CN113150136A - 新型冠状病毒n蛋白单克隆抗体的制备 - Google Patents
新型冠状病毒n蛋白单克隆抗体的制备 Download PDFInfo
- Publication number
- CN113150136A CN113150136A CN202110482613.3A CN202110482613A CN113150136A CN 113150136 A CN113150136 A CN 113150136A CN 202110482613 A CN202110482613 A CN 202110482613A CN 113150136 A CN113150136 A CN 113150136A
- Authority
- CN
- China
- Prior art keywords
- variable region
- chain variable
- protein
- seq
- novel coronavirus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title description 11
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000013604 expression vector Substances 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 9
- 239000002773 nucleotide Substances 0.000 claims description 31
- 125000003729 nucleotide group Chemical group 0.000 claims description 31
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 210000004027 cell Anatomy 0.000 claims description 18
- 230000014509 gene expression Effects 0.000 claims description 12
- 238000001976 enzyme digestion Methods 0.000 claims description 7
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims 10
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 12
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 12
- 238000012216 screening Methods 0.000 abstract description 12
- 238000004091 panning Methods 0.000 abstract description 6
- 101710141454 Nucleoprotein Proteins 0.000 description 32
- 239000000243 solution Substances 0.000 description 31
- 241000711573 Coronaviridae Species 0.000 description 20
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 18
- 239000007788 liquid Substances 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 238000005406 washing Methods 0.000 description 13
- 150000001413 amino acids Chemical group 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 239000011248 coating agent Substances 0.000 description 11
- 238000000576 coating method Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 229960000723 ampicillin Drugs 0.000 description 10
- 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 10
- 239000012528 membrane Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 239000000427 antigen Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 238000001042 affinity chromatography Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000003259 recombinant expression Methods 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 239000000499 gel Substances 0.000 description 6
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 108010037850 glycylvaline Proteins 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 229920001220 nitrocellulos Polymers 0.000 description 4
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 238000005507 spraying Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- AOHKLEBWKMKITA-IHRRRGAJSA-N Arg-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](CCCN=C(N)N)N AOHKLEBWKMKITA-IHRRRGAJSA-N 0.000 description 3
- QNFRBNZGVVKBNJ-PEFMBERDSA-N Asp-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N QNFRBNZGVVKBNJ-PEFMBERDSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 3
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 3
- NZGTYCMLUGYMCV-XUXIUFHCSA-N Ile-Lys-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N NZGTYCMLUGYMCV-XUXIUFHCSA-N 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- FEHQLKKBVJHSEC-SZMVWBNQSA-N Leu-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FEHQLKKBVJHSEC-SZMVWBNQSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- SPSSJSICDYYTQN-HJGDQZAQSA-N Met-Thr-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O SPSSJSICDYYTQN-HJGDQZAQSA-N 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 3
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 3
- BIBYEFRASCNLAA-CDMKHQONSA-N Thr-Phe-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 BIBYEFRASCNLAA-CDMKHQONSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- QUILOGWWLXMSAT-IHRRRGAJSA-N Tyr-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QUILOGWWLXMSAT-IHRRRGAJSA-N 0.000 description 3
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 3
- 238000003317 immunochromatography Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 108010044292 tryptophyltyrosine Proteins 0.000 description 3
- 108010073969 valyllysine Proteins 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 206010003445 Ascites Diseases 0.000 description 2
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 2
- WMLFFCRUSPNENW-ZLUOBGJFSA-N Asp-Ser-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O WMLFFCRUSPNENW-ZLUOBGJFSA-N 0.000 description 2
- 241001678559 COVID-19 virus Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SZQCDCKIGWQAQN-FXQIFTODSA-N Cys-Arg-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O SZQCDCKIGWQAQN-FXQIFTODSA-N 0.000 description 2
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 2
- 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 2
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 2
- GBYYQVBXFVDJPJ-WLTAIBSBSA-N Gly-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)CN)O GBYYQVBXFVDJPJ-WLTAIBSBSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- HXWALXSAVBLTPK-NUTKFTJISA-N Leu-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(C)C)N HXWALXSAVBLTPK-NUTKFTJISA-N 0.000 description 2
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 2
- VOOINLQYUZOREH-SRVKXCTJSA-N Met-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCSC)N VOOINLQYUZOREH-SRVKXCTJSA-N 0.000 description 2
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 2
- HJEBZBMOTCQYDN-ACZMJKKPSA-N Ser-Glu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HJEBZBMOTCQYDN-ACZMJKKPSA-N 0.000 description 2
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 2
- GJFYFGOEWLDQGW-GUBZILKMSA-N Ser-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GJFYFGOEWLDQGW-GUBZILKMSA-N 0.000 description 2
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 2
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 2
- 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 2
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 2
- JVTHIXKSVYEWNI-JRQIVUDYSA-N Thr-Asn-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JVTHIXKSVYEWNI-JRQIVUDYSA-N 0.000 description 2
- 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 2
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 2
- HUPLKEHTTQBXSC-YJRXYDGGSA-N Thr-Ser-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HUPLKEHTTQBXSC-YJRXYDGGSA-N 0.000 description 2
- YOPQYBJJNSIQGZ-JNPHEJMOSA-N Thr-Tyr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 YOPQYBJJNSIQGZ-JNPHEJMOSA-N 0.000 description 2
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 2
- 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 2
- UOXPLPBMEPLZBW-WDSOQIARSA-N Trp-Val-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 UOXPLPBMEPLZBW-WDSOQIARSA-N 0.000 description 2
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 210000003800 pharynx Anatomy 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- 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 1
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 1
- 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 1
- VCSABYLVNWQYQE-SRVKXCTJSA-N Ala-Lys-Lys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O VCSABYLVNWQYQE-SRVKXCTJSA-N 0.000 description 1
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 1
- REAQAWSENITKJL-DDWPSWQVSA-N Ala-Met-Asp-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O REAQAWSENITKJL-DDWPSWQVSA-N 0.000 description 1
- AWNAEZICPNGAJK-FXQIFTODSA-N Ala-Met-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O AWNAEZICPNGAJK-FXQIFTODSA-N 0.000 description 1
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 1
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 1
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 1
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 1
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 1
- JJHBEVZAZXZREW-LFSVMHDDSA-N Ala-Thr-Phe Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O JJHBEVZAZXZREW-LFSVMHDDSA-N 0.000 description 1
- QOIGKCBMXUCDQU-KDXUFGMBSA-N Ala-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N)O QOIGKCBMXUCDQU-KDXUFGMBSA-N 0.000 description 1
- PNIGSVZJNVUVJA-BQBZGAKWSA-N Arg-Gly-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O PNIGSVZJNVUVJA-BQBZGAKWSA-N 0.000 description 1
- JEOCWTUOMKEEMF-RHYQMDGZSA-N Arg-Leu-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JEOCWTUOMKEEMF-RHYQMDGZSA-N 0.000 description 1
- ISJWBVIYRBAXEB-CIUDSAMLSA-N Arg-Ser-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISJWBVIYRBAXEB-CIUDSAMLSA-N 0.000 description 1
- LFWOQHSQNCKXRU-UFYCRDLUSA-N Arg-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 LFWOQHSQNCKXRU-UFYCRDLUSA-N 0.000 description 1
- SLKLLQWZQHXYSV-CIUDSAMLSA-N Asn-Ala-Lys Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O SLKLLQWZQHXYSV-CIUDSAMLSA-N 0.000 description 1
- NUHQMYUWLUSRJX-BIIVOSGPSA-N Asn-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N NUHQMYUWLUSRJX-BIIVOSGPSA-N 0.000 description 1
- PPMTUXJSQDNUDE-CIUDSAMLSA-N Asn-Glu-Arg Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PPMTUXJSQDNUDE-CIUDSAMLSA-N 0.000 description 1
- HDHZCEDPLTVHFZ-GUBZILKMSA-N Asn-Leu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O HDHZCEDPLTVHFZ-GUBZILKMSA-N 0.000 description 1
- WLVLIYYBPPONRJ-GCJQMDKQSA-N Asn-Thr-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O WLVLIYYBPPONRJ-GCJQMDKQSA-N 0.000 description 1
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 1
- YSYTWUMRHSFODC-QWRGUYRKSA-N Asn-Tyr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O YSYTWUMRHSFODC-QWRGUYRKSA-N 0.000 description 1
- CBWCQCANJSGUOH-ZKWXMUAHSA-N Asn-Val-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O CBWCQCANJSGUOH-ZKWXMUAHSA-N 0.000 description 1
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 1
- YNCHFVRXEQFPBY-BQBZGAKWSA-N Asp-Gly-Arg Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N YNCHFVRXEQFPBY-BQBZGAKWSA-N 0.000 description 1
- TZOZNVLBTAFJRW-UGYAYLCHSA-N Asp-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N TZOZNVLBTAFJRW-UGYAYLCHSA-N 0.000 description 1
- KQBVNNAPIURMPD-PEFMBERDSA-N Asp-Ile-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O KQBVNNAPIURMPD-PEFMBERDSA-N 0.000 description 1
- WNGZKSVJFDZICU-XIRDDKMYSA-N Asp-Leu-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(=O)O)N WNGZKSVJFDZICU-XIRDDKMYSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 1
- GCACQYDBDHRVGE-LKXGYXEUSA-N Asp-Thr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC(O)=O GCACQYDBDHRVGE-LKXGYXEUSA-N 0.000 description 1
- ITGFVUYOLWBPQW-KKHAAJSZSA-N Asp-Thr-Val 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)C)C(O)=O ITGFVUYOLWBPQW-KKHAAJSZSA-N 0.000 description 1
- AWPWHMVCSISSQK-QWRGUYRKSA-N Asp-Tyr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O AWPWHMVCSISSQK-QWRGUYRKSA-N 0.000 description 1
- 102100031673 Corneodesmosin Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- MBILEVLLOHJZMG-FXQIFTODSA-N Cys-Gln-Glu Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CS)N MBILEVLLOHJZMG-FXQIFTODSA-N 0.000 description 1
- OZHXXYOHPLLLMI-CIUDSAMLSA-N Cys-Lys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OZHXXYOHPLLLMI-CIUDSAMLSA-N 0.000 description 1
- CMYVIUWVYHOLRD-ZLUOBGJFSA-N Cys-Ser-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CMYVIUWVYHOLRD-ZLUOBGJFSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-N 0.000 description 1
- ZNZPKVQURDQFFS-FXQIFTODSA-N Gln-Glu-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZNZPKVQURDQFFS-FXQIFTODSA-N 0.000 description 1
- FGYPOQPQTUNESW-IUCAKERBSA-N Gln-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N FGYPOQPQTUNESW-IUCAKERBSA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- PDXIOFXRBVDSHD-JBACZVJFSA-N Gln-Phe-Trp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)NC(=O)[C@H](CCC(=O)N)N PDXIOFXRBVDSHD-JBACZVJFSA-N 0.000 description 1
- SYZZMPFLOLSMHL-XHNCKOQMSA-N Gln-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)C(=O)O SYZZMPFLOLSMHL-XHNCKOQMSA-N 0.000 description 1
- DYFJZDDQPNIPAB-NHCYSSNCSA-N Glu-Arg-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O DYFJZDDQPNIPAB-NHCYSSNCSA-N 0.000 description 1
- CKRUHITYRFNUKW-WDSKDSINSA-N Glu-Asn-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CKRUHITYRFNUKW-WDSKDSINSA-N 0.000 description 1
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 1
- PAQUJCSYVIBPLC-AVGNSLFASA-N Glu-Asp-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PAQUJCSYVIBPLC-AVGNSLFASA-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
- DWBBKNPKDHXIAC-SRVKXCTJSA-N Glu-Leu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCC(O)=O DWBBKNPKDHXIAC-SRVKXCTJSA-N 0.000 description 1
- SJJHXJDSNQJMMW-SRVKXCTJSA-N Glu-Lys-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O SJJHXJDSNQJMMW-SRVKXCTJSA-N 0.000 description 1
- MFNUFCFRAZPJFW-JYJNAYRXSA-N Glu-Lys-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MFNUFCFRAZPJFW-JYJNAYRXSA-N 0.000 description 1
- JVZLZVJTIXVIHK-SXNHZJKMSA-N Glu-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N JVZLZVJTIXVIHK-SXNHZJKMSA-N 0.000 description 1
- UCZXXMREFIETQW-AVGNSLFASA-N Glu-Tyr-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O UCZXXMREFIETQW-AVGNSLFASA-N 0.000 description 1
- YQPFCZVKMUVZIN-AUTRQRHGSA-N Glu-Val-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQPFCZVKMUVZIN-AUTRQRHGSA-N 0.000 description 1
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 1
- ADZGCWWDPFDHCY-ZETCQYMHSA-N Gly-His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 ADZGCWWDPFDHCY-ZETCQYMHSA-N 0.000 description 1
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 1
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 1
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- JSLVAHYTAJJEQH-QWRGUYRKSA-N Gly-Ser-Phe Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JSLVAHYTAJJEQH-QWRGUYRKSA-N 0.000 description 1
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- 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 1
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 1
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 1
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 1
- FOKISINOENBSDM-WLTAIBSBSA-N Gly-Thr-Tyr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FOKISINOENBSDM-WLTAIBSBSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- WTUSRDZLLWGYAT-KCTSRDHCSA-N Gly-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)CN WTUSRDZLLWGYAT-KCTSRDHCSA-N 0.000 description 1
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 1
- DNAZKGFYFRGZIH-QWRGUYRKSA-N Gly-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 DNAZKGFYFRGZIH-QWRGUYRKSA-N 0.000 description 1
- NGBGZCUWFVVJKC-IRXDYDNUSA-N Gly-Tyr-Tyr Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 NGBGZCUWFVVJKC-IRXDYDNUSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101710114810 Glycoprotein Proteins 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
- QPSCMXDWVKWVOW-BZSNNMDCSA-N His-His-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QPSCMXDWVKWVOW-BZSNNMDCSA-N 0.000 description 1
- ZHHLTWUOWXHVQJ-YUMQZZPRSA-N His-Ser-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZHHLTWUOWXHVQJ-YUMQZZPRSA-N 0.000 description 1
- CWJQMCPYXNVMBS-STECZYCISA-N Ile-Arg-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N CWJQMCPYXNVMBS-STECZYCISA-N 0.000 description 1
- YKRIXHPEIZUDDY-GMOBBJLQSA-N Ile-Asn-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKRIXHPEIZUDDY-GMOBBJLQSA-N 0.000 description 1
- NPAYJTAXWXJKLO-NAKRPEOUSA-N Ile-Met-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)O)N NPAYJTAXWXJKLO-NAKRPEOUSA-N 0.000 description 1
- JODPUDMBQBIWCK-GHCJXIJMSA-N Ile-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O JODPUDMBQBIWCK-GHCJXIJMSA-N 0.000 description 1
- FBGXMKUWQFPHFB-JBDRJPRFSA-N Ile-Ser-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N FBGXMKUWQFPHFB-JBDRJPRFSA-N 0.000 description 1
- FXJLRZFMKGHYJP-CFMVVWHZSA-N Ile-Tyr-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N FXJLRZFMKGHYJP-CFMVVWHZSA-N 0.000 description 1
- ZGKVPOSSTGHJAF-HJPIBITLSA-N Ile-Tyr-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CO)C(=O)O)N ZGKVPOSSTGHJAF-HJPIBITLSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 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 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
- USTCFDAQCLDPBD-XIRDDKMYSA-N Leu-Asn-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N USTCFDAQCLDPBD-XIRDDKMYSA-N 0.000 description 1
- XVSJMWYYLHPDKY-DCAQKATOSA-N Leu-Asp-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O XVSJMWYYLHPDKY-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 1
- FAELBUXXFQLUAX-AJNGGQMLSA-N Leu-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C FAELBUXXFQLUAX-AJNGGQMLSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- 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 1
- LCNASHSOFMRYFO-WDCWCFNPSA-N Leu-Thr-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O LCNASHSOFMRYFO-WDCWCFNPSA-N 0.000 description 1
- 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 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- VSRXPEHZMHSFKU-IUCAKERBSA-N Lys-Gln-Gly Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O VSRXPEHZMHSFKU-IUCAKERBSA-N 0.000 description 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 1
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 1
- NCZIQZYZPUPMKY-PPCPHDFISA-N Lys-Ile-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NCZIQZYZPUPMKY-PPCPHDFISA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- WGILOYIKJVQUPT-DCAQKATOSA-N Lys-Pro-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WGILOYIKJVQUPT-DCAQKATOSA-N 0.000 description 1
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 1
- DIBZLYZXTSVGLN-CIUDSAMLSA-N Lys-Ser-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O DIBZLYZXTSVGLN-CIUDSAMLSA-N 0.000 description 1
- YCJCEMKOZOYBEF-OEAJRASXSA-N Lys-Thr-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YCJCEMKOZOYBEF-OEAJRASXSA-N 0.000 description 1
- UGCIQUYEJIEHKX-GVXVVHGQSA-N Lys-Val-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O UGCIQUYEJIEHKX-GVXVVHGQSA-N 0.000 description 1
- 101710145006 Lysis protein Proteins 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- UYAKZHGIPRCGPF-CIUDSAMLSA-N Met-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCSC)N UYAKZHGIPRCGPF-CIUDSAMLSA-N 0.000 description 1
- SJDQOYTYNGZZJX-SRVKXCTJSA-N Met-Glu-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SJDQOYTYNGZZJX-SRVKXCTJSA-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
- 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
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- WYBVBIHNJWOLCJ-UHFFFAOYSA-N N-L-arginyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCCN=C(N)N WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- NOFBJKKOPKJDCO-KKXDTOCCSA-N Phe-Ala-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NOFBJKKOPKJDCO-KKXDTOCCSA-N 0.000 description 1
- CUMXHKAOHNWRFQ-BZSNNMDCSA-N Phe-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 CUMXHKAOHNWRFQ-BZSNNMDCSA-N 0.000 description 1
- FSPGBMWPNMRWDB-AVGNSLFASA-N Phe-Cys-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N FSPGBMWPNMRWDB-AVGNSLFASA-N 0.000 description 1
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 1
- BIYWZVCPZIFGPY-QWRGUYRKSA-N Phe-Gly-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BIYWZVCPZIFGPY-QWRGUYRKSA-N 0.000 description 1
- WEDZFLRYSIDIRX-IHRRRGAJSA-N Phe-Ser-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=CC=C1 WEDZFLRYSIDIRX-IHRRRGAJSA-N 0.000 description 1
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 1
- JXQVYPWVGUOIDV-MXAVVETBSA-N Phe-Ser-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JXQVYPWVGUOIDV-MXAVVETBSA-N 0.000 description 1
- UNBFGVQVQGXXCK-KKUMJFAQSA-N Phe-Ser-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O UNBFGVQVQGXXCK-KKUMJFAQSA-N 0.000 description 1
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 1
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- HFZNNDWPHBRNPV-KZVJFYERSA-N Pro-Ala-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HFZNNDWPHBRNPV-KZVJFYERSA-N 0.000 description 1
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 1
- UUHXBJHVTVGSKM-BQBZGAKWSA-N Pro-Gly-Asn Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O UUHXBJHVTVGSKM-BQBZGAKWSA-N 0.000 description 1
- VYWNORHENYEQDW-YUMQZZPRSA-N Pro-Gly-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 VYWNORHENYEQDW-YUMQZZPRSA-N 0.000 description 1
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 1
- AFXCXDQNRXTSBD-FJXKBIBVSA-N Pro-Gly-Thr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O AFXCXDQNRXTSBD-FJXKBIBVSA-N 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- LGMBKOAPPTYKLC-JYJNAYRXSA-N Pro-Phe-Arg Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(=N)N)C(O)=O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 LGMBKOAPPTYKLC-JYJNAYRXSA-N 0.000 description 1
- LNICFEXCAHIJOR-DCAQKATOSA-N Pro-Ser-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LNICFEXCAHIJOR-DCAQKATOSA-N 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- HBZBPFLJNDXRAY-FXQIFTODSA-N Ser-Ala-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O HBZBPFLJNDXRAY-FXQIFTODSA-N 0.000 description 1
- QEDMOZUJTGEIBF-FXQIFTODSA-N Ser-Arg-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O QEDMOZUJTGEIBF-FXQIFTODSA-N 0.000 description 1
- WDXYVIIVDIDOSX-DCAQKATOSA-N Ser-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CCCN=C(N)N WDXYVIIVDIDOSX-DCAQKATOSA-N 0.000 description 1
- TUYBIWUZWJUZDD-ACZMJKKPSA-N Ser-Cys-Gln Chemical compound OC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCC(N)=O TUYBIWUZWJUZDD-ACZMJKKPSA-N 0.000 description 1
- KMWFXJCGRXBQAC-CIUDSAMLSA-N Ser-Cys-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N KMWFXJCGRXBQAC-CIUDSAMLSA-N 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- SQBLRDDJTUJDMV-ACZMJKKPSA-N Ser-Glu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SQBLRDDJTUJDMV-ACZMJKKPSA-N 0.000 description 1
- 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 1
- ZOPISOXXPQNOCO-SVSWQMSJSA-N Ser-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CO)N ZOPISOXXPQNOCO-SVSWQMSJSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 1
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 1
- NUEHQDHDLDXCRU-GUBZILKMSA-N Ser-Pro-Arg Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NUEHQDHDLDXCRU-GUBZILKMSA-N 0.000 description 1
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 1
- CUXJENOFJXOSOZ-BIIVOSGPSA-N Ser-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CO)N)C(=O)O CUXJENOFJXOSOZ-BIIVOSGPSA-N 0.000 description 1
- JURQXQBJKUHGJS-UHFFFAOYSA-N Ser-Ser-Ser-Ser Chemical compound OCC(N)C(=O)NC(CO)C(=O)NC(CO)C(=O)NC(CO)C(O)=O JURQXQBJKUHGJS-UHFFFAOYSA-N 0.000 description 1
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 206010042566 Superinfection Diseases 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- NJEMRSFGDNECGF-GCJQMDKQSA-N Thr-Ala-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O NJEMRSFGDNECGF-GCJQMDKQSA-N 0.000 description 1
- 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 1
- TWLMXDWFVNEFFK-FJXKBIBVSA-N Thr-Arg-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O TWLMXDWFVNEFFK-FJXKBIBVSA-N 0.000 description 1
- NAXBBCLCEOTAIG-RHYQMDGZSA-N Thr-Arg-Lys Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O NAXBBCLCEOTAIG-RHYQMDGZSA-N 0.000 description 1
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 1
- PAXANSWUSVPFNK-IUKAMOBKSA-N Thr-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N PAXANSWUSVPFNK-IUKAMOBKSA-N 0.000 description 1
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 1
- NCXVJIQMWSGRHY-KXNHARMFSA-N Thr-Leu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O NCXVJIQMWSGRHY-KXNHARMFSA-N 0.000 description 1
- YOOAQCZYZHGUAZ-KATARQTJSA-N Thr-Leu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YOOAQCZYZHGUAZ-KATARQTJSA-N 0.000 description 1
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 1
- KPNSNVTUVKSBFL-ZJDVBMNYSA-N Thr-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KPNSNVTUVKSBFL-ZJDVBMNYSA-N 0.000 description 1
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 1
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 1
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 1
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- GQHAIUPYZPTADF-FDARSICLSA-N Trp-Ile-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 GQHAIUPYZPTADF-FDARSICLSA-N 0.000 description 1
- YTCNLMSUXPCFBW-SXNHZJKMSA-N Trp-Ile-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O YTCNLMSUXPCFBW-SXNHZJKMSA-N 0.000 description 1
- KCZGSXPFPNKGLE-WDSOQIARSA-N Trp-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N KCZGSXPFPNKGLE-WDSOQIARSA-N 0.000 description 1
- UJGDFQRPYGJBEH-AAEUAGOBSA-N Trp-Ser-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N UJGDFQRPYGJBEH-AAEUAGOBSA-N 0.000 description 1
- DKKHULUSOSWGHS-UWJYBYFXSA-N Tyr-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N DKKHULUSOSWGHS-UWJYBYFXSA-N 0.000 description 1
- QOIKZODVIPOPDD-AVGNSLFASA-N Tyr-Cys-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QOIKZODVIPOPDD-AVGNSLFASA-N 0.000 description 1
- SLCSPPCQWUHPPO-JYJNAYRXSA-N Tyr-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 SLCSPPCQWUHPPO-JYJNAYRXSA-N 0.000 description 1
- AZZLDIDWPZLCCW-ZEWNOJEFSA-N Tyr-Ile-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O AZZLDIDWPZLCCW-ZEWNOJEFSA-N 0.000 description 1
- UPODKYBYUBTWSV-BZSNNMDCSA-N Tyr-Phe-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=C(O)C=C1 UPODKYBYUBTWSV-BZSNNMDCSA-N 0.000 description 1
- YYLHVUCSTXXKBS-IHRRRGAJSA-N Tyr-Pro-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YYLHVUCSTXXKBS-IHRRRGAJSA-N 0.000 description 1
- VPEFOFYNHBWFNQ-UFYCRDLUSA-N Tyr-Pro-Tyr Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 VPEFOFYNHBWFNQ-UFYCRDLUSA-N 0.000 description 1
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 1
- PWKMJDQXKCENMF-MEYUZBJRSA-N Tyr-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O PWKMJDQXKCENMF-MEYUZBJRSA-N 0.000 description 1
- RZAGEHHVNYESNR-RNXOBYDBSA-N Tyr-Trp-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RZAGEHHVNYESNR-RNXOBYDBSA-N 0.000 description 1
- GPLTZEMVOCZVAV-UFYCRDLUSA-N Tyr-Tyr-Arg Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=C(O)C=C1 GPLTZEMVOCZVAV-UFYCRDLUSA-N 0.000 description 1
- KSGKJSFPWSMJHK-JNPHEJMOSA-N Tyr-Tyr-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KSGKJSFPWSMJHK-JNPHEJMOSA-N 0.000 description 1
- JIODCDXKCJRMEH-NHCYSSNCSA-N Val-Arg-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N JIODCDXKCJRMEH-NHCYSSNCSA-N 0.000 description 1
- BMGOFDMKDVVGJG-NHCYSSNCSA-N Val-Asp-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BMGOFDMKDVVGJG-NHCYSSNCSA-N 0.000 description 1
- BCBFMJYTNKDALA-UFYCRDLUSA-N Val-Phe-Phe Chemical compound N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O BCBFMJYTNKDALA-UFYCRDLUSA-N 0.000 description 1
- JQTYTBPCSOAZHI-FXQIFTODSA-N Val-Ser-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N JQTYTBPCSOAZHI-FXQIFTODSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- 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 1
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 1
- OWFGFHQMSBTKLX-UFYCRDLUSA-N Val-Tyr-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N OWFGFHQMSBTKLX-UFYCRDLUSA-N 0.000 description 1
- AOILQMZPNLUXCM-AVGNSLFASA-N Val-Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN AOILQMZPNLUXCM-AVGNSLFASA-N 0.000 description 1
- STTYIMSDIYISRG-UHFFFAOYSA-N Valyl-Serine Chemical compound CC(C)C(N)C(=O)NC(CO)C(O)=O STTYIMSDIYISRG-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- -1 dried Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 108010010096 glycyl-glycyl-tyrosine Proteins 0.000 description 1
- 108010033719 glycyl-histidyl-glycine Proteins 0.000 description 1
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 1
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- ZBKIUFWVEIBQRT-UHFFFAOYSA-N gold(1+) Chemical compound [Au+] ZBKIUFWVEIBQRT-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 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
- 229930182823 kanamycin A Natural products 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 108010022588 methionyl-lysyl-proline Proteins 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010072637 phenylalanyl-arginyl-phenylalanine Proteins 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 1
- 108010079317 prolyl-tyrosine Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 108091069025 single-strand RNA Proteins 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 1
- 108010077037 tyrosyl-tyrosyl-phenylalanine Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 108010072644 valyl-alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明属于生物技术领域。本发明提供了一种重组蛋白，该重组蛋白氨基酸序列由新型冠状病毒N蛋白两个优势表位重复串联组成。本发明还涉及用该重组蛋白免疫小鼠建立噬菌体库，经淘选筛选得到对应新型冠状病毒N蛋白单链抗体scfv序列，将得到的scfv序列构建成完整鼠IgG1抗体序列表达载体，通过瞬转HEK293F细胞表达单克隆抗体，纯化单克隆抗体，通过胶体金正交实验确定两对最优单克隆抗体配对组合。
Description
技术领域
本发明属于生物技术领域。具体的说，本发明表达了一种新型冠状病毒N重组蛋白，涉及使用上述重组蛋白免疫小鼠建立噬菌体库，筛选得到特异性单链抗体scfv序列，还涉及将得到的scfv序列构建成真核表达载体表达新型冠状病毒N蛋白单克隆抗体，并应用于新型冠状病毒的感染诊断。
背景技术
2019新型冠状病毒，2020年1月12日，世界卫生组织正式将其命名为2019-nCoV。冠状病毒是一个大型病毒家族，在系统分类上属冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)。冠状病毒属的病毒是具外套膜(envelope)的正链单股RNA病毒，直径约80～120nm，其遗传物质是所有RNA病毒中最大的，只感染人、鼠、猪、猫、犬、禽类脊椎动物。冠状病毒粒子呈不规则形状，其蛋白分为：刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白)，和核衣壳蛋白(N蛋白)。N蛋白与病毒基因组RNA相互缠绕形成病毒核衣壳，在病毒RNA的合成过程中发挥着重要作用。同时，N蛋白相对保守，在病毒的结构蛋白中所占比例最大，感染早期机体就能产生抗N蛋白的高水平抗体。
目前，新型冠状病毒检测以核酸分子检测为主，需要从痰液、咽拭子、肺泡灌洗液等样本中提取核酸分子，然后再采用荧光PCR方法进行检测，总共需要3个小时左右。该方法虽然准确度高，但是需要专门的操作场地、专业的操作人员和设备，检测时间太长，要求较为苛刻，无法在基层医疗机构大面积使用。而新型冠状病毒疫情爆发后，疑似患者大量增加，急需一种检测产品能在短时间内快速鉴别筛查，而不仅仅是确诊。
免疫学检测方法因其操作简便，结果易于判定，适用于大量样本现场快速筛查等特点而得到非常广泛的应用。该方法主要是通过制备新型冠状病毒N蛋白的单克隆抗体来实现对新型冠状病毒的特异性检测。常规新型冠状病毒N蛋白单克隆抗体制备是将新型冠状病毒N蛋白单克隆细胞株制备Balb/c小鼠腹水，使用Protein A亲和层析法纯化单克隆抗体。但由于单只小鼠腹水产量不确定性且个体差异大，得到的新型冠状病毒N蛋白单克隆抗体批间差异大，使得检测准确性较差。
发明内容
设计目的：为解决传统制备单克隆抗体的不足之处，通过设计、合成重组新型冠状病毒N蛋白并通过建立噬菌体库和真核细胞表达来制备其单克隆抗体，比传统单克隆抗体制备大大缩短了时间，并且得到的单克隆抗体稳定性高，均一性好，大大减小了批间差异。
设计方案：为了实现上述设计目的。本申请：(1)以新型冠状病毒N蛋白为靶抗原，分析并选择该抗原两个优势抗原表位，序列比较结果显示所选择的两个抗原表位与其它蛋白序列无明显同源性。(2)为增强免疫效果并缩短单克隆抗体制备时间，将所选择的两个优势抗原表位串联，并在序列碳端加His标签，得到重组抗原氨基酸序列，将重组蛋白氨基酸序列转换为对应核苷酸序列。(3)化学合成上一步骤得到的核苷酸序列，并通过酶切连接，将合成得到的核苷酸片段插入表达载体pET-32a(+)，构建重组N蛋白表达载体。(4)重组N蛋白表达载体转化大肠杆菌ER2566感受态细胞，筛选得到重组蛋白表达菌株。(5)重组蛋白表达菌株大规模培养后，经超声破菌并低温离心，取溶液上清通过镍琼脂糖亲和层析柱亲和层析，洗脱得到纯化重组N蛋白。(6)重组N蛋白多次免疫Balb/c小鼠后，取其脾脏分离淋巴细胞用来建立单链抗体scfv噬菌体展示库。(7)将步骤(3)合成得到的核苷酸片段插入表达载体pTT5，构建重组N蛋白表达载体。(8)重组N蛋白表达载体转化大肠杆菌DH5α感受态细胞，筛选鉴定得到重组表达质粒。(9)重组表达质粒瞬转CHO细胞，7天后收集细胞上清，上清通过镍琼脂糖亲和层析纯化得到真核表达重组N蛋白。(10)使用真核表达重组N蛋白对单链抗体scfv噬菌体展示库进行多轮淘选筛选最终得到能与真核表达重组N蛋白结合的单链抗体scfv序列。(11)将scfv序列构建成完整鼠IgG1表达载体并使用HEK293F细胞表达单克隆抗体，使用Protein A亲和层析法纯化单克隆抗体，并分别标记胶体金颗粒。(12)利用胶体金免疫层析平台筛选显示3A2单抗包被与4B9胶体金标记单抗配对,2C6单抗包被与6D8胶体金标记单抗配对为检测新型冠状病毒N蛋白的两个优势组合。
具体实施方式
以下实施例虽然对本发明的设计思路作了比较详细的文字描述，但是这些文字描述，只是对本发明设计思路的简单文字描述，而不是对本发明设计思路的限制，任何不超出本发明设计思路的组合、增加或修改，均落入到本发明的保护范围内。
实施例1：新型冠状病毒N蛋白优势抗原表位选择和串联
以新型冠状病毒N蛋白为靶抗原，利用生物软件DNAssist2.0分析其抗原表位序列的亲水性及抗原性，选择A优势抗原表位和B优势抗原表位。序列比较结果显示所选择的A、B两个优势抗原表位序列与其它蛋白序列无明显同源性。将新型冠状病毒N蛋白的A、B两个优势抗原表位序列分别重复后再通过柔性片段(连续四个甘氨酸)连接，并在序列碳端加His标签，得到重组蛋白氨基酸序列。
实施例2：构建重组N蛋白表达载体
将编码重组蛋白的氨基酸序列转化为对应核苷酸序列，并在其上下游分别添加酶切位点BamHI和EcoRI对应的核苷酸序列后，由通用生物系统安徽有限公司合成。合成后的目的基因克隆于pMD25-T载体(宝生物工程大连有限公司)中。将含目的基因的pMD25-T载体和pET-32a(+)载体(德国Novagen公司)通过限制性内切酶BamHI和EcoRI(宝生物工程大连有限公司)分别于37℃双酶切12小时，酶切产物分别进行1％琼脂糖凝胶电泳，并分别切胶回收目的基因和pET-32a(+)载体(本发明所使用的胶回收试剂盒均来自爱思进生物技术杭州有限公司)。使用T4连接酶(宝生物工程大连有限公司)将回收的目的基因和pET-32a(+)载体按一定的比例于4℃连接12小时后，连接产物转化DH5α感受态细胞(杭州贤至生物科技有限公司)，并涂布于含氨苄青霉素抗性(50μg/mL)的LB平板，于37℃恒温培养12小时后，于平板上挑取单克隆菌株至含氨苄青霉素抗性(50μg/mL)的LB液体培养基，37℃恒温摇床培养12小时后，采用质粒纯化试剂盒(本发明所使用的质粒纯化试剂盒均来自爱思进生物技术杭州有限公司)提取质粒，经BamHI和EcoRI双酶切鉴定后得到正确的重组表达载体。
实施例3：构建重组N蛋白表达菌株
将构建好的重组表达载体转化E.coli ER2566感受态细胞，并涂布于含氨苄青霉素抗性(50μg/mL)的LB平板，于37℃过夜培养。第二日，挑取平板上单克隆菌株至含氨苄青霉素抗性(50μg/mL)的LB液体培养基，37℃恒温摇床培养8小时后，加诱导剂异丙基硫代-β-D-半乳糖苷(终浓度为1.0mmol/L)诱导表达4个小时后制备蛋白电泳样品。10％聚丙烯酰胺凝胶电泳结果表明重组蛋白成功表达，得到重组N蛋白表达菌株。
实施例4：纯化新型冠状病毒重组N蛋白
接种重组N蛋白表达菌株至LB液体培养基，加氨苄青霉素至终浓度为50μg/mL，37℃恒温摇床培养8小时后，用含50μg/mL氨苄青霉素的LB液体培养基将该菌按1：100比例稀释后，分装至细菌培养瓶中，置37℃恒温摇床培养至OD600＝0.8，加诱导剂异丙基硫代-β-D-半乳糖苷至终浓度为1.0mmol/L，继续培养诱导4小时。离心收集菌体后，4度低温超声破菌，低温离心后取上清通过镍琼脂糖亲和层析柱，经洗涤、洗脱最终得到原核表达重组N蛋白。
实施例5：单链抗体scfv噬菌体库构建
取4-6周龄雌性Balb/c小鼠，基础免疫每只小鼠皮下多点注射弗氏完全佐剂乳化的100μg重组N蛋白，共400μl/只。20天后进行第二次加强免疫，方法为取80μg重组N蛋白用弗氏不完全佐剂乳化，共400μl/只，皮下多点注射。第三次加强免疫在15天以后，方法与第二次加强免疫相同。20天后，取120μg重组N蛋白腹腔加强注射，于72小时后，眼眶取血，并处死小鼠，取其脾脏用鼠脾脏淋巴细胞分离试剂盒(天津市灏洋生物制品科技有限责任公司)分离鼠脾脏淋巴细胞。用RNA提取试剂盒(天根生化科技有限公司)从分离的淋巴细胞中提取总RNA，用反转录试剂盒(Takara)反转录合成cDNA，用鼠源单链抗体scfv通用简并引物扩增重链可变区以及轻链可变区基因，PCR产物分别进行1％琼脂糖凝胶电泳，并分别切胶回收目的基因，回收的目的基因通过overlap PCR链接成scfv，PCR产物进行1％琼脂糖凝胶电泳，并切胶回收目的基因经NotI和SfiI酶切后使用T4连接酶和pCANTAB5e(北京宝科维食安生物技术有限公司)载体按一定比例于4℃连接12小时后，连接产物经胶回收试剂盒回收以去除里面的酶及缓冲物质，回收产物用细菌电转化仪(biorad)分多次电转入大肠杆菌TG1电转感受态，并涂布于含氨苄青霉素抗性(50μg/mL)及2％葡萄糖的2×YT-AG平板，于30℃恒温培养12小时后，取适量的2×YT培养基用无菌玻璃棒将平板上的菌落全部刮取下来并收集菌体悬液，此为构建好的噬菌体抗体库。
实施例6：真核表达新型冠状病毒重组N蛋白
用限制性内切酶EcoRI和BamHI(宝生物工程大连有限公司)于37℃分别双酶切含新型冠状病毒重组N蛋白目的基因的pMD25-T载体和pTT5载体12小时，酶切产物分别行1％琼脂糖凝胶电泳，并分别切胶回收目的基因和pTT5载体(本发明所使用的胶回收试剂盒均来自爱思进生物技术杭州有限公司),使用T4连接酶(宝生物工程大连有限公司)将回收的目的基因和pTT5载体按一定比例于4℃连接12小时后，连接产物转化DH5α感受态细胞(杭州贤至生物科技有限公司)，并涂布于含氨苄青霉素抗性(50μg/mL)的LB平板，于37℃恒温培养12小时之后，于平板上挑取单克隆菌株至含氨苄青霉素抗性(50μg/mL)的LB液体培养基，37℃恒温摇床培养12小时后，采用质粒纯化试剂盒(本发明所使用的质粒纯化试剂盒均来自于爱思进生物技术杭州有限公司)提取质粒，经EcoRI和BamHI双酶切鉴定后得到正确的重组表达载体。
将构建好的重组表达载体转染CHO-K1细胞。转染前一天将CHO-K1细胞按照1×106/ml的密度传代以保证转染时细胞活力，转染当天将细胞密度调整为2×106/ml进行转染。根据转染体系每毫升中加入3.2ug重组表达载体，再根据转染体系每毫升中加入4.8ug转染试剂PEI(Polyscience),边加边摇匀。37℃，6％二氧化碳摇床转速120rpm培养4小时后，加入1％500mM VPA(sigma)以及1％30g/L L-盐酸半胱氨酸(索莱宝生物科技有限公司)，32℃，6％二氧化碳摇床转速120rpm培养6天后离心收集上清通过镍琼脂糖亲和层析柱(常州天地人和生物科技有限公司),20mM咪唑溶液去除杂蛋白，300mM咪唑溶液洗脱目的蛋白，收液后，4℃静置30分钟，转至截留分子量为8kD-10kD的透析袋中，于PBS(10mmol/L,pH7.4)中过夜透析。透析后立即取出并分装，于-20℃保存备用。20mM咪唑配制：咪唑1.36g，加10mmol/L,pH7.4 PBS溶液溶解定容至1000mL。300mM咪唑配制：咪唑10.2g，加10mmol/L，pH7.4 PBS溶液溶解定容至500mL。
实施例7：单链抗体scfv的淘选及筛选
从噬菌体抗体库中取一定量的菌液接种到2×YT-AG培养液中使OD600为0.3。37℃，250rpm振荡1h左右，使OD600达0.5后加入辅助噬菌体M13K07超感染，感染比例为M13K07/TG1＝20:1。37℃，250rpm振荡1h后3300g，4℃离心10min沉淀细菌，小心移弃上清。重悬细菌到含氨苄青霉素抗性(50μg/mL)及卡那青霉素抗性(50μg/mL)的2×YT-AK培养基中，30℃，250rpm振荡培养过夜。次日，10800g，4℃离心20min沉淀细菌。上清移入干净离心管中，加入1/5体积的PEG/NaCl，混合后冰浴2h。10800g，4℃离心20min沉淀细胞，小心移弃上清，扣干，将沉淀重悬于PBS中，用0.45μm膜过滤去除细菌碎片用于淘选步骤。将真核表达的新型冠状病毒重组N蛋白用包被液稀释至8μg/ml包被免疫管(Thermo)，每个免疫管4ml，4℃过夜包被。次日，弃去包被液和未吸附的抗原，无菌PBST洗涤3次，每个免疫管加入封闭液5ml，37℃孵育2h。弃去封闭液，无菌PBST洗涤3次后将经PEG沉淀获得的噬菌体加入到免疫管中，每个免疫管加入4ml，37℃孵育1h。弃去免疫管中的液体，用无菌PBST洗涤10次再用无菌PBS洗涤10次后加入1ml 100mM三乙胺将结合的噬菌体洗脱下来，再立即加入500μl 1MTris-HCl，pH 7.4进行中和。将中和后的噬菌体加入到一定量处于对数生长期的TG1大肠杆菌中进行超感染，此为第一轮淘选富集过程。经过3轮淘选，新型冠状病毒N蛋白特异性scfv得以富集。将最后一轮洗脱中和后的噬菌体侵染TG1大肠杆菌后涂布于2×YT-AG平板上，于30℃恒温培养12小时后随机挑取400-600个单克隆菌落于96孔深孔板中，用2×YT-AG培养基37℃，250rpm振荡2h后加入一定量M13K07辅助噬菌体进行超感染，37℃，250rpm振荡1h后离心去掉上清加入含氨苄青霉素抗性(50μg/mL)及卡那青霉素抗性(50μg/mL)的2×YT-AK培养基30℃，250rpm过夜培养。第二天进行单克隆ELISA筛选，筛选步骤如下：
包被：用包被液稀释真核表达的新型冠状病毒重组N蛋白至终浓度为1μg/mL，100μL/孔加入酶标板(深圳金灿华实业有限公司)，4℃过夜后通过DEM-3型洗板机(中山大学达安基因股份有限公司)用洗涤液洗涤1次；
封闭：以200μL/孔加入封闭液，37℃封闭2h，通过洗板机用洗涤液洗涤1次；
加样：加过夜诱导表达的细菌培养上清及对照血清，100μL/孔，37℃孵育1h，通过洗板机用洗涤液洗涤3次；
加酶标抗体：以100μL/孔加入新鲜稀释兔抗M13噬菌体HRP酶标二抗(购自北京义翘神州生物技术有限公司)，37℃孵育30分钟后，通过洗板机用洗涤液洗涤4次；
加显色液：每孔加显色液A和显色液B各50μL，37℃避光显色10分钟；
终止反应：以50μL/孔加入2M H2SO4；
结果判定：在酶标仪上，于450nm处，空白孔校零后读取OD值。以免疫小鼠血清作为阳性对照。结果显示有12个阳性克隆OD值较高，经测序得到7株scfv序列，分别为3A2，2A5，4B9，2C6，6D8，7E6，5G9。相关溶液配方如下：
包被液：Na2CO3 1.5g，NaHCO3 2.9g，加ddH2O定容至1000mL(pH9.6)。
封闭液：Na2HPO4.12H2O 2.68g，NaH2PO4.2H2O 0.39g，NaCl 8.5g，20g牛血清白蛋白，加ddH2O定容至1000mL(pH7.4)。
洗涤液：Na2HPO4.12H2O 2.68g，NaH2PO4.2H2O 0.39g，NaCl 8.5g，Tween-200.5mL，加ddH2O定容至1000mL(pH7.4)。
显色液A：200mg TMB溶于100mL无水乙醇，加ddH2O定容至1000mL。
显色液B：柠檬酸2.1g，Na2HPO4.12H2O 71g，加ddH2O定容至1000mL。
使用时：1mL显色液A+1mL显色液B+0.4μL 30％H2O2
终止液：2M H2SO4，21.7mL浓H2SO4加ddH2O定容至1000mL。
实施例8：真核表达载体构建及HEK293F细胞瞬转表达和纯化
将7株新型冠状病毒N蛋白单链抗体scfv序列分别构建成完整鼠IgG1抗体序列，即将scfv中重链可变区与轻链可变区通过PCR分别与鼠IgG1重链恒定区和轻链恒定区桥接，再分别插入到pcDNA3.1(德国Novagen公司)质粒中。分别通过PEI将构建好的重链质粒与轻链质粒共转染HEK293F细胞，37℃，5％二氧化碳，120rpm细胞摇床表达7天后离心沉淀，收集上清过0.45μm滤器。用50mL平衡缓冲液PBS(pH7.4)平衡琼脂糖亲和介质Protein A层析柱(南京金斯瑞生物科技有限公司)至电脑核酸蛋白检测仪(上海沪西分析仪器厂有限公司)显示吸光度为0。上清上样后加PBS洗涤至吸光度为0，然后用0.1M甘氨酸(pH3.0)洗脱，收集流出液并加入500mM Tris-HCl(pH8.5)缓冲液中和至pH7.0左右，得到纯化的单克隆抗体3A2，2A5，4B9，2C6，6D8，7E6，5G9。
实施例9：标记N蛋白单克隆抗体胶体金垫的制备
取5ml 0.01％胶体金溶液加入0.2mol/L碳酸钾溶液10uL，充分混匀后加入50ug N蛋白单克隆抗体，混匀，室温静置30min后，加入500ul 10％BSA(牛血清白蛋白)溶液进行封闭，封闭处理30min后，离心(10000rpm/min、20min)，弃上清后，沉淀用500ul复溶液充分溶解。溶解后的金溶液采用喷金划膜仪(上海金标生物科技有限公司)按照10ul/cm均匀喷涂于6mm宽的玻纤上，后置于电热鼓风干燥箱(上海一恒科学仪器有限公司)中37℃鼓风干燥2小时。
相关溶液配方如下:
0.01％胶体金溶液：1％氯金酸溶液1ml，1％柠檬酸溶液1.4ml，加超纯水加热溶解反应并定容至100ml。
1％氯金酸溶液：AuCL3.HCl.4H2O粉末1g加超纯水溶解并定容至100ml。
1％柠檬酸三钠溶液：柠檬酸三钠1g加超纯水溶解并定容至100ml。
0.2mol/L碳酸钾溶液：碳酸钾27.64g，加超纯水溶解并定容至1000ml。
复溶液：Tris 6.057g溶解于800ml超纯水中，用适量HCL调节pH至8.0，加超纯水定容到1000ml。
实施例10：硝酸纤维素膜(NC膜)的制备
新型冠状病毒N蛋白单克隆抗体(3A2，2A5，4B9，2C6，6D8，7E6，5G9)分别经包被液稀释后(终浓度为1mg/ml)，通过喷金划膜仪(上海金标生物科技有限公司)按照1ul/cm将其均匀包被于硝酸纤维素膜(Sartorius)，此为T线。通过喷金划膜仪(上海金标生物科技有限公司)将羊抗鼠溶液(终浓度为1mg/ml)按照1ul/cm均匀包被于硝酸纤维素膜，此为C线。划膜包被结束后，将硝酸纤维素膜置于电热恒温培养箱(上海一恒科学仪器有限公司)37℃静置12小时。
实施例11：胶体金免疫层析检测卡的制备
组装试纸条：在PVC底板上依次搭接粘贴：(1)喷涂N蛋白单克隆抗体(3A2，2A5，4B9，2C6，6D8，7E6，5G9)作为检测区和羊抗鼠IgG作为质控区的NC膜；(2)喷涂有胶体金标记N蛋白单克隆抗体(3A2，2A5，4B9，2C6，6D8，7E6，5G9)的金垫；(3)样本垫为一种经过1％Tween-20处理的玻璃纤维膜；(4)吸水纸，组装完成后剪切成4mm的宽度，装上试剂卡条壳并压紧，即得胶体金免疫层析检测卡。
实施例12：配对单抗筛选
真核表达的新型冠状病毒重组N蛋白和正常人咽拭子样本，经裂解液处理后，70μL/孔上样，室温放置15min，通过胶体金层析读数仪(上海捷浩科学仪器有限公司)分别读值并计算P/N值(真核表达的新型冠状病毒重组N蛋白检测值与阴性样本检测值的比值)，详见表1。
表1配对单抗P/N值统计
通过上表可知，3A2单抗包被与4B9胶体金标记单抗配对,2C6单抗包被与6D8胶体金标记单抗配对为检测新型冠状病毒N蛋白的两个优势组合。
SEQ ID NO1：新型冠状病毒N蛋白特异性单链抗体scfv-3A2轻链可变区氨基酸序列；
SEQ ID NO2：新型冠状病毒N蛋白特异性单链抗体scfv-3A2重链可变区氨基酸序列；
SEQ ID NO3：新型冠状病毒N蛋白特异性单链抗体scfv-4B9轻链可变区氨基酸序列；
SEQ ID NO4：新型冠状病毒N蛋白特异性单链抗体scfv-4B9重链可变区氨基酸序列；
SEQ ID NO5：新型冠状病毒N蛋白特异性单链抗体scfv-2C6轻链可变区氨基酸序列；
SEQ ID NO6：新型冠状病毒N蛋白特异性单链抗体scfv-2C6重链可变区氨基酸序列；
SEQ ID NO7：新型冠状病毒N蛋白特异性单链抗体scfv-6D8轻链可变区氨基酸序列；
SEQ ID NO8：新型冠状病毒N蛋白特异性单链抗体scfv-6D8重链可变区氨基酸序列；
SEQ ID NO9：新型冠状病毒N蛋白特异性单链抗体scfv-3A2轻链可变区核苷酸序列；
SEQ ID NO10：新型冠状病毒N蛋白特异性单链抗体scfv-3A2重链可变区核苷酸序列；
SEQ ID NO11：新型冠状病毒N蛋白特异性单链抗体scfv-4B9轻链可变区核苷酸序列；
SEQ ID NO12：新型冠状病毒N蛋白特异性单链抗体scfv-4B9重链可变区核苷酸序列；
SEQ ID NO13：新型冠状病毒N蛋白特异性单链抗体scfv-2C6轻链可变区核苷酸序列；
SEQ ID NO14：新型冠状病毒N蛋白特异性单链抗体scfv-2C6重链可变区核苷酸序列；
SEQ ID NO15：新型冠状病毒N蛋白特异性单链抗体scfv-6D8轻链可变区核苷酸序列；
SEQ ID NO16：新型冠状病毒N蛋白特异性单链抗体scfv-6D8重链可变区核苷酸序列；
序列表
<110> 杭州贤至生物科技有限公司
<120> 新型冠状病毒N蛋白单克隆抗体的制备
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Arg Tyr Ile
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Asn Val Ala Pro Gly Val Pro Phe Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Arg Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Glu Trp Ser Gly Tyr Pro Tyr Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 2
<211> 114
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gln Glu Ser Gly Thr Glu Val Val Lys Pro Gly Ala Ser Val Lys Leu
1 5 10 15
Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr Asp Ile Asp Trp
20 25 30
Val Arg Gln Thr Pro Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Phe
35 40 45
Pro Gly Glu Gly Ser Thr Glu Tyr Asn Glu Lys Phe Lys Gly Arg Ala
50 55 60
Thr Leu Ser Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Thr
65 70 75 80
Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Asp
85 90 95
Tyr Tyr Arg Arg Tyr Phe Asp Leu Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser
<210> 3
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Phe Ala Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Thr Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Gly Thr Tyr Ser Cys Gln His His Tyr Ala Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 4
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Tyr Pro Ser Asp Gly Arg Thr Asn Tyr Asn Glu Arg Phe
50 55 60
Thr Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Phe Cys
85 90 95
Ala Lys Lys Asp Tyr Gly Val Tyr Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser
115
<210> 5
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Asn Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Arg Gly Asn Thr Leu Pro Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 6
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr
20 25 30
Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Leu Pro Gly Gly Gly Glu Thr Asn Tyr Tyr Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Tyr Gly Asn Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser
115 120
<210> 7
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Ile Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Pro Ser Leu Glu Asn Gly Val Ser Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 8
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Tyr
20 25 30
Ala Met Ser Trp Ile Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Ser Gly Gly Ser Phe Thr Tyr Tyr Pro Ala Thr Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Asp Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Arg Gly Gly Tyr Ser Pro Gly Asn Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser
115
<210> 9
<211> 321
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gacatcgagc tgacacagag ccctgccatt atgagcgcct ctcctggcga aagagtgaca 60
atgacatgct ccgccagctc ttctatcaga tacatctatt ggtaccagca gaaacctgga 120
tctagcccaa gactgctgat ctacgacacc agcaacgtgg cccctggcgt tccttttaga 180
ttcagcggct ccggcagcgg caccagctac agcctgacca tcaaccggat ggaagccgag 240
gacgccgcta catactactg ccaggagtgg tccggctacc cttacacctt cggcggcgga 300
accaaactgg aaatcaagcg g 321
<210> 10
<211> 342
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caagagagcg gcacagaggt ggtgaagcct ggagccagcg tcaagctgtc ttgtaaagcc 60
tctggatata tcttcaccag ctacgacatc gactgggtgc ggcagacccc tgagcagggc 120
ctggaatgga tcggctggat cttccccggc gagggcagca cagagtacaa cgagaagttc 180
aagggcagag ctacactgag cgtggacaag tccagcagca ccgcctacat ggaactgacc 240
agactgacat ctgaagatag cgccgtgtac ttttgcgcca gaggcgatta ctaccggaga 300
tacttcgacc tgtggggcca gggcaccacc gtgaccgtgt cc 342
<210> 11
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gatatccaga tgacccaaag ccctgcttct ctgtctgctt ccgtgggcga caccgtgacc 60
atcacctgta gagcctccga gaacatctac tcctacctgg cttggtacca gcagaagcag 120
ggcaagtctc ctcaactgct ggtgtacaac gccaagacat tcgccgaggg cgtgccttct 180
cgcttcagcg gttccggctc tggcacccag ttctccctga agatcaccag cctgcagcct 240
gaggattttg gcacctactc ctgccagcat cactacgcca caccttacac cttcggcggc 300
ggaacaaagc tggaaatcaa gcgg 324
<210> 12
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
caggtgcagc tgcagcagcc aggcaccgaa gtcgtgaaac ctggcgcttc tgtcaaggtg 60
tcctgcaagg cttctggcta caccttcacc tcctactgga tgcactgggt caagcagaga 120
cctggacaag gcctggaatg gatcggcgag atctaccctt ctgacggcag aaccaactac 180
aacgagcgct ttaccagaaa ggctaccctg accgtggaca agtcctcctc taccgcctac 240
atgcagctga gcagcctgac ctctgaggac tccgccgtgt tcttctgcgc caaaaaggac 300
tacggcgtgt atggcttcgc ctactggggc cagggcacac tggtcaccgt gtcc 354
<210> 13
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gacatccaga tgacccagac cacctcctct ctgtccgcta gcctgggcga cagagtgacc 60
atcaattgca gggcctctca ggacatcagc aactacctga actggtacca gcagaagcct 120
gatggcaccg tcaagctgct gatctactac acctctcggc tgcatagcgg agtgccctct 180
agattctccg gctctggctc cggtaccgat tattccctga caatttccaa cctggaacaa 240
gaggacatcg ccacctactt ctgccagaga ggcaacaccc tgcctagaac ctttggaggc 300
ggcacaaaag tggaaatcaa gcgg 324
<210> 14
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caggtgcaac tgctgcagag cggcgccgag ctgatgaagc ctggcgcttc tgtgaagatc 60
tcttgcaagg ctacaggcta cacattctcc agatactgga tcgagtgggt caagcagcgg 120
cctggacatg gcctggaatg gatcggcgag atcctgcccg gcggaggcga gacaaactac 180
tacgagaagt tcaagggcaa ggccaccttc accgccgaca cctcttccaa caccgcttac 240
atgcagctga gcagtctgac ctccgaggac tccgccgtgt actactgcgc cagagaaggc 300
tacggcaact acggcggcta ctatttcgac tactggggcc agggcaccac cctgaccgtg 360
tcc 363
<210> 15
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gacatccaga tgacccagtc ttctagcagt tttagcattt ctctgggcga cagagtgaca 60
atctcctgca aggcctctga agatatctac aacagactgg cctggtacca gcaaaagccc 120
ggaaacgccc ctaggctgct gatctctggc gctccttccc tcgaaaatgg cgtctcctcc 180
agattctccg gctccggctc tggcaaggac tacaccctgt ccatcacctc cctgcagacc 240
gaggacgtgg ccacctacta ctgccagcag ttctggagca gcccttggac cttcggcggt 300
ggcaccaagc tggagatcaa gcgg 324
<210> 16
<211> 357
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gaagtgcagc tggtggaatc tggcgggggc ctggtgaagc ctggaggctc tctgaagctg 60
tcctgcgccg cttctggctt caccttcggc tcctacgcca tgtcttggat cagacagtct 120
cccgaaaaga gactggaatg ggtggccgag atctctagcg gaggctcctt cacctactat 180
cccgctaccg tgaccggccg cttcaccatc tctcgggaca acgccaagaa cacactgtac 240
ctggacatga cctccctccg gtccgaggac accgccatgt actactgcac cagaggcggc 300
tactcccctg gcaatgctat ggactactgg ggccagggca cctctgtcac tgtgtcc 357
Claims (17)
1.新型冠状病毒N蛋白特异性单链抗体scfv-3A2，包括轻链和重链，其特征在于：
所述轻链可变区的氨基酸序列如SEQ ID NO.1所示；
所述重链可变区的氨基酸序列如SEQ ID NO.2所示。
2.新型冠状病毒N蛋白特异性单链抗体scfv-4B9，包括轻链和重链，其特征在于：
所述轻链可变区的氨基酸序列如SEQ ID NO.3所示；
所述重链可变区的氨基酸序列如SEQ ID NO.4所示。
3.新型冠状病毒N蛋白特异性单链抗体scfv-2C6，包括轻链和重链，其特征在于：
所述轻链可变区的氨基酸序列如SEQ ID NO.5所示；
所述重链可变区的氨基酸序列如SEQ ID NO.6所示。
4.新型冠状病毒N蛋白特异性单链抗体scfv-6D8，包括轻链和重链，其特征在于：
所述轻链可变区的氨基酸序列如SEQ ID NO.7所示；
所述重链可变区的氨基酸序列如SEQ ID NO.8所示。
5.一种编码权利要求1所述的新型冠状病毒N蛋白特异性单链抗体scfv-3A2的基因，其特征在于：
编码轻链可变区的核苷酸序列如SEQ ID NO.9所示；
编码重链可变区的核苷酸序列如SEQ ID NO.10所示。
6.一种编码权利要求2所述的新型冠状病毒N蛋白特异性单链抗体scfv-4B9的基因，其特征在于：
编码轻链可变区的核苷酸序列如SEQ ID NO.11所示；
编码重链可变区的核苷酸序列如SEQ ID NO.12所示。
7.一种编码权利要求1所述的新型冠状病毒N蛋白特异性单链抗体scfv-2C6的基因，其特征在于：
编码轻链可变区的核苷酸序列如SEQ ID NO.13所示；
编码重链可变区的核苷酸序列如SEQ ID NO.14所示。
8.一种编码权利要求2所述的新型冠状病毒N蛋白特异性单链抗体scfv-6D8的基因，其特征在于：
编码轻链可变区的核苷酸序列如SEQ ID NO.15所示；
编码重链可变区的核苷酸序列如SEQ ID NO.16所示。
9.一种质粒载体，其特征在于该质粒载体含有权利要求5所述的轻链可变区核苷酸序列。
10.一种质粒载体，其特征在于该质粒载体含有权利要求5所述的重链可变区核苷酸序列。
11.一种质粒载体，其特征在于该质粒载体含有权利要求6所述的轻链可变区核苷酸序列。
12.一种质粒载体，其特征在于该质粒载体含有权利要求6所述的重链可变区核苷酸序列。
13.一种质粒载体，其特征在于该质粒载体含有权利要求7所述的轻链可变区核苷酸序列。
14.一种质粒载体，其特征在于该质粒载体含有权利要求7所述的重链可变区核苷酸序列。
15.一种质粒载体，其特征在于该质粒载体含有权利要求8所述的轻链可变区核苷酸序列。
16.一种质粒载体，其特征在于该质粒载体含有权利要求8所述的重链可变区核苷酸序列。
17.权利要求9、10、11、12、13、14、15、16所述的质粒载体用于真核表达新型冠状病毒N蛋白单克隆抗体，包括:
(a)通过PCR将权利要求5、6、7、8中所述轻链和重链核苷酸序列分别与鼠IgG1轻链恒定区和重链恒定区核苷酸序列桥接后酶切，并分别连接质粒载体，构建真核细胞表达载体；
(b)将步骤(a)中真核表达载体转染至HEK293F细胞表达得到新型冠状病毒N蛋白单克隆抗体；
(c)纯化单克隆抗体通过胶体金正交实验确定最佳单抗配对组合。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110482613.3A CN113150136B (zh) | 2021-04-30 | 2021-04-30 | 新型冠状病毒n蛋白单克隆抗体的制备 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110482613.3A CN113150136B (zh) | 2021-04-30 | 2021-04-30 | 新型冠状病毒n蛋白单克隆抗体的制备 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113150136A true CN113150136A (zh) | 2021-07-23 |
| CN113150136B CN113150136B (zh) | 2022-04-01 |
Family
ID=76873079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110482613.3A Active CN113150136B (zh) | 2021-04-30 | 2021-04-30 | 新型冠状病毒n蛋白单克隆抗体的制备 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113150136B (zh) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113527474A (zh) * | 2021-03-12 | 2021-10-22 | 中国人民解放军军事科学院军事医学研究院 | 一种抗新冠病毒n蛋白的单克隆抗体及其应用 |
| CN113603770A (zh) * | 2021-08-31 | 2021-11-05 | 厦门英博迈生物科技有限公司 | 一种新冠病毒核蛋白抗体和应用 |
| CN113912710A (zh) * | 2021-11-17 | 2022-01-11 | 杭州旭科生物技术有限公司 | 抗新型冠状病毒n蛋白的单克隆抗体及其应用 |
| CN114057881A (zh) * | 2021-12-28 | 2022-02-18 | 杭州贤至生物科技有限公司 | 抗氯胺酮特异性抗体、质粒载体及方法 |
| CN114751981A (zh) * | 2020-08-11 | 2022-07-15 | 优睿赛思(武汉)生物科技有限公司 | 抗SARS-CoV-2核衣壳蛋白的单克隆抗体及其应用 |
| CN116444657A (zh) * | 2022-01-10 | 2023-07-18 | 东莞市朋志生物科技有限公司 | 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 |
| CN117384279A (zh) * | 2022-07-05 | 2024-01-12 | 东莞市朋志生物科技有限公司 | 抗乙型流感病毒抗体、检测乙型流感病毒的试剂和试剂盒 |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269313A (zh) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | 一种用于检测新型冠状病毒的单克隆抗体及其制备试剂盒的用途 |
| CN111704666A (zh) * | 2020-04-22 | 2020-09-25 | 北京科卫临床诊断试剂有限公司 | 新型冠状病毒n蛋白的配对单克隆抗体及其应用 |
| US10822379B1 (en) * | 2020-03-12 | 2020-11-03 | University of Pittsburgh—of the Commonwealth System of Higher Education | Molecules that bind to SARS-CoV-2 |
| CN112079920A (zh) * | 2020-09-18 | 2020-12-15 | 北京华大蛋白质研发中心有限公司 | 一种用于检测SARS-CoV-2病毒N蛋白的单克隆抗体及其应用 |
| CN112111007A (zh) * | 2020-09-22 | 2020-12-22 | 通用生物系统（安徽）有限公司 | 一种新型冠状病毒核衣壳蛋白单克隆抗体的制备方法 |
| RU2744274C1 (ru) * | 2020-11-20 | 2021-03-04 | Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) | Моноклональное антитело к RBD фрагменту в составе S белка вируса SARS-CoV-2 |
| CN113912709A (zh) * | 2021-09-28 | 2022-01-11 | 深圳国家感染性疾病临床医学研究中心 | 新型冠状病毒单克隆抗体及其应用 |
-
2021
- 2021-04-30 CN CN202110482613.3A patent/CN113150136B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269313A (zh) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | 一种用于检测新型冠状病毒的单克隆抗体及其制备试剂盒的用途 |
| US10822379B1 (en) * | 2020-03-12 | 2020-11-03 | University of Pittsburgh—of the Commonwealth System of Higher Education | Molecules that bind to SARS-CoV-2 |
| CN111704666A (zh) * | 2020-04-22 | 2020-09-25 | 北京科卫临床诊断试剂有限公司 | 新型冠状病毒n蛋白的配对单克隆抗体及其应用 |
| CN112079920A (zh) * | 2020-09-18 | 2020-12-15 | 北京华大蛋白质研发中心有限公司 | 一种用于检测SARS-CoV-2病毒N蛋白的单克隆抗体及其应用 |
| CN112111007A (zh) * | 2020-09-22 | 2020-12-22 | 通用生物系统（安徽）有限公司 | 一种新型冠状病毒核衣壳蛋白单克隆抗体的制备方法 |
| RU2744274C1 (ru) * | 2020-11-20 | 2021-03-04 | Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) | Моноклональное антитело к RBD фрагменту в составе S белка вируса SARS-CoV-2 |
| CN113912709A (zh) * | 2021-09-28 | 2022-01-11 | 深圳国家感染性疾病临床医学研究中心 | 新型冠状病毒单克隆抗体及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| QUAN-XIN LONG: "Antibody responses to SARS-CoV-2 in patients with COVID-19", 《NATURE MEDICINE》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114751981A (zh) * | 2020-08-11 | 2022-07-15 | 优睿赛思(武汉)生物科技有限公司 | 抗SARS-CoV-2核衣壳蛋白的单克隆抗体及其应用 |
| CN114751981B (zh) * | 2020-08-11 | 2023-01-31 | 优睿赛思(武汉)生物科技有限公司 | 抗SARS-CoV-2核衣壳蛋白的单克隆抗体及其应用 |
| CN113527474B (zh) * | 2021-03-12 | 2023-11-07 | 中国人民解放军军事科学院军事医学研究院 | 一种抗新冠病毒n蛋白的单克隆抗体及其应用 |
| CN113527474A (zh) * | 2021-03-12 | 2021-10-22 | 中国人民解放军军事科学院军事医学研究院 | 一种抗新冠病毒n蛋白的单克隆抗体及其应用 |
| CN113603770A (zh) * | 2021-08-31 | 2021-11-05 | 厦门英博迈生物科技有限公司 | 一种新冠病毒核蛋白抗体和应用 |
| CN113603770B (zh) * | 2021-08-31 | 2022-03-08 | 厦门英博迈生物科技有限公司 | 一种新冠病毒核蛋白抗体和应用 |
| CN113912710A (zh) * | 2021-11-17 | 2022-01-11 | 杭州旭科生物技术有限公司 | 抗新型冠状病毒n蛋白的单克隆抗体及其应用 |
| CN113912710B (zh) * | 2021-11-17 | 2023-05-26 | 杭州旭科生物技术有限公司 | 抗新型冠状病毒n蛋白的单克隆抗体及其应用 |
| CN114057881A (zh) * | 2021-12-28 | 2022-02-18 | 杭州贤至生物科技有限公司 | 抗氯胺酮特异性抗体、质粒载体及方法 |
| CN114057881B (zh) * | 2021-12-28 | 2023-10-13 | 杭州贤至生物科技有限公司 | 抗氯胺酮特异性抗体、质粒载体及方法 |
| CN116444657B (zh) * | 2022-01-10 | 2023-10-31 | 东莞市朋志生物科技有限公司 | 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 |
| CN116444657A (zh) * | 2022-01-10 | 2023-07-18 | 东莞市朋志生物科技有限公司 | 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 |
| CN117384279A (zh) * | 2022-07-05 | 2024-01-12 | 东莞市朋志生物科技有限公司 | 抗乙型流感病毒抗体、检测乙型流感病毒的试剂和试剂盒 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113150136B (zh) | 2022-04-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113150136B (zh) | 新型冠状病毒n蛋白单克隆抗体的制备 | |
| CN112521492B (zh) | 乙肝表面抗原单克隆抗体的制备 | |
| CN112661849B (zh) | 一种艰难梭菌重组蛋白单克隆抗体的制备方法和应用 | |
| CN112812178B (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 | |
| CN110845582B (zh) | 一种猫细小病毒重组蛋白及其单克隆抗体的制备 | |
| CN110845608B (zh) | 一种番茄环斑病毒单克隆抗体及其制备方法 | |
| CN112500479B (zh) | 一种犬ⅱ型腺病毒重组蛋白单克隆抗体的制备 | |
| CN110885370B (zh) | 分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法 | |
| CN114106187B (zh) | 一种靶向ogt的特异性鲨鱼单域抗体及其制备方法和应用 | |
| CN112521497B (zh) | 肌红蛋白单克隆抗体的制备和应用 | |
| CN112608385B (zh) | 一种犬脑钠肽(bnp)单克隆抗体的制备 | |
| CN109306008B (zh) | 一种猪源性抗猪瘟病毒的单链抗体及其制备方法 | |
| CN113527474A (zh) | 一种抗新冠病毒n蛋白的单克隆抗体及其应用 | |
| CN116041526B (zh) | 一种鼠抗草鱼IgT单克隆抗体及其制备方法和应用 | |
| CN111606994A (zh) | 非融合型抗桥粒芯糖蛋白dsg3兔源单克隆抗体的制备方法 | |
| CN111349159A (zh) | 一种抗人血清白蛋白的纳米抗体及其应用 | |
| CN114213543B (zh) | 抗犬胰脂肪酶特异性单链抗体、质粒载体及方法 | |
| CN106008708B (zh) | 一种人乙型肝炎病毒x蛋白的单克隆抗体及用途 | |
| CN116041497A (zh) | 一种猫杯状病毒（fcv）重组蛋白单克隆抗体及制备方法 | |
| CN112646035B (zh) | Egfr的亲和力成熟结合蛋白及应用 | |
| CN110903359B (zh) | 一种空肠弯曲菌重组蛋白及其单克隆抗体的制备 | |
| CN111057154B (zh) | 基于驼源Fc片段的免疫原的制备及应用 | |
| CN113956353A (zh) | 抗猪急性腹泻综合征冠状病毒n蛋白单克隆抗体、该单克隆抗体的识别区域及其应用 | |
| CN113201069B (zh) | mcherry或mEOS纳米抗体及其制备方法和应用 | |
| CN114702574B (zh) | 白斑症病毒单链可变区重组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 |