CN114805562A - 抗新型冠状病毒人源化纳米抗体及其应用 - Google Patents
抗新型冠状病毒人源化纳米抗体及其应用 Download PDFInfo
- Publication number
- CN114805562A CN114805562A CN202210493578.XA CN202210493578A CN114805562A CN 114805562 A CN114805562 A CN 114805562A CN 202210493578 A CN202210493578 A CN 202210493578A CN 114805562 A CN114805562 A CN 114805562A
- Authority
- CN
- China
- Prior art keywords
- ser
- val
- gly
- leu
- thr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 60
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 21
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 20
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 20
- 239000013604 expression vector Substances 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 7
- 108010003723 Single-Domain Antibodies Proteins 0.000 claims description 29
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 18
- 108060003951 Immunoglobulin Proteins 0.000 claims description 6
- 102000018358 immunoglobulin Human genes 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 102000007562 Serum Albumin Human genes 0.000 claims description 2
- 108010071390 Serum Albumin Proteins 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 230000003472 neutralizing effect Effects 0.000 abstract description 20
- 108091005634 SARS-CoV-2 receptor-binding domains Proteins 0.000 abstract description 16
- 230000005847 immunogenicity Effects 0.000 abstract description 8
- 101000929928 Homo sapiens Angiotensin-converting enzyme 2 Proteins 0.000 abstract description 6
- 102000048657 human ACE2 Human genes 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 4
- 241000880493 Leptailurus serval Species 0.000 description 43
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 25
- 108010092854 aspartyllysine Proteins 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 23
- BUANFPRKJKJSRR-ACZMJKKPSA-N Ala-Ala-Gln Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CCC(N)=O BUANFPRKJKJSRR-ACZMJKKPSA-N 0.000 description 22
- SYIFFFHSXBNPMC-UWJYBYFXSA-N Ala-Ser-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N SYIFFFHSXBNPMC-UWJYBYFXSA-N 0.000 description 22
- DXQIQUIQYAGRCC-CIUDSAMLSA-N Arg-Asp-Gln Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)CN=C(N)N DXQIQUIQYAGRCC-CIUDSAMLSA-N 0.000 description 22
- DGFGDPVSDQPANQ-XGEHTFHBSA-N Arg-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCN=C(N)N)N)O DGFGDPVSDQPANQ-XGEHTFHBSA-N 0.000 description 22
- JOXIIFVCSATTDH-IHPCNDPISA-N Phe-Asn-Trp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N JOXIIFVCSATTDH-IHPCNDPISA-N 0.000 description 22
- RIAKPZVSNBBNRE-BJDJZHNGSA-N Ser-Ile-Leu Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O RIAKPZVSNBBNRE-BJDJZHNGSA-N 0.000 description 22
- ZZDYJFVIKVSUFA-WLTAIBSBSA-N Tyr-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O ZZDYJFVIKVSUFA-WLTAIBSBSA-N 0.000 description 22
- BVWPHWLFGRCECJ-JSGCOSHPSA-N Val-Gly-Tyr Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N BVWPHWLFGRCECJ-JSGCOSHPSA-N 0.000 description 22
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 22
- BUDNAJYVCUHLSV-ZLUOBGJFSA-N Ala-Asp-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O BUDNAJYVCUHLSV-ZLUOBGJFSA-N 0.000 description 21
- VNFSAYFQLXPHPY-CIQUZCHMSA-N Ala-Thr-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNFSAYFQLXPHPY-CIQUZCHMSA-N 0.000 description 21
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 21
- HTSSXFASOUSJQG-IHPCNDPISA-N Asp-Tyr-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HTSSXFASOUSJQG-IHPCNDPISA-N 0.000 description 21
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 21
- KTSZUNRRYXPZTK-BQBZGAKWSA-N Gly-Gln-Glu Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KTSZUNRRYXPZTK-BQBZGAKWSA-N 0.000 description 21
- HHRODZSXDXMUHS-LURJTMIESA-N Gly-Met-Gly Chemical compound CSCC[C@H](NC(=O)C[NH3+])C(=O)NCC([O-])=O HHRODZSXDXMUHS-LURJTMIESA-N 0.000 description 21
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 21
- AXZGZMGRBDQTEY-SRVKXCTJSA-N Leu-Gln-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O AXZGZMGRBDQTEY-SRVKXCTJSA-N 0.000 description 21
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 21
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 21
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 21
- 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 21
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 21
- LCRSGSIRKLXZMZ-BPNCWPANSA-N Pro-Ala-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LCRSGSIRKLXZMZ-BPNCWPANSA-N 0.000 description 21
- QEDMOZUJTGEIBF-FXQIFTODSA-N Ser-Arg-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O QEDMOZUJTGEIBF-FXQIFTODSA-N 0.000 description 21
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 21
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 21
- ZWSZBWAFDZRBNM-UBHSHLNASA-N Ser-Trp-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O ZWSZBWAFDZRBNM-UBHSHLNASA-N 0.000 description 21
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 21
- SLOYNOMYOAOUCX-BVSLBCMMSA-N Trp-Phe-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SLOYNOMYOAOUCX-BVSLBCMMSA-N 0.000 description 21
- OWFGFHQMSBTKLX-UFYCRDLUSA-N Val-Tyr-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N OWFGFHQMSBTKLX-UFYCRDLUSA-N 0.000 description 21
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 21
- 108010045350 alanyl-tyrosyl-alanine Proteins 0.000 description 21
- 108010008355 arginyl-glutamine Proteins 0.000 description 21
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 21
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 21
- 108010038745 tryptophylglycine Proteins 0.000 description 21
- FQCILXROGNOZON-YUMQZZPRSA-N Gln-Pro-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O FQCILXROGNOZON-YUMQZZPRSA-N 0.000 description 20
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val 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)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 20
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 20
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 19
- VKKYFICVTYKFIO-CIUDSAMLSA-N Arg-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N VKKYFICVTYKFIO-CIUDSAMLSA-N 0.000 description 18
- UGXYFDQFLVCDFC-CIUDSAMLSA-N Asn-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O UGXYFDQFLVCDFC-CIUDSAMLSA-N 0.000 description 18
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 18
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 16
- MMTOHPRBJKEZHT-BWBBJGPYSA-N Thr-Cys-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O MMTOHPRBJKEZHT-BWBBJGPYSA-N 0.000 description 15
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 12
- IESDGNYHXIOKRW-YXMSTPNBSA-N (2s)-2-[[(2s)-1-[(2s)-6-amino-2-[[(2s,3r)-2-amino-3-hydroxybutanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IESDGNYHXIOKRW-YXMSTPNBSA-N 0.000 description 11
- DIBLBAURNYJYBF-XLXZRNDBSA-N (2s)-2-[[(2s)-2-[[2-[[(2s)-6-amino-2-[[(2s)-2-amino-3-methylbutanoyl]amino]hexanoyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@H](NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 DIBLBAURNYJYBF-XLXZRNDBSA-N 0.000 description 11
- SKTGPBFTMNLIHQ-KKUMJFAQSA-N Arg-Glu-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SKTGPBFTMNLIHQ-KKUMJFAQSA-N 0.000 description 11
- HPSVTWMFWCHKFN-GARJFASQSA-N Arg-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O HPSVTWMFWCHKFN-GARJFASQSA-N 0.000 description 11
- SLKLLQWZQHXYSV-CIUDSAMLSA-N Asn-Ala-Lys Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O SLKLLQWZQHXYSV-CIUDSAMLSA-N 0.000 description 11
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 11
- LGCVSPFCFXWUEY-IHPCNDPISA-N Asn-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N LGCVSPFCFXWUEY-IHPCNDPISA-N 0.000 description 11
- PDECQIHABNQRHN-GUBZILKMSA-N Asp-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O PDECQIHABNQRHN-GUBZILKMSA-N 0.000 description 11
- SNDBKTFJWVEVPO-WHFBIAKZSA-N Asp-Gly-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SNDBKTFJWVEVPO-WHFBIAKZSA-N 0.000 description 11
- WSGVTKZFVJSJOG-RCOVLWMOSA-N Asp-Gly-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O WSGVTKZFVJSJOG-RCOVLWMOSA-N 0.000 description 11
- KTTCQQNRRLCIBC-GHCJXIJMSA-N Asp-Ile-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O KTTCQQNRRLCIBC-GHCJXIJMSA-N 0.000 description 11
- AHWRSSLYSGLBGD-CIUDSAMLSA-N Asp-Pro-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AHWRSSLYSGLBGD-CIUDSAMLSA-N 0.000 description 11
- YUELDQUPTAYEGM-XIRDDKMYSA-N Asp-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)O)N YUELDQUPTAYEGM-XIRDDKMYSA-N 0.000 description 11
- ZXCAQANTQWBICD-DCAQKATOSA-N Cys-Lys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N ZXCAQANTQWBICD-DCAQKATOSA-N 0.000 description 11
- CSMHMEATMDCQNY-DZKIICNBSA-N Gln-Val-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CSMHMEATMDCQNY-DZKIICNBSA-N 0.000 description 11
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 11
- GLWXKFRTOHKGIT-ACZMJKKPSA-N Glu-Asn-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O GLWXKFRTOHKGIT-ACZMJKKPSA-N 0.000 description 11
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 11
- BFEZQZKEPRKKHV-SRVKXCTJSA-N Glu-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CCCCN)C(=O)O BFEZQZKEPRKKHV-SRVKXCTJSA-N 0.000 description 11
- ALMBZBOCGSVSAI-ACZMJKKPSA-N Glu-Ser-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ALMBZBOCGSVSAI-ACZMJKKPSA-N 0.000 description 11
- MFYLRRCYBBJYPI-JYJNAYRXSA-N Glu-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O MFYLRRCYBBJYPI-JYJNAYRXSA-N 0.000 description 11
- ZALGPUWUVHOGAE-GVXVVHGQSA-N Glu-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZALGPUWUVHOGAE-GVXVVHGQSA-N 0.000 description 11
- GRIRDMVMJJDZKV-RCOVLWMOSA-N Gly-Asn-Val Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O GRIRDMVMJJDZKV-RCOVLWMOSA-N 0.000 description 11
- PABFFPWEJMEVEC-JGVFFNPUSA-N Gly-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)CN)C(=O)O PABFFPWEJMEVEC-JGVFFNPUSA-N 0.000 description 11
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 11
- WMKXFMUJRCEGRP-SRVKXCTJSA-N His-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N WMKXFMUJRCEGRP-SRVKXCTJSA-N 0.000 description 11
- 108010065920 Insulin Lispro Proteins 0.000 description 11
- PVMPDMIKUVNOBD-CIUDSAMLSA-N Leu-Asp-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O PVMPDMIKUVNOBD-CIUDSAMLSA-N 0.000 description 11
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 11
- AUNMOHYWTAPQLA-XUXIUFHCSA-N Leu-Met-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AUNMOHYWTAPQLA-XUXIUFHCSA-N 0.000 description 11
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 11
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 11
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 11
- YKIRNDPUWONXQN-GUBZILKMSA-N Lys-Asn-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKIRNDPUWONXQN-GUBZILKMSA-N 0.000 description 11
- KWUKZRFFKPLUPE-HJGDQZAQSA-N Lys-Asp-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWUKZRFFKPLUPE-HJGDQZAQSA-N 0.000 description 11
- 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 11
- WQDKIVRHTQYJSN-DCAQKATOSA-N Lys-Ser-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N WQDKIVRHTQYJSN-DCAQKATOSA-N 0.000 description 11
- IEVXCWPVBYCJRZ-IXOXFDKPSA-N Lys-Thr-His Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IEVXCWPVBYCJRZ-IXOXFDKPSA-N 0.000 description 11
- DLCAXBGXGOVUCD-PPCPHDFISA-N Lys-Thr-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DLCAXBGXGOVUCD-PPCPHDFISA-N 0.000 description 11
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 11
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 11
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 11
- MSHZERMPZKCODG-ACRUOGEOSA-N Phe-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MSHZERMPZKCODG-ACRUOGEOSA-N 0.000 description 11
- JDMKQHSHKJHAHR-UHFFFAOYSA-N Phe-Phe-Leu-Tyr Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)CC1=CC=CC=C1 JDMKQHSHKJHAHR-UHFFFAOYSA-N 0.000 description 11
- 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 11
- NXEYSLRNNPWCRN-SRVKXCTJSA-N Pro-Glu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NXEYSLRNNPWCRN-SRVKXCTJSA-N 0.000 description 11
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 11
- KWMUAKQOVYCQJQ-ZPFDUUQYSA-N Pro-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@@H]1CCCN1 KWMUAKQOVYCQJQ-ZPFDUUQYSA-N 0.000 description 11
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 11
- KBUAPZAZPWNYSW-SRVKXCTJSA-N Pro-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KBUAPZAZPWNYSW-SRVKXCTJSA-N 0.000 description 11
- 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 11
- PRKWBYCXBBSLSK-GUBZILKMSA-N Pro-Ser-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O PRKWBYCXBBSLSK-GUBZILKMSA-N 0.000 description 11
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 11
- VGNYHOBZJKWRGI-CIUDSAMLSA-N Ser-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO VGNYHOBZJKWRGI-CIUDSAMLSA-N 0.000 description 11
- MOVJSUIKUNCVMG-ZLUOBGJFSA-N Ser-Cys-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N)O MOVJSUIKUNCVMG-ZLUOBGJFSA-N 0.000 description 11
- HMRAQFJFTOLDKW-GUBZILKMSA-N Ser-His-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O HMRAQFJFTOLDKW-GUBZILKMSA-N 0.000 description 11
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 11
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-N 0.000 description 11
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 11
- PPCZVWHJWJFTFN-ZLUOBGJFSA-N Ser-Ser-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPCZVWHJWJFTFN-ZLUOBGJFSA-N 0.000 description 11
- ASJDFGOPDCVXTG-KATARQTJSA-N Thr-Cys-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O ASJDFGOPDCVXTG-KATARQTJSA-N 0.000 description 11
- KWQBJOUOSNJDRR-XAVMHZPKSA-N Thr-Cys-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N)O KWQBJOUOSNJDRR-XAVMHZPKSA-N 0.000 description 11
- DSLHSTIUAPKERR-XGEHTFHBSA-N Thr-Cys-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O DSLHSTIUAPKERR-XGEHTFHBSA-N 0.000 description 11
- NCXVJIQMWSGRHY-KXNHARMFSA-N Thr-Leu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O NCXVJIQMWSGRHY-KXNHARMFSA-N 0.000 description 11
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 11
- BEZTUFWTPVOROW-KJEVXHAQSA-N Thr-Tyr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O BEZTUFWTPVOROW-KJEVXHAQSA-N 0.000 description 11
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 11
- DQDXHYIEITXNJY-BPUTZDHNSA-N Trp-Gln-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N DQDXHYIEITXNJY-BPUTZDHNSA-N 0.000 description 11
- SCCKSNREWHMKOJ-SRVKXCTJSA-N Tyr-Asn-Ser Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O SCCKSNREWHMKOJ-SRVKXCTJSA-N 0.000 description 11
- RIVVDNTUSRVTQT-IRIUXVKKSA-N Tyr-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O RIVVDNTUSRVTQT-IRIUXVKKSA-N 0.000 description 11
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 11
- RKIGNDAHUOOIMJ-BQFCYCMXSA-N Val-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 RKIGNDAHUOOIMJ-BQFCYCMXSA-N 0.000 description 11
- XTDDIVQWDXMRJL-IHRRRGAJSA-N Val-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N XTDDIVQWDXMRJL-IHRRRGAJSA-N 0.000 description 11
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 11
- HPANGHISDXDUQY-ULQDDVLXSA-N Val-Lys-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HPANGHISDXDUQY-ULQDDVLXSA-N 0.000 description 11
- SBJCTAZFSZXWSR-AVGNSLFASA-N Val-Met-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N SBJCTAZFSZXWSR-AVGNSLFASA-N 0.000 description 11
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 11
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 11
- 108010087924 alanylproline Proteins 0.000 description 11
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 11
- 108010078144 glutaminyl-glycine Proteins 0.000 description 11
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 11
- 108010015792 glycyllysine Proteins 0.000 description 11
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 11
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 11
- 108010031719 prolyl-serine Proteins 0.000 description 11
- 108010070643 prolylglutamic acid Proteins 0.000 description 11
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 11
- 108010052774 valyl-lysyl-glycyl-phenylalanyl-tyrosine Proteins 0.000 description 11
- MKJBPDLENBUHQU-CIUDSAMLSA-N Asn-Ser-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O MKJBPDLENBUHQU-CIUDSAMLSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- BCADFFUQHIMQAA-KKHAAJSZSA-N Asn-Thr-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BCADFFUQHIMQAA-KKHAAJSZSA-N 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- OILNWMNBLIHXQK-ZLUOBGJFSA-N Ala-Cys-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O OILNWMNBLIHXQK-ZLUOBGJFSA-N 0.000 description 6
- 241000282836 Camelus dromedarius Species 0.000 description 6
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 6
- LAGPXKYZCCTSGQ-JYJNAYRXSA-N Leu-Glu-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LAGPXKYZCCTSGQ-JYJNAYRXSA-N 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000006386 neutralization reaction Methods 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 4
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000006167 equilibration buffer Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- LUTDBHBIHHREDC-IHRRRGAJSA-N Lys-Pro-Lys Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O LUTDBHBIHHREDC-IHRRRGAJSA-N 0.000 description 3
- JMNRXRPBHFGXQX-GUBZILKMSA-N Lys-Ser-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JMNRXRPBHFGXQX-GUBZILKMSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 3
- 108010034529 leucyl-lysine Proteins 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- YQPFCZVKMUVZIN-AUTRQRHGSA-N Glu-Val-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQPFCZVKMUVZIN-AUTRQRHGSA-N 0.000 description 2
- 239000012515 MabSelect SuRe Substances 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- HHWQMFIGMMOVFK-WDSKDSINSA-N Gln-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O HHWQMFIGMMOVFK-WDSKDSINSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- JDCQDJVYUXNCGF-SPOWBLRKSA-N Ile-Ser-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N JDCQDJVYUXNCGF-SPOWBLRKSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- SNVIOQXAHVORQM-WDSKDSINSA-N Ser-Gly-Gln Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O SNVIOQXAHVORQM-WDSKDSINSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- JNQZPAWOPBZGIX-RCWTZXSCSA-N Thr-Arg-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)O)CCCN=C(N)N JNQZPAWOPBZGIX-RCWTZXSCSA-N 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- -1 coatings Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明涉及抗体技术领域,具体而言,涉及一种抗新型冠状病毒人源化纳米抗体,该人源化纳米抗体的免疫原性风险低,能有效阻断野生型、Delta变异株和Omicron变异株新型冠状病毒SARS‑COV‑2RBD蛋白与人ACE2受体蛋白的结合,具有显著的新型冠状病毒中和活性;因此,本发明提供的人源化纳米抗体、核酸、表达载体、宿主细胞在制备治疗和/或预防新型冠状病毒的药物中具有很好的应用前景。
Description
技术领域
本发明属于抗体技术领域。更具体地,涉及抗新型冠状病毒人源化纳米抗体及其应用。
背景技术
单克隆抗体在癌症的检测及生物靶向治疗方面成功的应用,引起了肿瘤治疗的变革。然而,传统的单抗分子质量过大(150kD),难穿透组织,造成肿瘤区域的有效浓度较低,治疗效果不充分;另外,传统的抗体具有很高的免疫原性,而改造的抗体很难达到原来的亲和力,限制了其在临床上的广泛应用。
纳米抗体是目前最小的抗体分子,其分子量是普通抗体的1/10。纳米抗体除具备单克隆抗体的抗原反应性外,还拥有一些独特的功能特性,如分子质量小,稳定性强、可溶性好、易表达、免疫原性弱、穿透性强、靶向性强、人源化简单,制备成本低廉等,几乎完美克服了传统抗体开发周期长,稳定性较低,保存条件苛刻等缺陷。
新型冠状病毒(SARS-COV-2)自2019年爆发以来,在全世界各地广泛传播,造成了严重的经济损失、社会负担和其他负面影响;另外,陆续发现了多种类型的新型冠状病毒变异株,变异株是指在原有病毒的基因组基础上发生了某一个基因碱基的突变、或者某些碱基的缺失,发生突变或者碱基缺失会导致病毒的性质发生变化,病毒可能会出现比如传染性、宿主范围、传播力度、毒力、致病力、病情的严重程度、预后以及免疫原性的改变。这样就会导致临床整个发病过程以及流行病学发生变化,甚至对于免疫原性以及免疫的预防也可能会出现影响。
目前已研制出的瑞德西韦等广谱小分子抗病毒药物虽然对新型冠状病毒野生株由一定的疗效,但对新型冠状病毒野生株、Delta变异株和Omicron变异株无疗效和特异性,临床上缺乏新型冠状病毒特效药;因此,亟需开发对新型冠状病毒野生株和突变株均有特异性疗效的药物。
发明内容
本发明的目的是提供抗新型冠状病毒单域抗体,所述单域抗体的氨基酸序列如SEQ ID NO:5~14任一所示。
本发明的另一目的是提供抗新型冠状病毒人源化纳米抗体,所述抗体包含所述单域抗体。该人源化纳米抗体的免疫原性风险低,能有效阻断野生型、Delta变异株和Omicron变异株新型冠状病毒SARS-COV-2 RBD蛋白与人ACE2受体蛋白的结合,具有显著的新型冠状病毒中和活性。
本发明的另一目的是提供一种核酸,所述核酸编码所述单域抗体或所述人源化纳米抗体。
本发明还提供了一种表达载体,所述表达载体携带所述核酸。
本发明还提供了一种宿主细胞,所述宿主细胞携带所述核酸、包含所述表达载体、或其能够表达所述单域抗体、或其能够表达所述人源化纳米抗体。
本发明还提供了一种药物组合物,所述药物组合物含有所述单域抗体、所述人源化纳米抗体、所述核酸、所述表达载体或所述宿主细胞。
本发明还提供了所述单域抗体、所述人源化纳米抗体、所述核酸、所述表达载体、所述宿主细胞在制备治疗和/或预防新型冠状病毒的药物中的应用。
本发明还提供了一种治疗和/或预防新型冠状病毒的方法,其包括向新型冠状病毒感染主体施用有效剂量的所述药物组合物。
附图说明
图1是含有目的基因的质粒图。
图2是人源化纳米抗体对野生株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果图。
图3是人源化纳米抗体对Delta变异株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果图。
图4是人源化纳米抗体对Omicron变异株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
本发明提供了抗新型冠状病毒单域抗体,所述单域抗体的氨基酸序列如SEQ IDNO:5~14任一所示。
在一些实施方式中,所述单域抗体的氨基酸序列如SEQ ID NO:10所示。
本发明提供了抗新型冠状病毒人源化纳米抗体,所述抗体包含所述单域抗体。
本发明中,所述的“纳米抗体”是指缺失轻链的重链抗体(如:来源于骆驼体内),克隆其可变区得到的单域抗体,是最小的功能性抗原结合片段,相对分子质量(Mr)仅约为15000。纳米抗体具有分子质量小、稳定性强、可溶性好、易表达,免疫原性低等特点。
本发明也包括所述的纳米抗体的变体、衍生物和类似物。如本文所用,术语“变体”、“衍生物”和“类似物”是指基本上保持本发明的纳米抗体相同的生物学功能或活性的多肽。本发明的多肽变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的多肽,或(iii)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或多肽原序列,或融合多肽)。根据本文的定义这些变体、衍生物和类似物属于本领域熟练技术人员公知的范围。
此外,在所述的纳米抗体的氨基端或羧基端还可添加其它基本上不影响本发明所述的纳米抗体的活性、表达量和稳定性的氨基酸序列。这些添加的氨基酸序列有利于表达(如信号肽),有利于纯化(如6×His序列),或其它可促进所述的纳米抗体的活性、表达量或稳定性的序列。
在一些实施方式中,所述人源化纳米抗体还包含半衰期延长结构域。
在优选实施方式中,所述半衰期延长结构域选自免疫球蛋白Fc区或血清白蛋白结合结构域。
在优选实施方式中,所述半衰期延长结构域为免疫球蛋白Fc区。
在优选实施方式中,所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO:15所示。
在一些实施方式中,所述人源化纳米抗体的氨基酸序列如SEQ ID NO:16~25任一所示。
在优选实施方式中,所述人源化纳米抗体的氨基酸序列如SEQ ID NO:21所示。
本发明还提供了一种核酸,所述核酸编码所述单域抗体或所述人源化纳米抗体。
核酸通常是RNA或DNA,核酸分子可以是单链或双链的。当将核酸与另一个核酸序列置于功能关系中时,核酸是“有效连接的”。例如,如果启动子或增强子影响编码序列的转录,那么启动子或增强子有效地连接至所述编码序列。当其连入载体时采用DNA核酸。
本发明还提供了一种表达载体,所述表达载体携带所述核酸。
在本发明中,术语“载体”包括质粒、表达载体、克隆载体、病毒载体等。可选用本领域已知的各种载体。比如,选用市售的载体,然后将编码本发明纳米抗体的核苷酸序列可操作地连于表达调控序列,可以形成表达载体。
本发明还提供了一种宿主细胞,所述宿主细胞携带所述核酸、包含所述表达载体、或其能够表达所述单域抗体、或其能够表达所述人源化纳米抗体。
在本发明中,术语“宿主细胞”包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。用于表达纳米抗体的宿主细胞包括大肠杆菌、酵母细胞、昆虫细胞、COS细胞、CHO细胞等。在获得转化的宿主细胞后,可在适合表达本发明纳米抗体的条件下培养该细胞,从而表达出纳米抗体;然后再分离出表达的纳米抗体。
本发明还提供了一种药物组合物,所述药物组合物含有所述单域抗体、所述人源化纳米抗体、所述核酸、所述表达载体或所述宿主细胞。
在一些实施方式中,所述药物组合物还含有药学上可接受的载体。
本发明中,“药学上可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等。具体可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等以及它们的组合物中的任一种或几种。当然,“药学可接受的载体”还可包括微量的辅助物质,例如润湿剂或乳化剂、防腐剂或缓冲剂,用来延长抗体的保存限期或效力。
另外,所述单域抗体、所述人源化纳米抗体、所述核酸、所述表达载体、所述宿主细胞在制备治疗和/或预防新型冠状病毒的药物中的应用,也在本发明的保护范围之内。
本发明还提供了一种治疗和/或预防新型冠状病毒的方法,其包括向新型冠状病毒感染主体施用有效剂量的所述药物组合物。
本发明具有以下有益效果:
本发明提供了一种抗新型冠状病毒人源化纳米抗体,该人源化纳米抗体的免疫原性风险低,能有效阻断野生型、Delta变异株和Omicron变异株新型冠状病毒SARS-COV-2RBD蛋白与人ACE2受体蛋白的结合,具有显著的新型冠状病毒中和活性;因此,本发明提供的人源化纳米抗体、核酸、表达载体、宿主细胞在制备治疗和/或预防新型冠状病毒的药物中具有很好的应用前景。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1抗新型冠状病毒人源化纳米抗体的人源化设计
对抗新型冠状病毒驼源性纳米抗体(见CN202210089563.7,其CDR1、CDR2、CDR3的氨基酸序列依次如SEQ ID NO:1~3所示,VHH氨基酸序列如SEQ ID NO:4所示,VHH编号为RX011)进行人源化设计,通过CDR-grafting方法进行人源化改造;首先使用MOE软件对该驼源性纳米抗体进行同源建模,根据模型结构,结合发明人的经验分析能影响抗原结合区域构象稳定的关键组成氨基酸残基;随后检索人免疫球蛋白数据库,搜索人种系抗体库中与该驼源性纳米抗体同源性高的人IGVH序列作为人源化改造模板,分别将该驼源性纳米抗体与匹配的人IGVH序列进行比对,结合发明人的经验重点分析该驼源性纳米抗体上能影响抗原结合区域构象稳定的关键组成氨基酸残基与人IGVH序列不一致的位点,并在模型中观察是否能进行人源化替换;根据不同的人源化替换程度,共设计10条抗新型冠状病毒人源化纳米抗体的VHH氨基酸序列,VHH编号依次为RX012-RX021,其具体氨基酸序列如表1所示:
SEQ ID NO:1:RCTFNWDG
SEQ ID NO:2:ISWSGQEP
SEQ ID NO:3:AAAQYTGASYSILRDQVGYDY
SEQ ID NO:4:
QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSA
表1
为了方便验证,选择使用huFc恒定区作为表达标签,其具体氨基酸序列如表2所示:
表2
实施例2抗新型冠状病毒人源化纳米抗体的制备
蛋白瞬转表达:
含有目的基因的质粒图如图1所示,将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后,导入到宿主细胞Expi293,质粒在细胞内期间,质粒上的外源基因在细胞内发生转录翻译,从而得到目的蛋白。
Expi293在37℃、8%二氧化碳、130rpm条件培养,并在转染前通过细胞计数,将2E6的细胞接种至1L摇瓶中,培养体系约为300mL。配制转染复合物准备转染:首先将750μg目标质粒加入到含有15mL Opti-MEM试剂的50mL离心管中,轻轻混匀,标记为A管;将1.5mg转染试剂PEI加入到含有15mL Opti-MEM试剂的50mL离心管中,轻轻混匀后,室温孵育5min,标记为B管;将B管PEI稀释液逐滴加入到A管DNA稀释液中,轻轻混匀后,室温孵育15min,孵育结束后,将PEI-目标质粒复合物加入到Expi293细胞,置于37℃摇床中继续培养。直到D7-D10后收样。
复合物样品的纯化:
瞬转细胞表达液经过9000rpm/20min离心,收集上清,再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下,使用AKTA avant 150层析设备,用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX,GE),加载样品至层析柱,使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱,随后使用洗脱缓冲液(20mM NaAc,pH=3.4)洗脱目标蛋白,收集管中预先加入中和缓冲液(1M Tris,pH8.0),中和缓冲液的加入体积根据洗脱样品的预估含量而定,一般加入10%洗脱体积量。
采用常规方法进行抗体的制备,表达上清采用ProA亲和层析纯化。过程如下,使用AKTA avant 150层析设备,用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX,GE),加载样品至层析柱,使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱,随后使用洗脱缓冲液(20mM NaAc,pH=3.4)洗脱目标蛋白,收集管中预先加入中和缓冲液(1M Tris,pH8.0),中和缓冲液的加入体积根据洗脱样品的预估含量而定,一般加入10%洗脱体积量。
样品使用Biotek-Epoch-Take-3进行浓度测定,利用A280方法检测抗体浓度,即以消光系数E.C.=1.37(根据氨基酸序列预测),光程=0.05mm(Take-3板不同孔光程有细微差异,会自动校正),通过设备检测样品吸光值,按照Lambert-Beer law计算待测抗体的浓度。样品浓度过低则需要进行超滤浓缩,使用超滤浓缩管(Ultra-15 CentrifugalFilter Devices,30kD)按照说明书提供的通用操作方法,将样品浓度浓缩至>0.5mg/mL;收集浓缩端样品,0.22um无菌针头滤器除菌过滤(科百特,PES,0.22um,直径13mm)后分装冻存备用。
10条抗新型冠状病毒人源化纳米抗体的VHH氨基酸序列RX012-RX021直接与hIgG1-Fc(SEQ ID NO:15)连接,得到抗新型冠状病毒人源化纳米抗体,其编号依次为R1383-R1392;RX011直接与hIgG1-Fc(SEQ ID NO:15)连接,得到驼源性纳米抗体,编号为R1382。
表3
抗新型冠状病毒人源化纳米抗体表达量和纯度结果如表4所示,结果显示,人源化后抗体的表达量和纯度数据都很理想。
表4
实施例3人源化纳米抗体对野生株新型冠状病毒的中和活性
使用竞争法检测人源化纳米抗体对野生株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性,以实施例2中的R1382作为对照组,具体实验步骤如下:
包被条件:
病毒蛋白:野生株新型冠状病毒SARS-COV-2 RBD蛋白,2ug/mL;
包被液:50mm pH9.51 CB;
包被体积:100ul/孔;
包被温度:2℃-8℃;
包被时间:18小时;
封闭液:含1%BSA +1×PBS;
封闭温度:37℃;
封闭时间:3小时;
加样:加50uL待测抗体(所有抗体起始浓度是5ug/mL,并按照5倍进行系列梯度稀释),孵育30min,使用洗板液(1×PBS)清洗5次后,加入50uL ACE2蛋白,使用洗板液(1×PBS)清洗3次后,显色检测。
人源化纳米抗体对野生株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果如表5和图2所示,结果显示,实施例2制备得到的人源化纳米抗体能有效阻断野生型SARS-COV-2 RBD蛋白与人ACE2受体蛋白的结合,都有新型冠状病毒中和活性;其中,R1383、R1384、R1385、R1386、R1387、R1388显示出显著的野生型新型冠状病毒中和活性。
表5
实施例4人源化纳米抗体对Delta变异株新型冠状病毒的中和活性
使用竞争法检测人源化纳米抗体R1383-R1392对Delta变异株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性,以实施例2中的R1382作为对照组,具体实验步骤同实施例3。
人源化纳米抗体对Delta变异株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果如表6和图3所示,结果显示,实施例2制备得到的人源化纳米抗体能有效阻断Delta变异株新型冠状病毒SARS-COV-2 RBD蛋白与人ACE2受体蛋白的结合,都有新型冠状病毒中和活性;其中,R1385、R1386、R1387、R1388、R1389显示出显著的Delta变异株新型冠状病毒中和活性。
表6
编号 | Delta变异株新型冠状病毒SARS-COV-2 IC50 |
R1382 | 0.01369nM |
R1383 | 0.02232nM |
R1384 | 0.02133nM |
R1385 | 0.01237nM |
R1386 | 0.009197nM |
R1387 | 0.01038nM |
R1388 | 0.007211nM |
R1389 | 0.01255nM |
R1390 | 0.01807nM |
R1391 | 0.0204nM |
R1392 | 0.02351nM |
实施例5人源化纳米抗体对Omicron变异株新型冠状病毒的中和活性
使用竞争法检测人源化纳米抗体R1383-R1392对Omicron变异株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性,以实施例2中的R1382作为对照组,具体实验步骤同实施例3。
人源化纳米抗体对Omicron变异株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果如表7和图4所示,结果显示,实施例2制备得到的人源化纳米抗体能有效阻断Omicron变异株新型冠状病毒SARS-COV-2 RBD蛋白与人ACE2受体蛋白的结合,都有新型冠状病毒中和活性;其中,R1385、R1386、R1387、R1388、R1389显示出显著的Omicron变异株新型冠状病毒中和活性。
表7
编号 | Omicron变异株新型冠状病毒SARS-COV-2 IC50 |
R1382 | 0.17nM |
R1383 | 0.7067nM |
R1384 | 0.8007nM |
R1385 | 0.1458nM |
R1386 | 0.08825nM |
R1387 | 0.1157nM |
R1388 | 0.1174nM |
R1389 | 0.122nM |
R1390 | 0.2232nM |
R1391 | 0.2169nM |
R1392 | 0.2369nM |
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 广东菲鹏制药股份有限公司
<120> 抗新型冠状病毒人源化纳米抗体及其应用
<130> 2022
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> 人工序列
<400> 1
Arg Cys Thr Phe Asn Trp Asp Gly
1 5
<210> 2
<211> 8
<212> PRT
<213> 人工序列
<400> 2
Ile Ser Trp Ser Gly Gln Glu Pro
1 5
<210> 3
<211> 21
<212> PRT
<213> 人工序列
<400> 3
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
1 5 10 15
Val Gly Tyr Asp Tyr
20
<210> 4
<211> 128
<212> PRT
<213> 人工序列
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Pro Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ala
115 120 125
<210> 5
<211> 128
<212> PRT
<213> 人工序列
<400> 5
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 128
<212> PRT
<213> 人工序列
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 7
<211> 128
<212> PRT
<213> 人工序列
<400> 7
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 8
<211> 128
<212> PRT
<213> 人工序列
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 128
<212> PRT
<213> 人工序列
<400> 9
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 128
<212> PRT
<213> 人工序列
<400> 10
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 11
<211> 128
<212> PRT
<213> 人工序列
<400> 11
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 12
<211> 128
<212> PRT
<213> 人工序列
<400> 12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 13
<211> 128
<212> PRT
<213> 人工序列
<400> 13
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Pro Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 128
<212> PRT
<213> 人工序列
<400> 14
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 232
<212> PRT
<213> 人工序列
<400> 15
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 16
<211> 360
<212> PRT
<213> 人工序列
<400> 16
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 17
<211> 360
<212> PRT
<213> 人工序列
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 18
<211> 360
<212> PRT
<213> 人工序列
<400> 18
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 19
<211> 360
<212> PRT
<213> 人工序列
<400> 19
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 20
<211> 360
<212> PRT
<213> 人工序列
<400> 20
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 21
<211> 360
<212> PRT
<213> 人工序列
<400> 21
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 22
<211> 360
<212> PRT
<213> 人工序列
<400> 22
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 23
<211> 360
<212> PRT
<213> 人工序列
<400> 23
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 24
<211> 360
<212> PRT
<213> 人工序列
<400> 24
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Pro Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 25
<211> 360
<212> PRT
<213> 人工序列
<400> 25
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln
100 105 110
Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
130 135 140
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
180 185 190
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
195 200 205
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
210 215 220
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
225 230 235 240
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
260 265 270
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
275 280 285
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
305 310 315 320
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
325 330 335
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
340 345 350
Ser Leu Ser Leu Ser Pro Gly Lys
355 360
Claims (10)
1.抗新型冠状病毒单域抗体,其特征在于,所述单域抗体的氨基酸序列如SEQ ID NO:5~14任一所示。
2.抗新型冠状病毒人源化纳米抗体,其特征在于,所述抗体包含权利要求1所述单域抗体。
3.根据权利要求2所述人源化纳米抗体,其特征在于,所述人源化纳米抗体还包含半衰期延长结构域;
优选地,所述半衰期延长结构域选自免疫球蛋白Fc区或血清白蛋白结合结构域。
4.根据权利要求3所述人源化纳米抗体,其特征在于,所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO:15所示。
5.根据权利要求2~4任一项所述人源化纳米抗体,其特征在于,所述人源化纳米抗体的氨基酸序列如SEQ ID NO:16~25任一所示。
6.一种核酸,其特征在于,所述核酸编码权利要求1所述单域抗体或权利要求2~5任一项所述人源化纳米抗体。
7.一种表达载体,其特征在于,所述表达载体携带权利要求6所述核酸。
8.一种宿主细胞,其特征在于,所述宿主细胞携带权利要求6所述核酸、包含权利要求7所述表达载体、或其能够表达权利要求1所述单域抗体、或其能够表达权利要求2~5任一项所述人源化纳米抗体。
9.一种药物组合物,其特征在于,所述药物组合物含有权利要求1所述单域抗体、权利要求2~5任一项所述人源化纳米抗体、权利要求6所述核酸、权利要求7所述表达载体或权利要求8所述宿主细胞。
10.权利要求1所述单域抗体、权利要求2~5任一项所述人源化纳米抗体、权利要求6所述核酸、权利要求7所述表达载体、权利要求8所述宿主细胞在制备治疗和/或预防新型冠状病毒的药物中的应用。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210493578.XA CN114805562B (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
CN202310018333.6A CN117003859A (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210493578.XA CN114805562B (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310018333.6A Division CN117003859A (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114805562A true CN114805562A (zh) | 2022-07-29 |
CN114805562B CN114805562B (zh) | 2023-01-03 |
Family
ID=82510810
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310018333.6A Pending CN117003859A (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
CN202210493578.XA Active CN114805562B (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310018333.6A Pending CN117003859A (zh) | 2022-05-07 | 2022-05-07 | 抗新型冠状病毒人源化纳米抗体及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN117003859A (zh) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113388029A (zh) * | 2020-03-12 | 2021-09-14 | 中国科学院武汉病毒研究所 | 针对新型冠状病毒的中和性人源单克隆抗体及其应用 |
CN113461812A (zh) * | 2021-07-07 | 2021-10-01 | 北京鹭飞生物科技有限公司 | 人源化新冠病毒中和性抗体nCoV-00D及其应用 |
CN113512113A (zh) * | 2021-08-03 | 2021-10-19 | 浙江大学医学院附属第一医院 | 人源化广谱高中和活性抗新型冠状病毒单克隆抗体及应用 |
CN113563464A (zh) * | 2021-08-01 | 2021-10-29 | 中国疾病预防控制中心性病艾滋病预防控制中心 | 人源化高中和活性抗新型冠状病毒单克隆抗体及应用 |
CN113683687A (zh) * | 2020-05-19 | 2021-11-23 | 益科思特(北京)医药科技发展有限公司 | 抗新型冠状病毒Spike蛋白抗体及其应用 |
CN113881704A (zh) * | 2021-11-17 | 2022-01-04 | 浙江迪福润丝生物科技有限公司 | 一种包含新型冠状病毒双抗原靶标序列组合的重组新城疫病毒载体及相应疫苗株和疫苗 |
CN114426574A (zh) * | 2020-10-29 | 2022-05-03 | 中国科学院武汉病毒研究所 | 新型冠状病毒中和性人源单克隆抗体及其应用 |
-
2022
- 2022-05-07 CN CN202310018333.6A patent/CN117003859A/zh active Pending
- 2022-05-07 CN CN202210493578.XA patent/CN114805562B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113388029A (zh) * | 2020-03-12 | 2021-09-14 | 中国科学院武汉病毒研究所 | 针对新型冠状病毒的中和性人源单克隆抗体及其应用 |
CN113683687A (zh) * | 2020-05-19 | 2021-11-23 | 益科思特(北京)医药科技发展有限公司 | 抗新型冠状病毒Spike蛋白抗体及其应用 |
CN114426574A (zh) * | 2020-10-29 | 2022-05-03 | 中国科学院武汉病毒研究所 | 新型冠状病毒中和性人源单克隆抗体及其应用 |
CN113461812A (zh) * | 2021-07-07 | 2021-10-01 | 北京鹭飞生物科技有限公司 | 人源化新冠病毒中和性抗体nCoV-00D及其应用 |
CN113563464A (zh) * | 2021-08-01 | 2021-10-29 | 中国疾病预防控制中心性病艾滋病预防控制中心 | 人源化高中和活性抗新型冠状病毒单克隆抗体及应用 |
CN113512113A (zh) * | 2021-08-03 | 2021-10-19 | 浙江大学医学院附属第一医院 | 人源化广谱高中和活性抗新型冠状病毒单克隆抗体及应用 |
CN113881704A (zh) * | 2021-11-17 | 2022-01-04 | 浙江迪福润丝生物科技有限公司 | 一种包含新型冠状病毒双抗原靶标序列组合的重组新城疫病毒载体及相应疫苗株和疫苗 |
Also Published As
Publication number | Publication date |
---|---|
CN114805562B (zh) | 2023-01-03 |
CN117003859A (zh) | 2023-11-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2022184027A1 (zh) | 一种新型冠状病毒多价抗原、其制备方法和应用 | |
CN115043935B (zh) | 靶向新冠病毒的纳米抗体及其制备方法和应用 | |
CN113861288B (zh) | 新型冠状病毒SARS-CoV-2广谱中和抗体及其应用 | |
JPH07506486A (ja) | 免疫グロブリンEの高アフィニティレセプタのヒトβサブユニットの分離,特性化および用法 | |
CN113150137A (zh) | 一种ndm-1单克隆抗体的制备方法及其应用 | |
CN113461810B (zh) | 一种抗新型冠状病毒刺突蛋白的全人源单克隆抗体及其应用 | |
RU2478645C2 (ru) | Растворимый мутант рецептора фактора некроза опухоли | |
CN114409768A (zh) | SARS-CoV-2α、β、γ和δ突变株骆驼源高亲和力纳米抗体 | |
CN108179149B (zh) | S100b突变体及其应用 | |
CN114805562B (zh) | 抗新型冠状病毒人源化纳米抗体及其应用 | |
CN115057938B (zh) | 抗新型冠状病毒人源化多价结合蛋白及其应用 | |
US9840539B2 (en) | High affinity digoxigenin binding proteins | |
WO2022143611A1 (zh) | 靶向bcma的单域抗体 | |
CN113912716B (zh) | 针对α-突触核蛋白抗原的抗体及其应用 | |
CN113698487B (zh) | 抗人ace2单克隆抗体及其应用 | |
CN114891075A (zh) | 一种对新冠病毒s蛋白rbmfp结构域具有结合亲和力的多肽及其应用 | |
CN110760517B (zh) | 拮抗pd-1骆驼抗体类似物ap基因及蛋白和应用 | |
CN114539395A (zh) | SARS-CoV-2野生型毒株和α突变株骆驼源高亲和力纳米抗体 | |
CN108864258A (zh) | 具有抑制肿瘤功能的peg化多肽及其制备方法与应用 | |
CN115160434B (zh) | 人源化单域抗体及其应用和药物 | |
CN110894236A (zh) | 一种抗曲霉菌半乳甘露聚糖的单克隆抗体及其应用 | |
CN110759975B (zh) | 一种多肽以及具有强adcc效应的抗体和应用 | |
Sarratea et al. | Zika virus NS4B protein targets TANK-binding kinase 1 and inhibits type I interferon production | |
CN109897106A (zh) | 纳米抗体及其制备方法和应用 | |
CN114716541B (zh) | 抗冠状病毒的全人广谱中和抗体76e1及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40074953 Country of ref document: HK |