CN114671947B - 乙肝病毒不同亚型表面s蛋白高亲和力纳米抗体及其应用 - Google Patents
乙肝病毒不同亚型表面s蛋白高亲和力纳米抗体及其应用 Download PDFInfo
- Publication number
- CN114671947B CN114671947B CN202210206182.2A CN202210206182A CN114671947B CN 114671947 B CN114671947 B CN 114671947B CN 202210206182 A CN202210206182 A CN 202210206182A CN 114671947 B CN114671947 B CN 114671947B
- Authority
- CN
- China
- Prior art keywords
- seq
- antibody
- ser
- protein
- hbv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102100031673 Corneodesmosin Human genes 0.000 title claims abstract description 17
- 101710139375 Corneodesmosin Proteins 0.000 title claims abstract description 17
- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 14
- 239000000427 antigen Substances 0.000 claims abstract description 52
- 108091007433 antigens Proteins 0.000 claims abstract description 52
- 102000036639 antigens Human genes 0.000 claims abstract description 52
- 239000012634 fragment Substances 0.000 claims abstract description 43
- 230000027455 binding Effects 0.000 claims abstract description 42
- 208000015181 infectious disease Diseases 0.000 claims abstract description 19
- 108020001507 fusion proteins Proteins 0.000 claims description 22
- 102000037865 fusion proteins Human genes 0.000 claims description 22
- 238000001514 detection method Methods 0.000 claims description 21
- 239000012620 biological material Substances 0.000 claims description 15
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 150000007523 nucleic acids Chemical class 0.000 claims description 13
- 239000003814 drug Substances 0.000 claims description 11
- 230000014509 gene expression Effects 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 8
- 238000003745 diagnosis Methods 0.000 claims description 7
- 229920001184 polypeptide Polymers 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- 239000013603 viral vector Substances 0.000 claims description 5
- 108020004414 DNA Proteins 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 208000002672 hepatitis B Diseases 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 108020004511 Recombinant DNA Proteins 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 230000009261 transgenic effect Effects 0.000 claims description 3
- 230000002792 vascular Effects 0.000 claims description 3
- 238000001802 infusion Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 238000010254 subcutaneous injection Methods 0.000 claims 1
- 239000007929 subcutaneous injection Substances 0.000 claims 1
- 101000872838 Hepatitis B virus genotype C subtype adr (isolate China/NC-1/1988) Small envelope protein Proteins 0.000 abstract description 22
- 101001006139 Podospora anserina Heterokaryon incompatibility protein s Proteins 0.000 abstract description 22
- 241000282836 Camelus dromedarius Species 0.000 abstract description 7
- 201000007270 liver cancer Diseases 0.000 abstract description 3
- 208000014018 liver neoplasm Diseases 0.000 abstract description 3
- 208000006454 hepatitis Diseases 0.000 abstract description 2
- 231100000283 hepatitis Toxicity 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 23
- 102000004169 proteins and genes Human genes 0.000 description 18
- 238000002965 ELISA Methods 0.000 description 16
- 150000001413 amino acids Chemical group 0.000 description 15
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 14
- 239000007788 liquid Substances 0.000 description 14
- PIUPHASDUFSHTF-CIUDSAMLSA-N Gln-Pro-Asn Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O PIUPHASDUFSHTF-CIUDSAMLSA-N 0.000 description 13
- VFDRDMOMHBJGKD-UFYCRDLUSA-N Phe-Tyr-Arg Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N VFDRDMOMHBJGKD-UFYCRDLUSA-N 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 13
- 239000000243 solution Substances 0.000 description 11
- YCYXHLZRUSJITQ-SRVKXCTJSA-N Arg-Pro-Pro Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 YCYXHLZRUSJITQ-SRVKXCTJSA-N 0.000 description 10
- 108010029539 arginyl-prolyl-proline Proteins 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- PBSOQGZLPFVXPU-YUMQZZPRSA-N Arg-Glu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PBSOQGZLPFVXPU-YUMQZZPRSA-N 0.000 description 8
- 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 8
- HJGUQJJJXQGXGJ-FXQIFTODSA-N Cys-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CS)N HJGUQJJJXQGXGJ-FXQIFTODSA-N 0.000 description 8
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 8
- 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 8
- 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 8
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 8
- 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 8
- CNGOEHJCLVCJHN-SRVKXCTJSA-N Lys-Pro-Glu Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O CNGOEHJCLVCJHN-SRVKXCTJSA-N 0.000 description 8
- 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 8
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 8
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 8
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 8
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 8
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 7
- 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 7
- DTPOVRRYXPJJAZ-FJXKBIBVSA-N Gly-Arg-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N DTPOVRRYXPJJAZ-FJXKBIBVSA-N 0.000 description 7
- CQMFNTVQVLQRLT-JHEQGTHGSA-N Gly-Thr-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O CQMFNTVQVLQRLT-JHEQGTHGSA-N 0.000 description 7
- 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 7
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 7
- 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 7
- 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 7
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 7
- NKUGCYDFQKFVOJ-JYJNAYRXSA-N Tyr-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NKUGCYDFQKFVOJ-JYJNAYRXSA-N 0.000 description 7
- 108010008355 arginyl-glutamine Proteins 0.000 description 7
- 108010037850 glycylvaline Proteins 0.000 description 7
- 108010012058 leucyltyrosine Proteins 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- ADPACBMPYWJJCE-FXQIFTODSA-N Arg-Ser-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O ADPACBMPYWJJCE-FXQIFTODSA-N 0.000 description 6
- MRQQMVZUHXUPEV-IHRRRGAJSA-N Asp-Arg-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MRQQMVZUHXUPEV-IHRRRGAJSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 6
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 108010073969 valyllysine Proteins 0.000 description 6
- JSHWXQIZOCVWIA-ZKWXMUAHSA-N Asp-Ser-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JSHWXQIZOCVWIA-ZKWXMUAHSA-N 0.000 description 5
- BPAUXFVCSYQDQX-JRQIVUDYSA-N Asp-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)O)N)O BPAUXFVCSYQDQX-JRQIVUDYSA-N 0.000 description 5
- TVYMKYUSZSVOAG-ZLUOBGJFSA-N Cys-Ala-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O TVYMKYUSZSVOAG-ZLUOBGJFSA-N 0.000 description 5
- 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 5
- NHJKZMDIMMTVCK-QXEWZRGKSA-N Ile-Gly-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N NHJKZMDIMMTVCK-QXEWZRGKSA-N 0.000 description 5
- XDARBNMYXKUFOJ-GSSVUCPTSA-N Thr-Asp-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XDARBNMYXKUFOJ-GSSVUCPTSA-N 0.000 description 5
- NWECYMJLJGCBOD-UNQGMJICSA-N Thr-Phe-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O NWECYMJLJGCBOD-UNQGMJICSA-N 0.000 description 5
- SVGAWGVHFIYAEE-JSGCOSHPSA-N Trp-Gly-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 SVGAWGVHFIYAEE-JSGCOSHPSA-N 0.000 description 5
- 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 5
- LTSIAOZUVISRAQ-QWRGUYRKSA-N Tyr-Gly-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)O LTSIAOZUVISRAQ-QWRGUYRKSA-N 0.000 description 5
- GAKBTSMAPGLQFA-JNPHEJMOSA-N Tyr-Thr-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 GAKBTSMAPGLQFA-JNPHEJMOSA-N 0.000 description 5
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 5
- 108010047495 alanylglycine Proteins 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 108010078144 glutaminyl-glycine Proteins 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 4
- XYTNPQNAZREREP-XQXXSGGOSA-N Ala-Glu-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XYTNPQNAZREREP-XQXXSGGOSA-N 0.000 description 4
- 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 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- CCUSLCQWVMWTIS-IXOXFDKPSA-N His-Thr-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O CCUSLCQWVMWTIS-IXOXFDKPSA-N 0.000 description 4
- STAVRDQLZOTNKJ-RHYQMDGZSA-N Leu-Arg-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STAVRDQLZOTNKJ-RHYQMDGZSA-N 0.000 description 4
- CUXRXAIAVYLVFD-ULQDDVLXSA-N Leu-Arg-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CUXRXAIAVYLVFD-ULQDDVLXSA-N 0.000 description 4
- FPQMQEOVSKMVMA-ACRUOGEOSA-N Lys-Tyr-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CCCCN)N)O FPQMQEOVSKMVMA-ACRUOGEOSA-N 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 239000002250 absorbent Substances 0.000 description 4
- 230000002745 absorbent Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 108010003137 tyrosyltyrosine Proteins 0.000 description 4
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 3
- NHSDEZURHWEZPN-SXTJYALSSA-N Asp-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CC(=O)O)N NHSDEZURHWEZPN-SXTJYALSSA-N 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- FFYYUUWROYYKFY-IHRRRGAJSA-N His-Val-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O FFYYUUWROYYKFY-IHRRRGAJSA-N 0.000 description 3
- GRVMHFCZUIYNKQ-UFYCRDLUSA-N Phe-Phe-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O GRVMHFCZUIYNKQ-UFYCRDLUSA-N 0.000 description 3
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 description 3
- PNHABSVRPFBUJY-UMPQAUOISA-N Trp-Arg-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O PNHABSVRPFBUJY-UMPQAUOISA-N 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 108010059459 arginyl-threonyl-phenylalanine Proteins 0.000 description 3
- 239000002872 contrast media Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000007857 nested PCR Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- LJFNNUBZSZCZFN-WHFBIAKZSA-N Ala-Gly-Cys Chemical compound N[C@@H](C)C(=O)NCC(=O)N[C@@H](CS)C(=O)O LJFNNUBZSZCZFN-WHFBIAKZSA-N 0.000 description 2
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 2
- AQPVUEJJARLJHB-BQBZGAKWSA-N Arg-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N AQPVUEJJARLJHB-BQBZGAKWSA-N 0.000 description 2
- HMQDRBKQMLRCCG-GMOBBJLQSA-N Asp-Arg-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HMQDRBKQMLRCCG-GMOBBJLQSA-N 0.000 description 2
- UZNSWMFLKVKJLI-VHWLVUOQSA-N Asp-Ile-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O UZNSWMFLKVKJLI-VHWLVUOQSA-N 0.000 description 2
- OYSYWMMZGJSQRB-AVGNSLFASA-N Asp-Tyr-Gln 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](CCC(N)=O)C(O)=O OYSYWMMZGJSQRB-AVGNSLFASA-N 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- FCXJJTRGVAZDER-FXQIFTODSA-N Cys-Val-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O FCXJJTRGVAZDER-FXQIFTODSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 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 2
- DOQUICBEISTQHE-CIUDSAMLSA-N Gln-Pro-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O DOQUICBEISTQHE-CIUDSAMLSA-N 0.000 description 2
- SBCYJMOOHUDWDA-NUMRIWBASA-N Glu-Asp-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SBCYJMOOHUDWDA-NUMRIWBASA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- BGZIJZJBXRVBGJ-SXTJYALSSA-N Ile-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N BGZIJZJBXRVBGJ-SXTJYALSSA-N 0.000 description 2
- SQRLLZAQNOQCEG-KKUMJFAQSA-N Lys-Tyr-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 SQRLLZAQNOQCEG-KKUMJFAQSA-N 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 208000037581 Persistent Infection Diseases 0.000 description 2
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 2
- CVAUVSOFHJKCHN-BZSNNMDCSA-N Phe-Tyr-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=CC=C1 CVAUVSOFHJKCHN-BZSNNMDCSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- SVWQEIRZHHNBIO-WHFBIAKZSA-N Ser-Gly-Cys Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CS)C(O)=O SVWQEIRZHHNBIO-WHFBIAKZSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- GKMYGVQDGVYCPC-IUKAMOBKSA-N Thr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)N GKMYGVQDGVYCPC-IUKAMOBKSA-N 0.000 description 2
- WYKJENSCCRJLRC-ZDLURKLDSA-N Thr-Gly-Cys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)O WYKJENSCCRJLRC-ZDLURKLDSA-N 0.000 description 2
- WKQNLTQSCYXKQK-VFAJRCTISA-N Trp-Lys-Thr Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WKQNLTQSCYXKQK-VFAJRCTISA-N 0.000 description 2
- CGDZGRLRXPNCOC-SRVKXCTJSA-N Tyr-Cys-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CGDZGRLRXPNCOC-SRVKXCTJSA-N 0.000 description 2
- FJKXUIJOMUWCDD-FHWLQOOXSA-N Tyr-Gln-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N)O FJKXUIJOMUWCDD-FHWLQOOXSA-N 0.000 description 2
- HIINQLBHPIQYHN-JTQLQIEISA-N Tyr-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HIINQLBHPIQYHN-JTQLQIEISA-N 0.000 description 2
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 2
- DOFAQXCYFQKSHT-SRVKXCTJSA-N Val-Pro-Pro Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DOFAQXCYFQKSHT-SRVKXCTJSA-N 0.000 description 2
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-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](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000002073 nanorod Substances 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 2
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- NCQMBSJGJMYKCK-ZLUOBGJFSA-N Ala-Ser-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O NCQMBSJGJMYKCK-ZLUOBGJFSA-N 0.000 description 1
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- YNSGXDWWPCGGQS-YUMQZZPRSA-N Arg-Gly-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O YNSGXDWWPCGGQS-YUMQZZPRSA-N 0.000 description 1
- HNJNAMGZQZPSRE-GUBZILKMSA-N Arg-Pro-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O HNJNAMGZQZPSRE-GUBZILKMSA-N 0.000 description 1
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 1
- KVMPVNGOKHTUHZ-GCJQMDKQSA-N Asp-Ala-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KVMPVNGOKHTUHZ-GCJQMDKQSA-N 0.000 description 1
- YWLDTBBUHZJQHW-KKUMJFAQSA-N Asp-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)O)N YWLDTBBUHZJQHW-KKUMJFAQSA-N 0.000 description 1
- OZBXOELNJBSJOA-UBHSHLNASA-N Asp-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N OZBXOELNJBSJOA-UBHSHLNASA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- ZOLXQKZHYOHHMD-DLOVCJGASA-N Cys-Ala-Phe Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CS)N ZOLXQKZHYOHHMD-DLOVCJGASA-N 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- MLZRSFQRBDNJON-GUBZILKMSA-N Gln-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MLZRSFQRBDNJON-GUBZILKMSA-N 0.000 description 1
- ZCOJVESMNGBGLF-GRLWGSQLSA-N Glu-Ile-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZCOJVESMNGBGLF-GRLWGSQLSA-N 0.000 description 1
- MQVNVZUEPUIAFA-WDSKDSINSA-N Gly-Cys-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)CN MQVNVZUEPUIAFA-WDSKDSINSA-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
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- UMYZBHKAVTXWIW-GMOBBJLQSA-N Ile-Asp-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UMYZBHKAVTXWIW-GMOBBJLQSA-N 0.000 description 1
- JHNJNTMTZHEDLJ-NAKRPEOUSA-N Ile-Ser-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O JHNJNTMTZHEDLJ-NAKRPEOUSA-N 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- LOLUPZNNADDTAA-AVGNSLFASA-N Leu-Gln-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LOLUPZNNADDTAA-AVGNSLFASA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- PNHRPOWKRRJATF-IHRRRGAJSA-N Met-Tyr-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 PNHRPOWKRRJATF-IHRRRGAJSA-N 0.000 description 1
- GHQFLTYXGUETFD-UFYCRDLUSA-N Met-Tyr-Tyr Chemical compound CSCC[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 GHQFLTYXGUETFD-UFYCRDLUSA-N 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- SMCHPSMKAFIERP-FXQIFTODSA-N Pro-Asn-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@@H]1CCCN1 SMCHPSMKAFIERP-FXQIFTODSA-N 0.000 description 1
- VOHFZDSRPZLXLH-IHRRRGAJSA-N Pro-Asn-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VOHFZDSRPZLXLH-IHRRRGAJSA-N 0.000 description 1
- 102000051619 SUMO-1 Human genes 0.000 description 1
- 108700038981 SUMO-1 Proteins 0.000 description 1
- BRKHVZNDAOMAHX-BIIVOSGPSA-N Ser-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N BRKHVZNDAOMAHX-BIIVOSGPSA-N 0.000 description 1
- SFZKGGOGCNQPJY-CIUDSAMLSA-N Ser-Asp-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N SFZKGGOGCNQPJY-CIUDSAMLSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- VXMHQKHDKCATDV-VEVYYDQMSA-N Thr-Asp-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VXMHQKHDKCATDV-VEVYYDQMSA-N 0.000 description 1
- JMGJDTNUMAZNLX-RWRJDSDZSA-N Thr-Glu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JMGJDTNUMAZNLX-RWRJDSDZSA-N 0.000 description 1
- IJVNLNRVDUTWDD-MEYUZBJRSA-N Thr-Leu-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IJVNLNRVDUTWDD-MEYUZBJRSA-N 0.000 description 1
- GQPQJNMVELPZNQ-GBALPHGKSA-N Thr-Ser-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O GQPQJNMVELPZNQ-GBALPHGKSA-N 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- QNMIVTOQXUSGLN-SZMVWBNQSA-N Trp-Arg-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 QNMIVTOQXUSGLN-SZMVWBNQSA-N 0.000 description 1
- HQJOVVWAPQPYDS-ZFWWWQNUSA-N Trp-Gly-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQJOVVWAPQPYDS-ZFWWWQNUSA-N 0.000 description 1
- DYEGCOJHFNJBKB-UFYCRDLUSA-N Tyr-Arg-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 DYEGCOJHFNJBKB-UFYCRDLUSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 229940078916 carbamide peroxide Drugs 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 101150073438 nusA gene Proteins 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- A61P31/20—Antivirals for DNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- 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
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Urology & Nephrology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体及其应用。本发明涉及与乙肝病毒表面小S蛋白特异性结合的骆驼源纳米抗体及其抗原结合片段,具体涉及能以高亲和力结合HBV ADR亚型和ADW亚型表面小S蛋白的骆驼源纳米抗体或其抗原结合片段,其能够用于检测、诊断由HBV引起的感染,对治疗由HBV感染引起的肝炎肝癌及其并发症具有重要意义。
Description
技术领域
本发明属于生物技术、免疫检测和生物医药领域,具体地说,涉及乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体及其应用。
背景技术
乙型肝炎病毒(Hepatitis B Virus,HBV)是一种嗜肝病毒属DNA病毒。具有强烈的嗜肝性、高变异性、致癌和难以清除等特点。HBV主要通过血液、体液和母婴途径传播,潜伏期一般为30~180天。急性感染症状包括呕吐、黄疸病、疲劳、小便黄赤和急性腹痛等,此外,机体免疫反应低下或病毒变异导致的免疫逃逸则会使其转为慢性感染,逐步发展为肝硬化和肝癌等并发症。
HBV的表现型是其血清亚型(Subtype),由外膜主蛋白上的一些残基决定,包含众多基因型(HBV-A、HBV-C和HBV-D),包括ADW、ADY、AYW、ADR亚型。ADR亚型主要分布在东亚地区,与这一地区流行的C基因型一致。而ADW亚型在我国较为少见,但在世界范围内广泛分布,与基因型D型一致。
纳米抗体(Nanobody)是只含有重链抗体抗原结合域VHH的单域抗体,与传统多克隆抗体、单克隆抗体和单链抗体相比具有诸多明显优势,比如体积小,可以穿过常规抗体无法进入的组织和器官(如鞘膜、脊髓、大脑等)中;稳定性强,无需冷链运输和冷藏保存;免疫原性低,易于进行人源化改造。
发明内容
本发明的目的是提供乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体及其应用。
本发明构思如下:HBV外膜上的可识别抗原为S蛋白,一般分为大、中、小三种表面S蛋白。本发明将HBV表面小S蛋白作为靶标,通过构建噬菌体展示纳米抗体免疫文库、生物淘选研制出可识别HBV的骆驼源高亲和力纳米抗体,为乙型肝炎机理的进一步研究奠定基础,也为乙肝临床检测提供潜在的新选择。
为了实现本发明目的,第一方面,本发明提供一种乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体或其抗原结合片段,其高变区CDR1、CDR2和CDR3分别选自SEQ ID NO:9-15、SEQ ID NO:16-22和SEQ ID NO:23-30任一所示的氨基酸序列。
所述抗原结合片段为Fv、Fab、Fab′、scFv、F(ab′)2、多价化或多特异片段。
进一步地,本发明的乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体或其抗原结合片段,其氨基酸序列如SEQ ID NO:1-8任一所示;或者
SEQ ID NO:1-8任一所示序列经截短得到的氨基酸序列;或者,
将SEQ ID NO:1-8任一所示序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的抗体或抗原结合片段。
本发明的纳米抗体与HBV具有纳摩尔级别的亲和力。
第二方面,本发明提供一种基因工程抗体,其包含所述的抗体或其抗原结合片段。
其中,所述基因工程抗体包括但不限于人源化抗体、嵌合抗体、多价化或多特异性抗体。
第三方面,本发明提供一种融合蛋白,其包含所述的抗体或其抗原结合片段或所述的基因工程抗体。
进一步地,所述融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。
第四方面,本发明提供一种偶联物,其包含所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白。
进一步地,所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP,或其组合。
第五方面,本发明提供一种核酸分子,其编码所述的抗体或其抗原结合片段,或编码所述的基因工程抗体,或编码所述的融合蛋白,或编码所述的偶联物。
其中,所述核酸分子可以是RNA、DNA或cDNA。
第六方面,本发明提供含有所述核酸分子的生物材料,所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第七方面,本发明提供一种药物组合物,其活性成分为所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物。
所述药物组合物可以是外用型、皮下注射型、血管输入型,或其组合。
优选地,所述药物组合物还包括药用赋形剂或载体。
第八方面,本发明提供所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物在制备用于预防、治疗和/或诊断HBV感染、相关科学研究的生物制品中的应用。
第九方面,本发明提供所述的抗体或其抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物的以下任一应用:
1)用于制备预防或治疗由HBV感染以及由其感染所致相关疾病的药物;
2)用于制备HBV检测试剂或试剂盒;
3)检测HBV抗原;
4)阻断HBV感染;
5)消杀HBV颗粒;
6)诊断HBV引起的相关疾病;
7)治疗HBV引起的相关疾病;
8)进行HBV相关的基础科学研究。
借由上述技术方案,本发明至少具有下列优点及有益效果:
(一)本发明提供的HBV纳米抗体,有效克服目前HBV抗体种类少、稳定较差、生产成本高、临床效果不理想等缺点,具有体积小、亲和力高、稳定性强等优点,并且可以低成本、大批量快速生产。
(二)本发明提供的纳米抗体不仅可以作为初期感染阻断、早期感染诊断、中晚期感染治疗的潜在药物,还可以用于体内成像和体外快速检测,例如生产生物活体成像造影剂、ELISA检测/诊断试剂盒、胶体金检测/诊断试剂盒。
(三)基于本发明抗体建立的ELISA检测方法能准确灵敏地检测样品中是否含有HBV。样品的前处理过程简单,耗时少,能同时检测大量的样品,样品检测成本远低于传统的核酸检测方法。
(四)将本发明抗体应用于胶体金检测/诊断试剂盒可以迅速且准确检测样品中是否含有HBV,为解决HBV感染的快速现场诊断提供有力工具。
(五)将本发明抗体应用于乙肝相关基础科学研究可以大幅提高实验灵敏度和精确性,有望解决常规抗体自身无法克服的缺陷如体积大、稳定性差等导致的技术难题。
附图说明
图1为本发明较佳实施例中纳米抗体与HBV ADR和ADW亚型小S蛋白的结合曲线。
图2为本发明较佳实施例中纳米抗体与HBV ADR和ADW亚型小S蛋白的亲和力曲线(以抗体A1为例)。
图3为本发明较佳实施例中所述抗体的序列及其CDR区。
图4为本发明中所使用的pComb3Xss的质粒图谱。
具体实施方式
本发明旨在提供一种针对HBV的高亲和力抗体,该抗体可有效检测HBV,并对其具有潜在的阻断和治疗作用。
具体地,本发明涉及与乙肝病毒表面小S蛋白特异性结合的骆驼源纳米抗体及其抗原结合片段,具体涉及能以高亲和力结合HBV ADR亚型和ADW亚型表面小S蛋白的骆驼源纳米抗体或其抗原结合片段,其能够用于检测、诊断由HBV引起的感染,并且为预防和治疗由HBV感染引起的肝炎、肝癌及其并发症打下坚实基础。
在本发明的具体实施方案中,提供了抗HBV纳米抗体,其可以与HBV的包膜S蛋白结合,亲和力达到了纳摩尔级别。
在本发明的具体实施方案中,可以进行多种基于抗原/抗体反应的酶联免疫分析检测方法的建立和检测产品的开发。
在本发明的具体实施方案中,可以进行同种或多种基于纳米抗体的多价化等基因工程改造。
在本发明的具体实施方案中,提供了针对HBV的纳米抗体,所述纳米抗体包含如下的氨基酸序列和功能特性:
i)SEQ ID NO:1-8所示的氨基酸序列;或者所述抗体可具有SEQ ID NO:9-15任一所示的高度可变区CDR1氨基酸序列;SEQ ID NO:16-22任一所示的高度可变区CDR2氨基酸序列;和SEQ ID NO:23-30任一所示的高度可变区CDR3氨基酸序列;
ii)所述纳米抗体与HBV具有纳摩尔级别的亲和力。
本发明还提供一种含有所述编码所述抗体的核酸分子的生物材料,所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程菌。
本发明还提供所述抗体的以下任一应用:
1)用于HBV相关的科学研究;
2)用于HBV包膜S蛋白的检测;
3)用于研制HBV检测试剂或ELISA检测试剂。
本发明中,在进行分析检测时,向包被有HBV表面抗原的酶标板的各孔中加入不同浓度所述纳米抗体,由于每个孔中的固相抗原含量均一致,因此当结合在固相抗原上的抗体少,加入的酶标二抗与被结合的纳米抗体结合量少,最后加入底物液和显色液,显色反应浅,用酶标仪检测的OD值低;反之,当所述纳米抗体和固相抗原结合多时,则所测的OD值高,根据加入的纳米抗体量和对应孔的OD值绘制纳米抗体和HBV的结合曲线。
具体地,本发明采用如下技术方案:
本发明提供一种抗体或其抗原结合片段,其氨基酸序列包含由SEQ ID NO:9-15任一所示的CDR1、由SEQ ID NO:16-22任一所示的CDR2、由SEQ ID NO:23-30任一所示的CDR3。
优选地,所述抗原结合片段例如为Fv、Fab、Fab′、scFv、F(ab′)2、多价化或多特异片段。
进一步地,其氨基酸序列如SEQ ID NO:1-8任一所示;
或者所述抗体或抗原结合片段是包含将SEQ ID NO:1-8任一所示序列自N末端起第1~123位氨基酸进行截短所获得的序列的抗体,或者将SEQ ID NO:1-8任一所示序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的抗体或抗原结合片段。
本发明提供一种基因工程抗体,其包含所述的抗体或抗原结合片段;优选地,所述基因工程抗体为人源化抗体、嵌合抗体、多价化或多特异性抗体。
本发明提供一种融合蛋白,其包含所述的抗体或抗原结合片段或所述的基因工程抗体;优选地,融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。
本发明提供一种偶联物,其包含所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白;优选地,所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP,或其组合。
本发明提供一种核酸分子,其编码所述的抗体或抗原结合片段、如所述的基因工程抗体、如所述的融合蛋白或所述的偶联物,其中所述核酸分子为RNA、DNA或cDNA。
本发明提供一种表达载体,其包含所述的核酸分子;
任选地,所述表达载体可以是DNA、RNA、病毒载体、质粒、表达盒、转座子、其他基因转移系统、或其组合;
优选地,所述表达载体包括病毒载体,如噬菌体载体、慢病毒、腺病毒、AAV病毒、逆转录病毒、其他蛋白表达系统、或其组合。
本发明提供一种宿主细胞,其包含所述的表达载体;其中,所述宿主细胞是用于表达外源蛋白的宿主细胞,例如原核表达细胞、真核表达细胞、转基因细胞系;优选地,所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞、植物细胞、动物细胞。
本发明提供一种组织样本或培养物,其通过培养所述的宿主细胞获得。
本发明提供一种蛋白或抗原结合片段,其从所述的组织样本或培养物中分离获得。
本发明提供一种制备所述抗体或抗原结合片段、所述基因工程抗体、所述融合蛋白或所述偶联物或从所述生物材料中获得的培养物的方法,包括从所述的组织样本或培养物中分离/回收目的蛋白或多肽。
本发明提供一种药物组合物,其包含所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物作为活性成分;例如,所述药物组合物为粘膜或表皮外用型药物、皮下注射型药物、血管输入型药物、或其组合;优选地,所述药物还包括药用赋形剂或载体。
本发明提供所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物在制备或从所述生物材料中获得的培养物用于预防、治疗和/或诊断HBV感染的产品或药物中的用途。
本发明提供所述的抗体或抗原结合片段或所述的基因工程抗体或所述的融合蛋白或所述的偶联物或从所述生物材料中获得的培养物在制备用于以下功能的产品中的应用:
1)检测HBV抗原;
2)阻断HBV感染;
3)消杀HBV颗粒;
4)诊断HBV引起的相关疾病;
5)治疗HBV引起的相关疾病;
6)进行HBV相关的基础科学研究。
在本发明的具体实施方案中,所述HBV包括ADR和ADW血清亚型。
在本发明的具体实施方案中,所述标签多肽中所包含纯化标签、检测标签、鉴定标签、偶联标签、功能验证标签等功能多肽,例如His标签、HA标签、Flag标签、c-Myc标签、Avi标签等。
在本发明的具体实施方案中,所述融合蛋白中所包含的检测蛋白包括荧光蛋白、荧光素标记蛋白、过氧化物酶等功能蛋白,例如FPs蛋白、HRP蛋白、Alexa Fluor标记蛋白、FITC标记蛋白等。
在本发明的具体实施方案中,所述融合蛋白中所包含的辅助蛋白为用于辅助折叠、辅助表达、辅助溶解、屏蔽毒性蛋白等功能的蛋白,例如GST蛋白、MBP蛋白、SUMO蛋白、NusA蛋白。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
根据本发明的一些优选实施例,所述纳米抗体可以按如下方法进行制备:将HBV表面小S蛋白作为免疫原免疫实验动物骆驼,提取外周血淋巴细胞的总RNA,经反转录及巢式PCR,克隆出纳米抗体重链(VHH)基因片段,通过酶切连接,将基因片段克隆至噬菌粒载体,高效电转化至大肠杆菌,经辅助噬菌体拯救,构建得到噬菌体纳米抗体库,筛选出HBV纳米抗体,将其进行表达纯化,得到灵敏度高的HBV纳米抗体,并且与流行的突变毒株有较高的交叉反应。制备的纳米抗体分子小,可溶性强,耐高温,易纯化,易表达。
根据本发明的一些优选实施例,所述HBV ADR亚型和ADW亚型表面小S蛋白作为免疫原和包被抗原HBV均购自上海普欣生物技术有限公司。
所述酶标板为96孔酶标板,包被抗原的包被浓度为1ug/mL。
所述酶标记的二抗为辣根过氧化物酶标记的抗HA标签抗体,浓度为0.1μg/mL。购自Abcam公司,商品编号:ab1265。
所述显色液A液由过氧化脲1g、柠檬酸10.3g、Na2HPO4·12H2O 35.8g、吐温-20 100μL和蒸馏水1000mL配制而成,pH5。
所述显色液B液由四甲基联苯胺700mg、DMSO 40mL、柠檬酸10.3g和蒸馏水1000mL配制而成,pH值2.4。
所述反应终止液为2M的硫酸液。
实施例1HBV纳米抗体库的构建
取200ug HBV表面小S蛋白(上海普欣生物技术有限公司,货号671-01、671-02)与等体积完全弗氏佐剂混合,充分乳化后注射至骆驼,以后每隔两周加强免疫一次,其中在加强免疫中使用不完全弗氏佐剂与免疫原的混合液,颈背部皮下多点免疫,共免疫5次。从第三次免疫开始,每次免疫后一周从颈锁静脉采血并检测血清效价。
从第5次免疫后的外周血中分离白细胞,提取总RNA,经反转录PCR及巢式PCR,克隆得到VHH基因片段,用限制性内切酶SfiI修饰粘性末端,通过T4连接酶将VHH基因片段连接至噬菌粒pComb3Xss(由University of California,Davis的Bruce DHammock教授惠赠,结构见图4),高效电转化至大肠杆菌ER2738(购自英国NEB公司),构建HBsAg的噬菌体纳米抗体库。经测定,初级库容量达109cfu,加入辅助噬菌体(感染复数为20:1)M13KO7(购自NEB公司,货号:N0315S)进行拯救,得到噬菌体纳米抗体库,库容量为1012pfu/mL,库的多样性较好。
反转录PCR:
反转录试剂盒采用PrimeScriptTM RT-PCR Kit,购自Takara公司,商品编号:AK2701。
反转录体系如下:
65℃反应5min。取出置于冰上,按以下体系加样,进行cDNA第一链合成。
30℃10min;42℃1h;72℃5min。
巢式PCR:(购自TAKATA公司,货号:6210A)
第一轮PCR:
反应体系如下:
反应程序如下:
第二轮PCR:
反应体系如下:
反应程序如下:
巢式PCR引物序列如下(5′-3′):
GSP-RT:CGCCATCAATRTACCAGTTGA
LP-leader:GTGGTCCTGGCTGCTCTW
R:CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG
F:CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC
其中,R表示碱基A/G,W表示碱基A/T,K表示碱基G/T。
实施例2HBV纳米抗体的筛选
在96孔酶标板的第1个孔包被HBV表面小S蛋白抗原,包被浓度为1ug/mL,4℃过夜;次日,倒出包被液,用PBST洗涤3次,将酶标板第1、2两个孔用BSA封闭,室温孵育2h;倒出封闭液,用PBST洗涤3次;将实施例1获得的噬菌体纳米抗体库加入第1个孔,反应2h;倒出液体,在洁净的吸水纸上拍干,用PBST洗涤5次;将100μL HBV S蛋白抗原添加到第1个孔中,反应1h;吸出第1个孔中的液体,加入第2个孔,反应1h,除去与BSA结合的噬菌体;收集洗脱液,取5μL用于滴度测定,其余用于扩增。
将噬菌体洗脱液加入新鲜的大肠杆菌ER2738菌液(实验室保存,也可商购获得,例如购自NEB公司),37℃,静置15min;加入羧苄青霉素和SB培养基,37℃,220rpm,培养2h;加入辅助噬菌体M13KO7(感染复数MOI=20:1)(购自NEB公司,货号:N0315S)和卡那霉素,培养过夜;次日,离心取上清,加入PEG-NaCl溶液沉淀纯化噬菌体。
将扩增产物进行下一轮筛选,保证每轮筛选的加入量相同,抗原包被浓度及S蛋白竞争洗脱浓度按2倍递减,计算每轮的滴度,挑取单克隆进行扩增及ELISA鉴定。经3轮淘选得到阳性单克隆。
实施例3HBV纳米抗体的表达
提取阳性单克隆质粒,转化至大肠杆菌TOP10F’感受态细胞(购自ThermoFishier),复苏后涂布于固体培养基过夜培养。次日,挑取单个克隆于SB-羧苄培养基中培养,加入IPTG诱导过夜表达;次日,用高压均浆仪裂解细胞,滤膜过滤后用镍柱纯化,即利用组氨酸标签与镍柱中氯化镍的亲和层析对纳米抗体进行分离纯化,得到高纯度的抗HBV纳米抗体,即抗体A1-A8,经氨基酸测序分析,所得纳米抗体的氨基酸序列如SEQ ID NO:1-8所示,抗体的CDR区见图3。
实施例4纳米抗体与HBV表面小S蛋白的结合曲线
将HBV ADR亚型表面小S蛋白和HBV ADW亚型表面小S蛋白(上海普欣生物技术有限公司)分别包被于96孔酶标板上,每个孔包被浓度为1ug/mL,4℃过夜反应;次日,甩出孔中的液体,用含0.05%吐温的PBST洗3次,将酶标板倒置在吸水纸上拍干;加入封闭液,37℃孵育30分钟,甩出孔中的液体,用0.05%PBST洗3次,将酶标板倒置在吸水纸上拍干;分别加入100μL不同稀释倍数的实施例3所得的纳米抗体液,37℃孵育30分钟;甩出孔中的液体,用PBST洗3次,将酶标板倒置在吸水纸上拍干;加入酶标二抗(辣根过氧化物酶标记的抗HA标签抗体,购自Roche公司),37℃孵育30分钟;甩出孔中的液体,用PBST洗板3次,拍干;取A液和B液等体积混匀,每孔加100μL,避光显色10~15分钟,加入终止液终止反应,酶标仪上测定各孔在波长为450nm处的OD值。根据抗体浓度和对应孔中的OD值绘制纳米抗体和HBV S蛋白的结合曲线(图1)。
实施例5纳米抗体与HBV表面小S蛋白的亲和力曲线
亲和力检测使用的是亲和素探针,利用Octec red 96仪器进行检测,所述亲和力检测方法是本领域常规技术操作,具体操作如下。向黑色无结合力的96孔板第一列的8个孔中加入0.02%吐温-20的PBST;再向第二列8个孔中加入浓度为15ug/ml生物素标记的HBVADR亚型表面小S蛋白或HBV ADW亚型表面小S蛋白。第三、五、七、九、十一列中加入PBST,第四、六、八、十列中加入倍比稀释的本发明所述的纳米抗体,其中每列的第8个孔加入PBST,第十二列中加入甘氨酸2.0,所述上述液体均200ul每孔。主要程序如下:
1)先将8根亲和素探针(streptavidin-sensor,购自FORTEBIO,货号:18-5019)浸入到第一列PBST中进行平衡60s;
2)再将亲和素探针浸入到HBV表面小S蛋白稀释液中结合3min;
3)再回到第一、三列PBST中进行两次平衡;
4)平衡后的探针浸入到第四列纳米抗体稀释液中,进行抗原抗体的特异性结合,结合3min;
5)再回到第三列PBST中进行解离,解离10min。
6)解离后探针在第十二列甘氨酸2.0中再生5s,将结合的纳米抗体完全洗脱下来;
7)再回到第十一列PBST中进行中和5s;
8)重复步骤6)、7);
9)再将探针浸入第五列PBST中进行平衡;重复步骤4)~8)依次检测其他纳米抗体与HBV表面小S蛋白结合能力;
10)最后将实验数据导入到excel表中。
结果见图2和表1,结果显示八个纳米抗体对HBV ADR亚型表面小S蛋白(ADR-S)的亲和力范围为2.01-8.33nM,对HBV ADW亚型表面小S蛋白(ADW-S)的亲和力范围为4.72-9.95nM。
表1纳米抗体与HBV ADR亚型表面小S蛋白和HBV ADW亚型表面小S蛋白的亲和力常数KD(M)
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 中国科学院生物物理研究所
<120> 乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体及其应用
<130> KHP221111794.8
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ser Ser Thr Asp Thr Trp Arg Thr Tyr
20 25 30
Cys Met Ser Ser Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Ile Asp Ile Trp Gly Arg Thr Phe Phe Val Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Ser Asp His Thr Leu Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys Val Ala Gly
85 90 95
Arg Pro Pro Phe Tyr Arg Ser Gly Cys Gln Pro Asn Asp Tyr Thr Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ala
115 120
<210> 2
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Asp Thr Leu Arg Thr Tyr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Thr Asp Ile Trp Arg Arg Thr Thr Phe Val Asp Ser Trp Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Ser Asp His Val Leu Tyr Leu Gln Leu
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Ser Cys Ala Ala Ser
85 90 95
Val Pro Pro Phe Tyr Arg Tyr Gly Gly Gln Pro Asn Asp Tyr Gln Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Ile Ser Ala
115 120
<210> 3
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Asp Thr Trp Arg Thr Tyr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Gly Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Thr Glu Ile Ile Gly Arg Thr Thr Phe Val Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Ser Asp His Thr Leu Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Ser Cys Val Ala Gly
85 90 95
Arg Pro Pro Phe Tyr Arg Tyr Gly Cys Gln Pro Asp Asp Tyr Thr Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 4
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ser Ser Ile Asp Thr Leu Arg Arg Tyr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Lys Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Ile Asp Arg Ile Gly Arg Thr Phe Phe Val Asp Ser Val Lys
50 55 60
Asp Lys Phe Thr Ile Ser Arg Ser Asp His Val Leu Tyr Leu Gln Met
65 70 75 80
Ser Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys Ala Ala Gly
85 90 95
Arg Pro Pro Phe Tyr Cys Thr Gly Cys Gln Pro Asn Asp Tyr Thr Tyr
100 105 110
Arg Gly Gln Gly Thr Gln Val Thr Ile Ser Ser
115 120
<210> 5
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Ile Asp Thr Leu Arg Thr Tyr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Thr Asp Ile Ile Gly Arg Thr Thr Phe Val Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Ser Asp His Thr Leu Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys Ala Ala Gly
85 90 95
Arg Pro Pro Phe Tyr Arg Tyr Gly Cys Gln Pro Asn Asp Tyr Thr Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 6
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Ile Asp Thr Trp Lys Thr Tyr
20 25 30
Cys Met Ser Ser Phe Arg Gln Ala Gly Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Thr Asp Arg Ile Gly Arg Thr Thr Phe Val Thr Ser Trp Lys
50 55 60
Asp Arg Arg Thr Ile Ser Arg Ser Asp His Thr Leu Tyr Leu Gln Leu
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys Ala Ala Gly
85 90 95
Phe Pro Pro Phe Tyr Arg Ala Gly Cys Gln Pro Asn Asp Tyr Gln Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 7
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Asp Thr Leu Arg Tyr Tyr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Thr
35 40 45
Ala Glu Ile Asp Ile Ile Gly Arg Thr Thr Phe Val Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Ser Ser His Val Leu Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Ser Cys Ala Phe Gly
85 90 95
Arg Pro Pro Phe Tyr Arg Tyr Cys Cys Gln Pro Asn Asp Ala Thr Tyr
100 105 110
Arg Gly Ala Gly Thr Gln Val Thr Val Ser Ala
115 120
<210> 8
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Ile Asp Thr Leu Arg Tyr Tyr
20 25 30
Cys Met Ser Ser Phe Arg Ser Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Glu Ile Asp Ile Ile Gly Arg Thr Phe Phe Val Val Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Ser Asp His Thr Leu Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala
85 90 95
Arg Pro Asn Phe Tyr Arg Tyr Gly Cys Gln Pro Asn Asp Tyr Thr Tyr
100 105 110
Arg Gly Ala Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 9
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Thr Asp Thr Trp Arg Thr Tyr Cys
1 5
<210> 10
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Glu Asp Thr Leu Arg Thr Tyr Cys
1 5
<210> 11
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ile Asp Thr Leu Arg Arg Tyr Cys
1 5
<210> 12
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ile Asp Thr Leu Arg Thr Tyr Cys
1 5
<210> 13
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Ile Asp Thr Trp Lys Thr Tyr Cys
1 5
<210> 14
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Thr Asp Thr Leu Arg Tyr Tyr Cys
1 5
<210> 15
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ile Asp Thr Leu Arg Tyr Tyr Cys
1 5
<210> 16
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Ile Asp Ile Trp Gly Arg Thr
1 5
<210> 17
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Thr Asp Ile Trp Arg Arg Thr
1 5
<210> 18
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Thr Glu Ile Ile Gly Arg Thr
1 5
<210> 19
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Ile Asp Arg Ile Gly Arg Thr
1 5
<210> 20
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Thr Asp Ile Ile Gly Arg Thr
1 5
<210> 21
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Thr Asp Arg Ile Gly Arg Thr
1 5
<210> 22
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Ile Asp Ile Ile Gly Arg Thr
1 5
<210> 23
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Val Ala Gly Arg Pro Pro Phe Tyr Arg Ser Gly Cys Gln Pro Asn Asp
1 5 10 15
Tyr Thr Tyr
<210> 24
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Ala Ala Ser Val Pro Pro Phe Tyr Arg Tyr Gly Gly Gln Pro Asn Asp
1 5 10 15
Tyr Gln Tyr
<210> 25
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Val Ala Gly Arg Pro Pro Phe Tyr Arg Tyr Gly Cys Gln Pro Asp Asp
1 5 10 15
Tyr Thr Tyr
<210> 26
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ala Ala Gly Arg Pro Pro Phe Tyr Cys Thr Gly Cys Gln Pro Asn Asp
1 5 10 15
Tyr Thr Tyr
<210> 27
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Ala Ala Gly Arg Pro Pro Phe Tyr Arg Tyr Gly Cys Gln Pro Asn Asp
1 5 10 15
Tyr Thr Tyr
<210> 28
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Ala Ala Gly Phe Pro Pro Phe Tyr Arg Ala Gly Cys Gln Pro Asn Asp
1 5 10 15
Tyr Gln Tyr
<210> 29
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Ala Phe Gly Arg Pro Pro Phe Tyr Arg Tyr Cys Cys Gln Pro Asn Asp
1 5 10 15
Ala Thr Tyr
<210> 30
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ala Ala Ala Arg Pro Asn Phe Tyr Arg Tyr Gly Cys Gln Pro Asn Asp
1 5 10 15
Tyr Thr Tyr
Claims (11)
1.乙肝病毒不同亚型表面S蛋白高亲和力纳米抗体或其抗原结合片段,其特征在于,其高变区CDR1、CDR2和CDR3分别选自:
(1)SEQ ID NO:9、SEQ ID NO:16和SEQ ID NO:23;或
(2)SEQ ID NO:10、SEQ ID NO:17和SEQ ID NO:24;或
(3)SEQ ID NO:9、SEQ ID NO:18和SEQ ID NO:25;或
(4)SEQ ID NO:11、SEQ ID NO:19和SEQ ID NO:26;或
(5)SEQ ID NO:12、SEQ ID NO:20和SEQ ID NO:27;或
(6)SEQ ID NO:13、SEQ ID NO:21和SEQ ID NO:28;或
(7)SEQ ID NO:14、SEQ ID NO:22和SEQ ID NO:29;或
(8)SEQ ID NO:15、SEQ ID NO:22和SEQ ID NO:30;
所述抗原结合片段为Fv、Fab、Fab'、F(ab')2。
2.根据权利要求1所述的纳米抗体或其抗原结合片段,其特征在于,其氨基酸序列如SEQ ID NO:1-8任一所示。
3.基因工程抗体,其特征在于,其包含权利要求1或2所述的抗体或其抗原结合片段;
其中,所述基因工程抗体为人源化抗体、嵌合抗体。
4.融合蛋白,其特征在于,由权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体,以及标签多肽组成。
5.核酸分子,其特征在于,其编码权利要求1或2所述的抗体或其抗原结合片段,或编码权利要求3所述的基因工程抗体,或编码权利要求4所述的融合蛋白;
其中,所述核酸分子为RNA或DNA。
6.根据权利要求5所述的核酸分子,其特征在于,所述核酸分子为cDNA。
7.含有权利要求5或6所述核酸分子的生物材料,其特征在于,所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
8.药物组合物,其特征在于,活性成分为权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或从权利要求7所述生物材料中获得的培养物;
所述药物组合物为外用型、皮下注射型、血管输入型,或其组合。
9.根据权利要求8所述的药物组合物,其特征在于,所述药物组合物还包括药用赋形剂或载体。
10.权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或从权利要求7所述生物材料中获得的培养物在制备用于预防、治疗和/或诊断HBV感染的生物制品中的应用。
11.权利要求1或2所述的抗体或其抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或从权利要求7所述生物材料中获得的培养物的以下任一应用:
1)用于制备预防或治疗乙肝病毒感染的药物;
2)用于制备乙肝病毒检测试剂或试剂盒。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210206182.2A CN114671947B (zh) | 2022-02-28 | 2022-02-28 | 乙肝病毒不同亚型表面s蛋白高亲和力纳米抗体及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210206182.2A CN114671947B (zh) | 2022-02-28 | 2022-02-28 | 乙肝病毒不同亚型表面s蛋白高亲和力纳米抗体及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114671947A CN114671947A (zh) | 2022-06-28 |
CN114671947B true CN114671947B (zh) | 2024-04-09 |
Family
ID=82071896
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210206182.2A Active CN114671947B (zh) | 2022-02-28 | 2022-02-28 | 乙肝病毒不同亚型表面s蛋白高亲和力纳米抗体及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114671947B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117250356B (zh) * | 2023-05-23 | 2024-02-20 | 安徽千诚生物技术有限公司 | 一种定量检测可溶性st2蛋白的胶乳增强免疫比浊试剂盒及其制备方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6146629A (en) * | 1996-06-11 | 2000-11-14 | Xtl Biopharmaceuticals Limited | Human monoclonal antibody against Hepatitis B virus surface antigen (HBVsAg) |
CN112661852A (zh) * | 2019-10-15 | 2021-04-16 | 中以海德人工智能药物研发股份有限公司 | 针对乙肝病毒的双特异性抗体及其应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2860188B1 (en) * | 2012-06-11 | 2020-10-21 | Xiamen University | Polypeptides and antibodies for treating hbv infection and related diseases |
-
2022
- 2022-02-28 CN CN202210206182.2A patent/CN114671947B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6146629A (en) * | 1996-06-11 | 2000-11-14 | Xtl Biopharmaceuticals Limited | Human monoclonal antibody against Hepatitis B virus surface antigen (HBVsAg) |
CN112661852A (zh) * | 2019-10-15 | 2021-04-16 | 中以海德人工智能药物研发股份有限公司 | 针对乙肝病毒的双特异性抗体及其应用 |
Non-Patent Citations (3)
Title |
---|
Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice;Benedikte Serruys et al;Hepatology;第49卷(第1期);全文 * |
三个HIV-1广谱中和表位与HBV S抗原融合表达可诱导小鼠特异性抗体应答;李学仁;陈红;王文;邓瑶;齐香荣;高瑛瑛;孟昕;谭文杰;阮力;;病毒学报(第04期);全文 * |
乙型肝炎病毒PreS1基因的原核表达及免疫学性质鉴定;张君;慕生枝;陈维贤;黄英;黄爱龙;唐霓;;免疫学杂志(第01期);全文 * |
Also Published As
Publication number | Publication date |
---|---|
CN114671947A (zh) | 2022-06-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2023527927A (ja) | 新型コロナウイルス(sars-cov-2)スパイクタンパク質結合分子及びその使用 | |
ES2774192T3 (es) | Variantes mejoradas de unión de anti-albúmina sérica | |
WO2016173558A1 (zh) | 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用 | |
WO2023125520A1 (zh) | SARS-CoV-2α、β、γ和δ突变株骆驼源高亲和力纳米抗体 | |
CN113150129A (zh) | 抗新冠病毒SARS-CoV-2表面S2蛋白的单链抗体及其应用 | |
CN112500480A (zh) | 针对新型冠状病毒的纳米抗体及其应用 | |
WO2021147984A1 (zh) | 抗angptl3抗体及其应用 | |
CN114671947B (zh) | 乙肝病毒不同亚型表面s蛋白高亲和力纳米抗体及其应用 | |
WO2023142309A1 (zh) | 抗tslp纳米抗体及其应用 | |
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 | |
CN116396381A (zh) | 人腺相关病毒(aav)单域抗体制备及其应用 | |
CN114539395B (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 | |
WO2023216623A1 (zh) | SARS-CoV-2α、γ、δ和ο突变株骆驼源高亲和力纳米抗体 | |
WO2022011717A1 (zh) | 针对新型冠状病毒的纳米抗体及其应用 | |
CN114702573B (zh) | 乙肝病毒表面s蛋白高亲和力纳米抗体及其应用 | |
WO2023137994A1 (zh) | 结直肠癌相关脆弱拟杆菌毒素蛋白激活体的特异性纳米抗体Nb3.27及其应用 | |
CN114478761B (zh) | 绿色荧光蛋白鲨源纳米抗体、制备方法及其应用 | |
CN114591423B (zh) | 新冠病毒n蛋白的特异性抗体及其制备方法与应用 | |
CN114539394B (zh) | SARS-CoV-2α突变株和β突变株骆驼源高亲和力纳米抗体 | |
CN115975015A (zh) | 一种小反刍兽疫病毒(pprv)f蛋白纳米抗体和纳米抗体的制备、纯化及中和试验方法 | |
US20150240230A1 (en) | Method for producing and obtaining variable domains of anti-digoxin monoclonal antibody fab fragment using the molecular biology cloning technique | |
CN114591435B (zh) | 胃蛋白酶原ii的特异性抗体及其制备方法与应用 | |
CN114591436B (zh) | 胃蛋白酶原i的特异性抗体及其制备方法与应用 | |
CN114702590B (zh) | 抗c-MET纳米抗体、编码核酸及其应用 | |
WO2023035272A1 (zh) | 一种il17抗体及其制备方法和应用 |
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 |