bp nucleotide sequence; preferably comprises SEQ ID NO:5, or a nucleotide sequence identical to SEQ ID NO:5 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.
11. The targeting vector according to claim 10, wherein said targeting vector further comprises a 5 'arm and/or a 3' arm; the 5' arm has at least 90% homology with the sequence having NCBI accession number NC _ 000083.7; preferably, the 5' arm sequence is as set forth in SEQ ID NO:3 is shown in the figure;
the 3' arm has at least 90% homology with the sequence having NCBI accession number NC _ 000083.7; preferably, the 3' arm sequence is as shown in SEQ ID NO:4, respectively.
12. A method for constructing a non-human animal humanized with a CD70 gene, wherein the non-human animal expresses a human or humanized CD70 protein in vivo, and/or the genome of the non-human animal comprises the human or humanized CD70 gene;
preferably, the humanized CD70 protein is selected from the group consisting of the humanized CD70 protein of any one of claims 1 to 4;
preferably, the humanized CD70 gene is selected from the group consisting of the humanized CD70 gene of any one of claims 6 to 9.
13. The method of claim 12, wherein the non-human animal has reduced or absent expression of endogenous CD70 protein.
14. The method of constructing a recombinant vector according to any one of claims 12-13, which comprises introducing into the CD70 locus of a non-human animal any one of the following nucleotide sequences:
a) A nucleotide sequence encoding the humanized CD70 protein of any one of claims 1 to 4;
b) A nucleotide sequence encoding all or part of the extracellular region of the human CD70 protein, preferably encoding a polypeptide comprising SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; alternatively, the code comprises a nucleotide sequence identical to SEQ ID NO:2 from position 39-193 or 48-193, is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;
c) The humanized CD70 gene of any one of claims 6-9; or,
d) Part of No. 1 exon, all of No. 2 exon and part of No. 3 exon of human CD70 gene, wherein the part of No. 1 exon at least comprises 10bp nucleotide sequence, and the part of No. 3 exon at least comprises 100bp nucleotide sequence; preferably comprises SEQ ID NO:5, or a nucleotide sequence identical to SEQ ID NO:5 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.
15. The method of any one of claims 12-14, wherein the human or humanized CD70 gene, nucleotide sequence encoding human or humanized CD70 protein, is regulated in the non-human animal by endogenous regulatory elements.
16. The method of any one of claims 14-15, wherein said introducing is substitution or insertion; preferably, said introduced non-human animal CD70 locus replaces a corresponding region of a non-human animal; preferably, part of exon 1, all of exon 2, and part of exon 3 of the non-human animal CD70 gene are replaced; it is further preferred that introns 1-2 and/or introns 2-3 of the non-human animal are also replaced.
17. The method of any one of claims 12 to 16, wherein the targeting vector of any one of claims 10 to 11 is used for the construction of a non-human animal.
18. The method according to any one of claims 12 to 17, wherein the method comprises mating a non-human animal humanized with a CD70 gene with another genetically modified non-human animal, in vitro fertilization or direct gene editing, and screening to obtain a polygenetically modified non-human animal;
preferably, the additional gene is selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB and/or LAG 3.
19. Construction method according to any one of claims 12 to 18, characterized in that the human or humanized CD70 gene or other gene is homozygous or heterozygous for the endogenous modified locus.
20. A cell, tissue or organ which expresses the humanized CD70 protein of any one of claims 1 to 4 or which comprises the humanized CD70 gene of any one of claims 6 to 9 in its genome or which is derived from a non-human animal obtained by the construction method of any one of claims 12 to 19.
21. A tumor-bearing tumor tissue, wherein the tumor tissue expresses the humanized CD70 protein of any one of claims 1 to 4, or the genome of the tumor tissue comprises the human or humanized CD70 gene of any one of claims 6 to 9, or the tumor tissue is derived from the non-human animal obtained by the construction method of any one of claims 12 to 19.
22. Use of a humanized CD70 protein according to any of claims 1 to 4, a humanized CD70 gene according to any of claims 6 to 9, or a non-human animal obtained by the construction method according to any of claims 12 to 19, comprising:
a) Use in the development of products involving CD 70-related immune processes in human cells;
b) Use as a model system in association with CD70 for pharmacological, immunological, microbiological and medical research;
c) To the production and use of animal experimental disease models for the study of CD 70-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;
d) The application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human CD70 signal channel regulator is studied in vivo; or,
e) The research on the CD70 gene function, the research on the medicine and the drug effect aiming at the human CD70 target site, and the research on the application of the medicine for the immune-related diseases related to the CD70 and the anti-tumor medicine.
23. The humanized CD70 protein according to any one of claims 3 to 4, the humanized CD70 gene according to any one of claims 8 to 9, or the method of construction according to any one of claims 12 to 19, wherein the non-human animal is a rat or a mouse.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110604475 | 2021-05-31 | ||
CN2021106044751 | 2021-05-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115260303A true CN115260303A (en) | 2022-11-01 |
Family
ID=83759059
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210609446.9A Pending CN115260303A (en) | 2021-05-31 | 2022-05-31 | Construction method and application of CD70 gene humanized non-human animal |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN115260303A (en) |
WO (1) | WO2022253219A1 (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103596979A (en) * | 2011-03-16 | 2014-02-19 | 阿尔金-X公司 | Antibodies to CD70 |
WO2018113774A1 (en) * | 2016-12-23 | 2018-06-28 | Beijing Biocytogen Co., Ltd | Genetically modified non-human animal with human or chimeric cd27 |
CN109021106A (en) * | 2018-08-30 | 2018-12-18 | 浙江蓝盾药业有限公司 | A kind of humanization CD70 antibody LD70 and the preparation method and application thereof |
CN110859968A (en) * | 2019-11-21 | 2020-03-06 | 四川安可康生物医药有限公司 | Genetic biopharmaceuticals to activate systemic immune response to tumors |
CN111793646A (en) * | 2020-09-08 | 2020-10-20 | 北京百奥赛图基因生物技术有限公司 | Construction method and application of non-human animal subjected to IL1R1 gene humanization transformation |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4701374B2 (en) * | 2000-08-04 | 2011-06-15 | 大学共同利用機関法人自然科学研究機構 | NaV2 channel gene-deficient non-human animal |
MX2009006277A (en) * | 2006-12-14 | 2009-07-24 | Medarex Inc | Human antibodies that bind cd70 and uses thereof. |
CN108239659B (en) * | 2016-12-23 | 2020-03-03 | 百奥赛图江苏基因生物技术有限公司 | Preparation method and application of humanized gene modified animal model |
WO2020125639A1 (en) * | 2018-12-17 | 2020-06-25 | Biocytogen Jiangsu Co., Ltd. | Genetically modified non-human animal with human or chimeric genes |
CN112779285B (en) * | 2019-11-11 | 2023-01-06 | 百奥赛图(北京)医药科技股份有限公司 | Construction method and application of humanized IL-10 and IL-10RA gene modified animal |
-
2022
- 2022-05-31 CN CN202210609446.9A patent/CN115260303A/en active Pending
- 2022-05-31 WO PCT/CN2022/096213 patent/WO2022253219A1/en active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103596979A (en) * | 2011-03-16 | 2014-02-19 | 阿尔金-X公司 | Antibodies to CD70 |
WO2018113774A1 (en) * | 2016-12-23 | 2018-06-28 | Beijing Biocytogen Co., Ltd | Genetically modified non-human animal with human or chimeric cd27 |
CN109021106A (en) * | 2018-08-30 | 2018-12-18 | 浙江蓝盾药业有限公司 | A kind of humanization CD70 antibody LD70 and the preparation method and application thereof |
CN110859968A (en) * | 2019-11-21 | 2020-03-06 | 四川安可康生物医药有限公司 | Genetic biopharmaceuticals to activate systemic immune response to tumors |
CN111793646A (en) * | 2020-09-08 | 2020-10-20 | 北京百奥赛图基因生物技术有限公司 | Construction method and application of non-human animal subjected to IL1R1 gene humanization transformation |
Non-Patent Citations (1)
Title |
---|
NAKAE R.ET AL: "CD70 antigen isoform 1 [Homo sapiens]", GENBANK, 26 April 2021 (2021-04-26), pages 1 - 3 * |
Also Published As
Publication number | Publication date |
---|---|
WO2022253219A1 (en) | 2022-12-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111304246B (en) | Humanized cytokine animal model, preparation method and application | |
CN111057721B (en) | Preparation method and application of humanized IL-4 and/or IL-4R alpha modified animal model | |
CN112779285B (en) | Construction method and application of humanized IL-10 and IL-10RA gene modified animal | |
CN111793646B (en) | Construction method and application of non-human animal subjected to IL1R1 gene humanization transformation | |
CN112300265B (en) | Construction method and application of IL33 gene humanized non-human animal | |
CN113881681B (en) | CCR8 gene humanized non-human animal and construction method and application thereof | |
CN113046390B (en) | Humanized non-human animal of CSF1R gene, construction method and application thereof | |
CN112501204B (en) | IL21R gene humanized non-human animal and construction method and application thereof | |
CN112501205B (en) | Construction method and application of CEACAM1 gene humanized non-human animal | |
CN112553252B (en) | Construction method and application of TNFR2 gene humanized non-human animal | |
CN112501206B (en) | Construction method and application of PSMA (PSMA) gene humanized non-human animal | |
CN114835799A (en) | Construction method and application of IL1RAP gene humanized non-human animal | |
CN115011606A (en) | Construction method and application of CD37 gene humanized non-human animal | |
CN113264996A (en) | Humanized non-human animal and preparation method and application thereof | |
CN115260303A (en) | Construction method and application of CD70 gene humanized non-human animal | |
CN113388640B (en) | CCR4 gene humanized non-human animal and construction method and application thereof | |
CN115010800B (en) | Construction method and application of PVRIG gene humanized non-human animal | |
CN114853871B (en) | Humanized non-human animal of CSF1 and/or CSF1R gene, construction method and application thereof | |
CN112501203B (en) | Construction method and application of IL17RB gene humanized non-human animal | |
CN111926038B (en) | Construction method and application of CSF2RB gene humanized modified non-human animal | |
CN112501202B (en) | CXCR4 gene humanized non-human animal and construction method and application thereof | |
CN115010799A (en) | Construction method and application of BCMA gene humanized non-human animal | |
CN114276433A (en) | Non-human animal humanized with CD38 gene and construction method and application thereof | |
CN113831403A (en) | Construction method and application of humanized non-human animal of STING gene | |
CN114990128A (en) | Construction method and application of CD20 gene humanized non-human animal |
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 |