CN112048483B - 1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备 - Google Patents
1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备 Download PDFInfo
- Publication number
- CN112048483B CN112048483B CN202010872788.0A CN202010872788A CN112048483B CN 112048483 B CN112048483 B CN 112048483B CN 202010872788 A CN202010872788 A CN 202010872788A CN 112048483 B CN112048483 B CN 112048483B
- Authority
- CN
- China
- Prior art keywords
- type
- monoclonal antibody
- capsid protein
- antibody
- pastv
- 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
- 108090000565 Capsid Proteins Proteins 0.000 title claims abstract description 32
- 102100023321 Ceruloplasmin Human genes 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 26
- 241000472152 Mamastrovirus 3 Species 0.000 claims abstract description 19
- 238000001262 western blot Methods 0.000 claims abstract description 15
- 238000002965 ELISA Methods 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 238000003745 diagnosis Methods 0.000 claims abstract description 8
- 241000699660 Mus musculus Species 0.000 claims abstract description 5
- 244000309743 astrovirus Species 0.000 claims description 9
- 238000011997 immunoflourescence assay Methods 0.000 claims description 6
- 238000003149 assay kit Methods 0.000 claims description 2
- 230000003248 secreting effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 6
- 230000028327 secretion Effects 0.000 abstract description 5
- 241000486679 Antitype Species 0.000 abstract description 3
- 230000001900 immune effect Effects 0.000 abstract description 3
- 238000003748 differential diagnosis Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 23
- 102000004169 proteins and genes Human genes 0.000 description 21
- 230000014509 gene expression Effects 0.000 description 17
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 230000003053 immunization Effects 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 239000000835 fiber Substances 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 230000009465 prokaryotic expression Effects 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 6
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000283707 Capra Species 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 4
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001378 electrochemiluminescence detection Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 210000004988 splenocyte Anatomy 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 210000002845 virion Anatomy 0.000 description 3
- 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 2
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 2
- BHFOJPDOQPWJRN-XDTLVQLUSA-N Ala-Tyr-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCC(N)=O)C(O)=O BHFOJPDOQPWJRN-XDTLVQLUSA-N 0.000 description 2
- HOBNTSHITVVNBN-ZPFDUUQYSA-N Asp-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N HOBNTSHITVVNBN-ZPFDUUQYSA-N 0.000 description 2
- OREPWMPAUWIIAM-ZPFDUUQYSA-N Gln-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N OREPWMPAUWIIAM-ZPFDUUQYSA-N 0.000 description 2
- BRFJMRSRMOMIMU-WHFBIAKZSA-N Gly-Ala-Asn Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O BRFJMRSRMOMIMU-WHFBIAKZSA-N 0.000 description 2
- WSWWTQYHFCBKBT-DVJZZOLTSA-N Gly-Thr-Trp Chemical compound C[C@@H](O)[C@H](NC(=O)CN)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O WSWWTQYHFCBKBT-DVJZZOLTSA-N 0.000 description 2
- NBWATNYAUVSAEQ-ZEILLAHLSA-N His-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O NBWATNYAUVSAEQ-ZEILLAHLSA-N 0.000 description 2
- FMEICTQWUKNAGC-YUMQZZPRSA-N Leu-Gly-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O FMEICTQWUKNAGC-YUMQZZPRSA-N 0.000 description 2
- GMMLGMFBYCFCCX-KZVJFYERSA-N Met-Thr-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O GMMLGMFBYCFCCX-KZVJFYERSA-N 0.000 description 2
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- ZSDXEKUKQAKZFE-XAVMHZPKSA-N Ser-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N)O ZSDXEKUKQAKZFE-XAVMHZPKSA-N 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 2
- AKHDFZHUPGVFEJ-YEPSODPASA-N Thr-Val-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AKHDFZHUPGVFEJ-YEPSODPASA-N 0.000 description 2
- FFCRCJZJARTYCG-KKUMJFAQSA-N Tyr-Cys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N)O FFCRCJZJARTYCG-KKUMJFAQSA-N 0.000 description 2
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 2
- RTJPAGFXOWEBAI-SRVKXCTJSA-N Val-Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RTJPAGFXOWEBAI-SRVKXCTJSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 108010077245 asparaginyl-proline Proteins 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011841 epidemiological investigation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 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 2
- 239000012160 loading buffer Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FXKNPWNXPQZLES-ZLUOBGJFSA-N Ala-Asn-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O FXKNPWNXPQZLES-ZLUOBGJFSA-N 0.000 description 1
- NFDVJAKFMXHJEQ-HERUPUMHSA-N Ala-Asp-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N NFDVJAKFMXHJEQ-HERUPUMHSA-N 0.000 description 1
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 1
- LDLSENBXQNDTPB-DCAQKATOSA-N Ala-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LDLSENBXQNDTPB-DCAQKATOSA-N 0.000 description 1
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- GXXWTNKNFFKTJB-NAKRPEOUSA-N Arg-Ile-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O GXXWTNKNFFKTJB-NAKRPEOUSA-N 0.000 description 1
- KZXPVYVSHUJCEO-ULQDDVLXSA-N Arg-Phe-Lys Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=CC=C1 KZXPVYVSHUJCEO-ULQDDVLXSA-N 0.000 description 1
- AZHXYLJRGVMQKW-UMPQAUOISA-N Arg-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCCN=C(N)N)N)O AZHXYLJRGVMQKW-UMPQAUOISA-N 0.000 description 1
- HZPSDHRYYIORKR-WHFBIAKZSA-N Asn-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O HZPSDHRYYIORKR-WHFBIAKZSA-N 0.000 description 1
- PAXHINASXXXILC-SRVKXCTJSA-N Asn-Asp-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N)O PAXHINASXXXILC-SRVKXCTJSA-N 0.000 description 1
- WIDVAWAQBRAKTI-YUMQZZPRSA-N Asn-Leu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIDVAWAQBRAKTI-YUMQZZPRSA-N 0.000 description 1
- XEGZSHSPQNDNRH-JRQIVUDYSA-N Asn-Tyr-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XEGZSHSPQNDNRH-JRQIVUDYSA-N 0.000 description 1
- SVFOIXMRMLROHO-SRVKXCTJSA-N Asp-Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SVFOIXMRMLROHO-SRVKXCTJSA-N 0.000 description 1
- MVRGBQGZSDJBSM-GMOBBJLQSA-N Asp-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)O)N MVRGBQGZSDJBSM-GMOBBJLQSA-N 0.000 description 1
- RVMXMLSYBTXCAV-VEVYYDQMSA-N Asp-Pro-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMXMLSYBTXCAV-VEVYYDQMSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710193695 Capsid protein precursor Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- UWMDGPFFTKDUIY-HJGDQZAQSA-N Gln-Pro-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWMDGPFFTKDUIY-HJGDQZAQSA-N 0.000 description 1
- MRVYVEQPNDSWLH-XPUUQOCRSA-N Gln-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(N)=O MRVYVEQPNDSWLH-XPUUQOCRSA-N 0.000 description 1
- FITIQFSXXBKFFM-NRPADANISA-N Gln-Val-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FITIQFSXXBKFFM-NRPADANISA-N 0.000 description 1
- PBEQPAZRHDVJQI-SRVKXCTJSA-N Glu-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)O)N PBEQPAZRHDVJQI-SRVKXCTJSA-N 0.000 description 1
- WATXSTJXNBOHKD-LAEOZQHASA-N Glu-Asp-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O WATXSTJXNBOHKD-LAEOZQHASA-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
- RQZGFWKQLPJOEQ-YUMQZZPRSA-N Gly-Arg-Gln Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)CN)CN=C(N)N RQZGFWKQLPJOEQ-YUMQZZPRSA-N 0.000 description 1
- CIMULJZTTOBOPN-WHFBIAKZSA-N Gly-Asn-Asn Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CIMULJZTTOBOPN-WHFBIAKZSA-N 0.000 description 1
- KQDMENMTYNBWMR-WHFBIAKZSA-N Gly-Asp-Ala Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O KQDMENMTYNBWMR-WHFBIAKZSA-N 0.000 description 1
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 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
- OVPYIUNCVSOVNF-ZPFDUUQYSA-N Ile-Gln-Pro Natural products CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O OVPYIUNCVSOVNF-ZPFDUUQYSA-N 0.000 description 1
- URWXDJAEEGBADB-TUBUOCAGSA-N Ile-His-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N URWXDJAEEGBADB-TUBUOCAGSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 description 1
- PJYSOYLLTJKZHC-GUBZILKMSA-N Leu-Asp-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O PJYSOYLLTJKZHC-GUBZILKMSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- OVZLLFONXILPDZ-VOAKCMCISA-N Leu-Lys-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OVZLLFONXILPDZ-VOAKCMCISA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- CANPXOLVTMKURR-WEDXCCLWSA-N Lys-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN CANPXOLVTMKURR-WEDXCCLWSA-N 0.000 description 1
- PLOUVAYOMTYJRG-JXUBOQSCSA-N Lys-Thr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PLOUVAYOMTYJRG-JXUBOQSCSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- BQHLZUMZOXUWNU-DCAQKATOSA-N Met-Pro-Glu Chemical compound CSCC[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N BQHLZUMZOXUWNU-DCAQKATOSA-N 0.000 description 1
- FIZZULTXMVEIAA-IHRRRGAJSA-N Met-Ser-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 FIZZULTXMVEIAA-IHRRRGAJSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 101150001779 ORF1a gene Proteins 0.000 description 1
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- IDUCUXTUHHIQIP-SOUVJXGZSA-N Phe-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O IDUCUXTUHHIQIP-SOUVJXGZSA-N 0.000 description 1
- DNAXXTQSTKOHFO-QEJZJMRPSA-N Phe-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 DNAXXTQSTKOHFO-QEJZJMRPSA-N 0.000 description 1
- MRWOVVNKSXXLRP-IHPCNDPISA-N Phe-Ser-Trp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O MRWOVVNKSXXLRP-IHPCNDPISA-N 0.000 description 1
- OBVCYFIHIIYIQF-CIUDSAMLSA-N Pro-Asn-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O OBVCYFIHIIYIQF-CIUDSAMLSA-N 0.000 description 1
- SFECXGVELZFBFJ-VEVYYDQMSA-N Pro-Asp-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SFECXGVELZFBFJ-VEVYYDQMSA-N 0.000 description 1
- NOXSEHJOXCWRHK-DCAQKATOSA-N Pro-Cys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@@H]1CCCN1 NOXSEHJOXCWRHK-DCAQKATOSA-N 0.000 description 1
- RPLMFKUKFZOTER-AVGNSLFASA-N Pro-Met-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@@H]1CCCN1 RPLMFKUKFZOTER-AVGNSLFASA-N 0.000 description 1
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- OHKFXGKHSJKKAL-NRPADANISA-N Ser-Glu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OHKFXGKHSJKKAL-NRPADANISA-N 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- IHAPJUHCZXBPHR-WZLNRYEVSA-N Thr-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N IHAPJUHCZXBPHR-WZLNRYEVSA-N 0.000 description 1
- VRUFCJZQDACGLH-UVOCVTCTSA-N Thr-Leu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VRUFCJZQDACGLH-UVOCVTCTSA-N 0.000 description 1
- GIAMKIPJSRZVJB-IHPCNDPISA-N Trp-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N GIAMKIPJSRZVJB-IHPCNDPISA-N 0.000 description 1
- JBBYKPZAPOLCPK-JYJNAYRXSA-N Tyr-Arg-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O JBBYKPZAPOLCPK-JYJNAYRXSA-N 0.000 description 1
- DWJQKEZKLQCHKO-SRVKXCTJSA-N Tyr-Asn-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N)O DWJQKEZKLQCHKO-SRVKXCTJSA-N 0.000 description 1
- DAOREBHZAKCOEN-ULQDDVLXSA-N Tyr-Leu-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O DAOREBHZAKCOEN-ULQDDVLXSA-N 0.000 description 1
- ZPFLBLFITJCBTP-QWRGUYRKSA-N Tyr-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O ZPFLBLFITJCBTP-QWRGUYRKSA-N 0.000 description 1
- CPTQYHDSVGVGDZ-UKJIMTQDSA-N Val-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N CPTQYHDSVGVGDZ-UKJIMTQDSA-N 0.000 description 1
- BEGDZYNDCNEGJZ-XVKPBYJWSA-N Val-Gly-Gln Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O BEGDZYNDCNEGJZ-XVKPBYJWSA-N 0.000 description 1
- KSFXWENSJABBFI-ZKWXMUAHSA-N Val-Ser-Asn Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KSFXWENSJABBFI-ZKWXMUAHSA-N 0.000 description 1
- PQSNETRGCRUOGP-KKHAAJSZSA-N Val-Thr-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(N)=O PQSNETRGCRUOGP-KKHAAJSZSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 244000309457 enveloped RNA virus Species 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010085203 methionylmethionine Proteins 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108010093296 prolyl-prolyl-alanine Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000006490 viral transcription Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from 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
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/12011—Astroviridae
- C12N2770/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- G01N2333/08—RNA viruses
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一株杂交瘤细胞株,保藏编号为CCTCC NO:C2020107,分类命名Mus musculus B1F5。该杂交瘤细胞株分泌得到的抗1型PAstV衣壳蛋白单克隆抗体B1F5,经鉴定为IgG1,κ型,能特异性识别1型PAstV,其识别的抗原表位如SEQ.ID.NO.19所示。同时,还建立了相应杂交瘤细胞株的制备方法。据此,该抗体可作为鉴别诊断1型PAstV的特异性抗体,用于建立和制备1型猪星状病毒的ELISA、Western Blot、IFA等生物诊断方法及其试剂盒。研究表明,本发明杂交瘤抗体分泌水平稳定,特异性好,生物学特性优良,为1型PAstV免疫学检测方法研究奠定了基础。
Description
技术领域
本发明属于星状病毒技术领域,尤其涉及一种1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备。
背景技术
猪星状病毒(Porcine Astrovirus,PAstV)是一种单股、正链、无囊膜包被的RNA病毒。该病毒在1980年首次检测出来,发现PAstV与轮状病毒和杯状病毒混合感染的现象,并导致感染猪群出现腹泻、厌食等症状。PAstV在猪群中的流行十分普遍,流行病学调查发现,在加拿大、美国等地猪星状病毒的流行率分别高达79%和62%;在国内的流行病学调查发现,1型PAstV在广西猪群的流行率高达40.3%,其中猪场阳性率更是高达82.8%。已有研究表明,星状病毒可以通过粪-口途径进行传播,城市污水中也可以检测到星状病毒,其传播途径十分广泛。PAstV感染动物主要引起水样腹泻等临床症状,动物致病性试验发现,PAstV可以使感染猪出现轻微腹泻,增重减少等,受感染猪肠系膜淋巴结肿大,肠绒毛萎缩变短,肠道出现炎性反应等。
星状病毒的基因组大小约6.2-7.7kb,包含3个开放性阅读框(open readingframes,ORFs),其中位于基因组5′端的ORF1a和ORF1b负责编码病毒转录和复制相关的非结构蛋白(non-structure protein,nsp);而位于基因组3′端的ORF2通过转录形成亚基因组RNA而编码病毒唯一的结构蛋白,即衣壳蛋白(capsid protein,capsid)。cap蛋白大小约为80-90kDa,由病毒基因组ORF2位置转录的亚基因组RNA翻译得到病毒衣壳蛋白前体VP90,经细胞内半胱天冬酶和细胞外胰酶的一系列水解切割后,形成具有感染性的成熟病毒粒子,其中VP27和VP25主要由衣壳蛋白C端编码,组成病毒粒子纤突部分spike结构。
发明内容
本发明要解决的技术问题是提供一种1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备,为进一步建立1型PAstV的免疫学检测方法奠定了基础。
为解决上述技术问题,本发明采用以下技术方案:
一株杂交瘤细胞株,保藏编号为CCTCC NO:C2020107,分类命名Mus musculusB1F5。
上述杂交瘤细胞分泌的抗1型猪星状病毒衣壳蛋白的单克隆抗体B1F5。
单克隆抗体B1F5能与1型猪星状病毒衣壳蛋白抗原纤突结构域特异性结合。
单克隆抗体B1F5亚型为IgG1,κ型。
上述单克隆抗体B1F5在生物诊断鉴定中的应用。
生物诊断针对1型猪星状病毒,生物诊断包括ELISA、Western Blot、IFA。
猪星状病毒生物诊断鉴定试剂盒,包含权利要求2所述的单克隆抗体B1F5。
上述杂交瘤细胞株的制备方法,用spike-His作为抗原免疫小鼠;收集免疫后的小鼠的脾细胞,并将所述脾细胞与骨髓瘤细胞进行融合得到融合细胞;采用HAT培养基对所述融合细胞进行选择培养;以及用spike-His蛋白抗原检测所述融合细胞的培养上清中的抗体,筛选获得一株稳定分泌单克隆抗体B1F5的杂交瘤细胞B1F5。
1型猪星状病毒衣壳蛋白抗原表位,具有序列表SEQ.ID.NO.19的氨基酸序列。
特异性结合权利要求9所述抗原表位的抗1型猪星状病毒衣壳蛋白的多克隆抗体或单克隆抗体。
发明人针对1型猪星状病毒检测存在的问题,选定纤突结构域作为靶抗原,运用融合杂交瘤技术获得了一株杂交瘤细胞株,保藏编号为CCTCC NO:C2020107,分类命名Musmusculus B1F5。该杂交瘤细胞株分泌得到的抗1型PAstV衣壳蛋白单克隆抗体B1F5,经鉴定为IgG1,κ型,能特异性识别1型PAstV,其识别的抗原表位如SEQ.ID.NO.19所示。WesternBlot检测结果显示B1F5可与1型PAstV衣壳蛋白发生特异性反应,尤其可识别其纤突结构域。同时,发明人还建立了相应杂交瘤细胞株的制备方法。据此,该抗体可作为鉴别诊断1型PAstV的特异性抗体,用于建立和制备1型猪星状病毒的ELISA、Western Blot、IFA等生物诊断方法及其试剂盒。研究表明,本发明杂交瘤抗体分泌水平稳定,特异性好,生物学特性优良,为1型PAstV的免疫学检测方法研究奠定了基础。
附图说明
图1是实施例1中PAstV衣壳蛋白截短体原核表达蛋白spike-His鉴定结果图。
图2是实施例1中PAstV衣壳蛋白单克隆抗体的亚型鉴定结果图。
图3是实施例2中PAstV衣壳蛋白单克隆抗体的IFA检测结果图。
图4是实施例2中针对PAstV衣壳蛋白单克隆抗体B1F5检测PAstV细胞毒的westernblot鉴定结果图。
图5是实施例3中PAstV衣壳蛋白纤突结构域各截短蛋白表达图。
图6是实施例3中单克隆抗体B1F5识别的抗原表位western blot鉴定图。
具体实施方式
研究设计原理
以pET-32a(+)为载体构建表达1型PAstV衣壳蛋白截短体,经镍柱纯化后作为免疫原免疫小鼠,按常规免疫程序免疫4次后取小鼠脾淋巴细胞与骨髓瘤细胞sp2/0融合,经筛选、克隆、传代和反复冻存、复苏后获得杂交瘤细胞系B1F5,能稳定分泌抗1型猪星状病毒衣壳蛋白的单克隆抗体B1F5。
具体研究试验如下:
实施例1抗1型PAstV衣壳蛋白单克隆抗体制备方法
1材料
1.1毒株、细胞和实验动物
PAstV-GX1株、PK15细胞、SP2/0细胞均由广西大学动物科学技术学院保存;6-8周雌性Balb/c小鼠购自北京维通利华公司。
1.2主要试剂及抗体
胶回收试剂盒及质粒提取试剂盒购自OMEGA公司;反转录试剂盒、蛋白预染Marker、限制性内切酶BamH I和HindⅢ购自购自TaKaRa公司;弗氏完全佐剂、弗氏不完全佐剂购自Sigma公司;His标签纯化试剂盒、BCA蛋白浓度测定试剂盒购自碧云天公司;6*His-Tag Antibody、HRP标记山羊抗鼠IgG抗体、Alexa Fluor 488标记的山羊抗鼠IgG抗体购自Proteintech公司,抗体亚型鉴定试纸条购自Roche。
2方法及结果
2.1PAstV-GX1 spike基因原核表达质粒的构建
以GenBank公布的PAstV-GX1株序列(GenBank登录号:KF787112)设计扩增ORF2中纤突结构域spike domain序列(上游引物引入限制性酶切位点BamHI,下游引物引入限制性酶切位点HindIII),引物由生工生物工程有限公司合成。提取PAstV-GX1毒株总RNA,扩增PAstV-GX1 spike基因(碱基序列见SEQ.ID.NO.2,氨基酸序列见SEQ.ID.NO.1),回收目的片段,经BamH I和HindⅢ双酶切后连接至经同样双酶切处理的原核表达载体pET-32a(+),连接产物转化于大肠杆菌BL21(DE3)感受态菌株中,经涂板、挑菌、摇菌,提质粒,PCR、测序验证正确的即为阳性重组表达质粒pET32a-spike。(由广州生物工程公司进行测序验证)。
2.2蛋白的表达、鉴定与纯化
将上述鉴定正确的阳性重组菌株接种于含有氨苄抗性的培养基中,37℃振荡培养至OD600为0.6-1.0,加入终浓度为1mmol/L的IPTG,在37℃恒温振荡诱导6h后,收集诱导表达菌液经8000rpm 4℃离心,收集菌体,使用PBS重悬,取少量加入5×SDS-PAGE上样缓冲液裂解处理,进行SDS-PAGE电泳及考马斯亮蓝染色检测spike-His蛋白的表达(图1)。
将获得的表达菌体进行超声破碎,之后收集处理后的可溶性表达产物进行Ni柱纯化,将可溶性蛋白在4℃条件下与Ni-NTA亲和层析柱结合过夜,收集流穿液,使用washbuffer对亲和层析清洗3次,最后用适量洗脱液洗脱目的蛋白,并对流穿液及各部分洗脱液进行SDS-PAGE及考马斯亮蓝检测,并进行BCA蛋白浓度测定,分装保存于-80℃。
2.3小鼠免疫
小鼠免疫以上述纯化的spike-His重组蛋白免疫4-6周雌性Balb/c小鼠,共免疫3次,每次免疫时间间隔两周,首免免疫100ug/只,用等量弗式完全佐剂与蛋白乳化,第二、三次免疫剂量为50ug/只,取等量弗式不完全佐剂进行乳化,免疫途径为腹腔免疫。融合前3-4天进行加强免疫,加强免疫剂量为50ug/只,不添加佐剂,通过尾静脉注射。
2.4细胞融合
融合前一天,取一只正常鼠取其腹腔巨噬细胞作为饲养细胞,按照常规方法取细胞铺于96孔细胞板中待用。免疫鼠进行眼球采血,分离血清保存,处死小鼠,在无菌条件下取免疫小鼠的脾脏,分离脾细胞,按照脾细胞与SP2/0细胞8:1的比例用PEG2000进行融合,融合后的细胞铺于前一天铺好的饲养细胞中,置于37℃CO2培养箱中培养。
2.5阳性杂交瘤细胞株的筛选及克隆
将纯化后的spike-His蛋白和PET-32a空载体诱导产物分别包被酶标板,剂量为100ng/孔,4℃包被过夜,用PBST清洗后加入5%脱脂奶粉封闭,37℃封闭3h,PBST清洗后备用。将培养1周后的融合板换液,3-4天后可观察孔中杂交瘤细胞的形成,取上清液加入到包被的酶标板中进行ELISA试验,筛选阳性杂交瘤细胞株。
对反应阳性的杂交瘤细胞扩大培养,以有限稀释法对阳性克隆进行亚克隆3次,每次亚克隆后都利用ELISA实验对单克隆孔进行检测,筛选稳定分泌的单克隆扩大培养,最终获得一株稳定分泌的单克隆抗体细胞株,命名为B1F5。
为了鉴定杂交瘤细胞株分泌抗体的稳定性,将细胞冻存后复苏并进行传代,ELISA检测杂交瘤细胞分泌抗体的稳定性。
该杂交瘤已送至中国典型培养物保藏中心保藏,保藏信息如下
杂交瘤细胞株B1F5(Mus musculus B1F5),保藏编号为CCTCC NO:C2020107,保藏日期:2020年7月13日,保藏地址为:武汉·武汉大学,邮编430072,保藏单位:中国典型培养物保藏中心。
杂交瘤细胞B1F5在含有10%胎牛血清的DMEM培养基中,在含有5%CO2,37℃条件下培养,细胞生长状态良好,抗体分泌稳定。
2.6单克隆抗体western blot的鉴定
通过western blot实验对单克隆抗体的反应特异性进行鉴定。将PEC-spike表达的spike-His重组蛋白转印至PVDF膜,以杂交瘤细胞株B1F5分泌的上清液作为一抗HRP标记山羊抗鼠IgG为二抗,ECL显色。
试验结果证实,本发明所制备的单克隆抗体B1F5能够与spike-His发生特异性反应。
2.7单克隆抗体的亚型鉴定
经Roche抗体亚类鉴定试剂盒(购自Roche 11493027001Mouse MonoclonalAntibody Isotyping Kit)鉴定,抗1型PAstV衣壳蛋白的单克隆抗体B1F5亚类为IgG1,κ型(图2)。
实施例2单克隆抗体的应用
1.单克隆抗体IFA鉴定1型PAstV
将生长状态良好的PK15细胞接种96孔板中,待汇合度生长至80%左右接种适量PAstV-GX1,在37℃CO2培养箱中继续24h后弃掉培养基,用PBS清洗两次,加入冰甲醇,-20℃固定10min,弃掉冰甲醇后用PBS清洗两次,加入2%BSA,37℃封闭30min,用于后续IFA检测。将筛选纯化得到的阳性克隆株B1F5分泌得抗体作为一抗,以AF488标记的驴抗鼠IgG荧光二抗进行IFA检测,将反应板置于导致荧光显微镜下观察结果。
试验结果显示,抗1型PAstV衣壳蛋白的单克隆抗体B1F5可特异性识别1型PAstV感染的PK15细胞(图3)。
2.单克隆抗体western blot鉴定1型PAstV尤其其纤突结构域
将生长状态良好的PK15细胞接种6孔板中,待汇合度生长至80%左右接种适量PAstV-GX1,赶作1h后分别加入含有0.5μg/mg TPCK的DMEM培养基和不含TPCK的DMEM培养基,在37℃CO2培养箱中继续48h后收集细胞毒,加入5×SDS-PAGE上样缓冲液,沸水煮5min后进行SDS-PAGE电泳,电泳结束后转印至PVDF膜上,封闭后将筛选纯化得到的阳性克隆株B1F5分泌的抗体作为一抗,以HRP标记的山羊抗鼠IgG二抗进行western blot检测,ECL显色。
试验结果显示,抗1型PAstV衣壳蛋白的单克隆抗体B1F5可特异性识别1型PAstV衣壳蛋白前体VP90,尤其病毒粒子的纤突结构域spike domain VP25(图4)。
实施例3单克隆抗体抗原表位的鉴定
1.方法
1.1 1型PAstV衣壳蛋白纤突结构域的分段扩增和克隆
将PAstV-GX1衣壳蛋白纤突结构域进行截断表达,将其分为2个相互重叠的小片段,分别命名S1、S2。针对每个片段设计引物(表1),引物由生工生物工程有限公司(上海)合成,在两端引物XhoI和EcoRI限制性酶切位点,以ORF2全长质粒为模板,对两个片段进行扩增,经胶回收、酶切后连接至线性化的pGEX6p-1原核表达载体中,转化至BL21大肠杆菌感受态中,经涂板、挑菌、摇菌,提质粒,PCR、测序验证正确的即为阳性重组表达质粒。(由广州生物工程公司进行测序验证)。
1.2 S1-GST、S2-GST的表达鉴定
将S1、S2的阳性菌种及pGEX6p-1空载体菌种扩大培养,在含有氨苄抗性的液体培养基中37℃震荡培养至OD600约0.4-0.6时,加入终浓度为1mM的IPTG诱导剂,37℃诱导6-8h后8000rpm离心2min,收集菌体,加入适量PBS重悬,加入加入5×SDS-PAGE上样缓冲液裂解处理,进行SDS-PAGE电泳及考马斯亮蓝染色检测重组融合蛋白S1-GST、S2-GST蛋白的表达。
1.3单克隆抗体B1F5识别表位的初步鉴定
将鉴定的成功表达的S1-GST、S2-GST蛋白及pGEX6p-1空载体蛋白进行SDS-PAGE电泳,电泳结束后将蛋白转印至PVDF膜,封闭后将得到的单克隆抗体B1F5作为一抗,以HRP标记的山羊抗鼠IgG二抗进行western blot检测,ECL显色,初步鉴定单克隆抗体B1F5识别的区域。
1.4单克隆抗体B1F5识别抗原表位的鉴定
为了进一步鉴定B1F5识别的抗原表位,对上一步骤中识别的区域进行截短表达,方法步骤按照实施例3方法1.1进行,抗原表位的鉴定方法按照实施例3方法1.3进行。
表1 PAstV 1衣壳蛋白纤突结构域各截短基因扩增引物序列及位置
2.结果
2.1 PAstV 1衣壳蛋白纤突结构域各截短体的表达
将纤突结构域spike蛋白截短分成相互重叠的基因片段,连接至pGEX 6p-1原核表达载体,经诱导表达获得重组融合蛋白S1-GST、S2-GST,表达产物经SDS-PAGE及考马斯亮蓝染色,结果显示S1-GST、S2-GST均成功表达(图5)。
2.2单克隆抗体B1F5抗原表位初步鉴定
以截短表达的衣壳蛋白纤突结构域(420-669aa)spike蛋白作为检测抗原,western blot对B1F5识别的抗原进行初步鉴定,结果显示单克隆抗体识别S1(420-587aa),不识别S2(502-669aa)(图6)。
2.3 S1截短体的表达
将S1蛋白截短分成相互重叠的基因片段,连接至pGEX 6p-1原核表达载体,经诱导表达获得重组融合蛋白S1-1-GST、S1-2-GST,表达产物经SDS-PAGE及考马斯亮蓝染色,结果显示S1-1-GST、S1-2-GST均成功表达(图5)。
2.4单克隆抗体B1F5识别区域的鉴定
以截短表达表达的S1-1-GST、S1-2-GST蛋白作为检测抗原,western blot对B1F5识别的抗原进一步鉴定,结果显示单克隆抗体识别S1-2(480-589aa),不识别S1-1(420-520aa)(图6)。
2.5 S1-2截短体的表达
将S1-2蛋白截短分成相互重叠的基因片段,连接至pGEX 6p-1原核表达载体,经诱导表达获得重组融合蛋白1-2-1-GST、1-2-2GST、1-2-3GST,表达产物经SDS-PAGE及考马斯亮蓝染色,结果显示1-2-1-GST、1-2-2GST、1-2-3-GST均成功表达(图5)。
2.6单克隆抗体B1F5抗原表位的鉴定
以截短表达表达的1-2-1-GST、1-2-2GST、1-2-3GST蛋白作为检测抗原,westernblot鉴定单克隆抗体B1F5识别的抗原表位,结果显示单克隆抗体识别1-2-2-GST(500-549aa),不识别1-2-1-GST(480-529aa)、1-2-3GST(520-589aa)(图6)。分析该结果确定B1F5识别的抗原表位位于衣壳蛋白的500-549aa区域(SEQ.ID.NO.19)。
序列表
<110> 广西大学
<120> 1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备
<160> 19
<170> SIPOSequenceListing 1.0
<210> 2
<211> 250
<212> PRT
<213> 1型猪星状病毒(Porcine Astrovirus 1)
<400> 2
Gly Glu Thr Pro Val Thr Phe Lys Ala Tyr Arg Met Met Pro Glu Asp
1 5 10 15
Thr Ile Tyr Leu Arg Phe Lys Pro Asp Thr Leu Ser Val Val Ser Asn
20 25 30
Phe Gln Pro Ala Lys Arg Pro Met Leu Ala Lys Thr Tyr Ser Gly Asp
35 40 45
Thr Leu Thr Val Gly Gln Gly Asn Asn Lys Thr Ala Ile His Thr Val
50 55 60
Val Arg Ile Ser Asp Pro Thr Trp Phe Ser Ala Asp Trp Asp Pro Ile
65 70 75 80
Ser Thr Pro Gln Pro Ile Ala Glu Ile Tyr Cys Lys Ala Gly Thr Thr
85 90 95
Thr Val Gly Asp Ile Leu Ala Ala Tyr Gln Val His Gly Leu Gly Asn
100 105 110
His Thr Thr Thr Ala Tyr Val Val Arg Met Thr Ala Gly Ala Asn Pro
115 120 125
Gln Val Ser Ala Gly Ile Val Thr Asn Lys Gly Thr Asn Asp Tyr Asp
130 135 140
Leu Lys Thr Ala Asn Ser Asn Ala Gly Phe Ser Trp Asn Leu Gly Ser
145 150 155 160
Gly Thr Trp Tyr Leu Met Met Ser Phe Gly Asp Ala Leu Gly Ser Leu
165 170 175
Gly Thr Trp Arg Trp Thr Pro Asn Glu Leu Ser Ala Asn Tyr Thr Ile
180 185 190
Tyr Asn Cys Glu Ile Ile Pro Cys Leu Leu Leu Ala Asn Asp Asp Phe
195 200 205
His Ile Val Ile Pro Thr Lys Asn Ala Leu Val Pro Leu Val Ala Arg
210 215 220
Glu Arg His Leu Asp Gln Gly Arg Gln Val Gln Ile Gln Pro Thr Glu
225 230 235 240
Pro Pro Ala Ser Glu Val Glu Asp Val Gly
245 250
<210> 1
<211> 750
<212> DNA
<213> 1型猪星状病毒(Porcine Astrovirus 1)
<400> 1
ggagaaacac cagtcacgtt taaggcctac cgcatgatgc cagaagatac catatatctt 60
agatttaagc ctgacaccct gagcgtagtc tcaaactttc aaccagcaaa gcggcctatg 120
cttgccaaaa cgtatagtgg tgacaccttg acagttgggc aggggaacaa taaaacagca 180
attcatactg ttgttagaat ttctgacccc acttggttta gtgctgactg ggaccctata 240
tctacacccc agccaattgc agagatttac tgtaaggcag gcaccacgac tgttggtgac 300
atcctcgcgg cctaccaggt acatgggctt gggaatcaca caaccacagc atatgttgtt 360
aggatgaccg ctggtgcaaa cccccaagtg tcagccggga ttgttactaa taaagggact 420
aatgattatg atttgaaaac cgctaattca aatgctggtt tctcatggaa tttaggatca 480
ggtacgtggt acctcatgat gtcctttggt gatgctttgg gtagtcttgg cacctggcgt 540
tggacaccca acgagttgtc tgccaactat acaatataca attgtgagat tataccatgt 600
cttctcttgg caaatgatga cttccacata gtgataccaa caaagaacgc attggttccg 660
cttgtggcgc gggagcgtca ccttgatcag ggccgacagg ttcagattca acccaccgaa 720
cctccagcct ctgaagtgga ggacgtaggt 750
<210> 3
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctggattca tgggagaaac accagtcacg ttaag 35
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
acaagcttac ctacgtcctc cacttcagag 30
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccggaattca tgggagaaac accagtc 27
<210> 6
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccgctcgagt taggacatca tgaggtacca c 31
<210> 7
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccggaattca tgccccagcc aattgc 26
<210> 8
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccgctcgagt taacctacgt cctccacttc ag 32
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccggaattca tgggagaaac accagtc 27
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccgctcgagt tagtcaccaa cagtcgtggt g 31
<210> 11
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccggaattca tgattcatac tgttgttag 29
<210> 12
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccgctcgagt taaccaaagg acatcatg 28
<210> 13
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccggaattca tgattcatac tgttgttag 29
<210> 14
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccgctcgagt taaagcccat gtacctgg 28
<210> 15
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ccggaattca tgtctacacc ccagccaatt g 31
<210> 16
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccgctcgagt tacacttggg ggtttgcac 29
<210> 17
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ccggaattca tgatcctcgc ggcctaccag g 31
<210> 18
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccgctcgagt taaccaaagg acatcatg 28
<210> 19
<211> 50
<212> PRT
<213> 1型猪星状病毒(Porcine Astrovirus 1)
<400> 19
Ser Thr Pro Gln Pro Ile Ala Glu Ile Tyr Cys Lys Ala Gly Thr Thr
1 5 10 15
Thr Val Gly Asp Ile Leu Ala Ala Tyr Gln Val His Gly Leu Gly Asn
20 25 30
His Thr Thr Thr Ala Tyr Val Val Arg Met Thr Ala Gly Ala Asn Pro
35 40 45
Gln Val
50
Claims (8)
1.一株杂交瘤细胞株,保藏编号为CCTCC NO:C2020107,分类命名Mus musculus B1F5。
2.权利要求1所述的杂交瘤细胞分泌的抗1型猪星状病毒衣壳蛋白的单克隆抗体B1F5。
3.根据权利要求2所述的单克隆抗体B1F5,其特征在于:所述单克隆抗体B1F5能与1型猪星状病毒衣壳蛋白抗原纤突结构域特异性结合。
4.根据权利要求3所述的单克隆抗体B1F5,其特征在于:所述单克隆抗体B1F5亚型为IgG1,κ型。
5.权利要求2所述单克隆抗体B1F5在生物诊断鉴定试剂盒中的应用。
6.根据权利要求5所述的应用,其特征在于:所述生物诊断针对1型猪星状病毒,生物诊断包括ELISA、Western Blot、IFA。
7.一种猪星状病毒生物诊断鉴定试剂盒,其特征在于包含权利要求2所述的单克隆抗体B1F5。
8.一种1型猪星状病毒衣壳蛋白抗原表位肽,其特征在于为序列表SEQ.ID.NO.19的氨基酸序列。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010872788.0A CN112048483B (zh) | 2020-08-26 | 2020-08-26 | 1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010872788.0A CN112048483B (zh) | 2020-08-26 | 2020-08-26 | 1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112048483A CN112048483A (zh) | 2020-12-08 |
CN112048483B true CN112048483B (zh) | 2022-09-02 |
Family
ID=73599287
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010872788.0A Active CN112048483B (zh) | 2020-08-26 | 2020-08-26 | 1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112048483B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113388632B (zh) * | 2021-06-29 | 2022-06-28 | 广西大学 | 牛星状病毒elisa抗体检测的引物、试剂盒及应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014153168A2 (en) * | 2013-03-14 | 2014-09-25 | The Trustees Of Columbia University In The City Of New York | Porcine astrovirus sequences and uses thereof |
CN111551750A (zh) * | 2020-06-17 | 2020-08-18 | 广西大学 | 猪星状病毒间接elisa检测试剂盒 |
-
2020
- 2020-08-26 CN CN202010872788.0A patent/CN112048483B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014153168A2 (en) * | 2013-03-14 | 2014-09-25 | The Trustees Of Columbia University In The City Of New York | Porcine astrovirus sequences and uses thereof |
CN111551750A (zh) * | 2020-06-17 | 2020-08-18 | 广西大学 | 猪星状病毒间接elisa检测试剂盒 |
Non-Patent Citations (1)
Title |
---|
Construction of a reverse genetic system for porcine astrovirus;Qin, Yifeng;《ARCHIVES OF VIROLOGY》;20180630;第163卷(第6期);第1511-1518页 * |
Also Published As
Publication number | Publication date |
---|---|
CN112048483A (zh) | 2020-12-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111153991A (zh) | 一种人SARS-CoV-2单克隆抗体及其制备方法和应用 | |
CN109796531B (zh) | 猪Delta冠状病毒N蛋白单克隆抗体及其抗原表位和应用 | |
CN112175086B (zh) | 一种抗猪流行性腹泻病毒nsp13蛋白的单克隆抗体及应用 | |
CN112552396B (zh) | 抗非洲猪瘟病毒p54蛋白单克隆抗体、制备方法及应用 | |
CN112961222B (zh) | 2019新型冠状病毒n蛋白线性表位肽和单克隆抗体及应用 | |
CN112940087B (zh) | 一种SARS-CoV和SARS-CoV-2共同抗原表位肽及其应用 | |
CN107058239B (zh) | 一种抗猪瘟病毒e2蛋白单克隆抗体细胞株及其应用 | |
CN113480642B (zh) | 抗非洲猪瘟病毒CD2v蛋白单克隆抗体、制备方法及应用 | |
CN112921005B (zh) | 一株杂交瘤细胞株及其产生的犬细小病毒vp2蛋白单克隆抗体和应用 | |
CN112812178A (zh) | PCV3 Cap蛋白抗原表位肽、抗PCV3 Cap蛋白的单克隆抗体及其制备方法和应用 | |
CN113416245A (zh) | 一种可结合SARS-CoV-2病毒RBD蛋白的中和抗体及其应用 | |
CN115724958A (zh) | 抗诺如病毒gⅱ基因组衣壳蛋白vp1的单克隆抗体及其应用 | |
CN112048483B (zh) | 1型PAstV衣壳蛋白的抗原表位、单克隆抗体及其制备 | |
CN105801691B (zh) | 一种hpv16e7单克隆抗体及其制备方法和应用 | |
CN116836270B (zh) | 一种抗蓝舌病毒vp7蛋白的单克隆抗体及制备方法与用途 | |
CN105753980B (zh) | 一种hpv18 e6单克隆抗体及其制备方法和应用 | |
CN114703147A (zh) | 一种抗SARS-CoV-2广谱中和性单克隆抗体及其杂交瘤细胞株、检测试剂盒和应用 | |
CN115991744A (zh) | 人鼻病毒的通用亲和表位多肽、抗体及其用途 | |
CN107619435B (zh) | 一种猪瘟病毒e2蛋白的抗原表位、抗体的制备及应用 | |
CN112458060B (zh) | 1型PAstV的单克隆抗体及其制备和ELISA应用 | |
CN113087790B (zh) | 抗非洲猪瘟p72蛋白单域抗体及应用 | |
CN116769019B (zh) | 一种ASFVp30蛋白单克隆抗体及其应用 | |
CN114805564B (zh) | 特异性识别SARS-CoV-2 S蛋白NTD区域的单克隆抗体及应用 | |
CN115786274B (zh) | 一种罗非鱼Igλ单克隆抗体及应用 | |
CN116854810A (zh) | 特异性结合猪圆环病毒3型抗原表位的单克隆抗体及其应用 |
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 |