CN111732654B - 一种抗SARS-CoV-2的单克隆抗体1E10 - Google Patents
一种抗SARS-CoV-2的单克隆抗体1E10 Download PDFInfo
- Publication number
- CN111732654B CN111732654B CN202010566786.9A CN202010566786A CN111732654B CN 111732654 B CN111732654 B CN 111732654B CN 202010566786 A CN202010566786 A CN 202010566786A CN 111732654 B CN111732654 B CN 111732654B
- Authority
- CN
- China
- Prior art keywords
- seq
- monoclonal antibody
- amino acid
- acid sequence
- cov
- 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
- 241001678559 COVID-19 virus Species 0.000 title claims abstract description 26
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 14
- 239000000427 antigen Substances 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 101800000904 Spike protein S1 Proteins 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 150000007523 nucleic acids Chemical group 0.000 claims description 2
- 230000009385 viral infection Effects 0.000 claims description 2
- 230000000890 antigenic effect Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 150000001413 amino acids Chemical group 0.000 description 19
- 230000027455 binding Effects 0.000 description 10
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 241000700605 Viruses Species 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 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
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000010530 Virus Neutralization Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 208000025721 COVID-19 Diseases 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 2
- 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 2
- MCIXMYKSPQUMJG-SRVKXCTJSA-N Phe-Ser-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MCIXMYKSPQUMJG-SRVKXCTJSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108091005609 SARS-CoV-2 Spike Subunit S1 Proteins 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
- 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
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 108010054812 diprotin A Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 2
- JNTMAZFVYNDPLB-PEDHHIEDSA-N (2S,3S)-2-[[[(2S)-1-[(2S,3S)-2-amino-3-methyl-1-oxopentyl]-2-pyrrolidinyl]-oxomethyl]amino]-3-methylpentanoic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JNTMAZFVYNDPLB-PEDHHIEDSA-N 0.000 description 1
- 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
- XQJAFSDFQZPYCU-UWJYBYFXSA-N Ala-Asn-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N XQJAFSDFQZPYCU-UWJYBYFXSA-N 0.000 description 1
- AWAXZRDKUHOPBO-GUBZILKMSA-N Ala-Gln-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O AWAXZRDKUHOPBO-GUBZILKMSA-N 0.000 description 1
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-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
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 1
- GIVATXIGCXFQQA-FXQIFTODSA-N Arg-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N GIVATXIGCXFQQA-FXQIFTODSA-N 0.000 description 1
- FFEUXEAKYRCACT-PEDHHIEDSA-N Arg-Ile-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(O)=O FFEUXEAKYRCACT-PEDHHIEDSA-N 0.000 description 1
- 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 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
- LFAUVOXPCGJKTB-DCAQKATOSA-N Arg-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N LFAUVOXPCGJKTB-DCAQKATOSA-N 0.000 description 1
- JPPLRQVZMZFOSX-UWJYBYFXSA-N Asn-Tyr-Ala Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 JPPLRQVZMZFOSX-UWJYBYFXSA-N 0.000 description 1
- 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 1
- WOPJVEMFXYHZEE-SRVKXCTJSA-N Asp-Phe-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O WOPJVEMFXYHZEE-SRVKXCTJSA-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
- 241000283707 Capra Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 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 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 1
- RBWKVOSARCFSQQ-FXQIFTODSA-N Gln-Gln-Ser Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O RBWKVOSARCFSQQ-FXQIFTODSA-N 0.000 description 1
- IKFZXRLDMYWNBU-YUMQZZPRSA-N Gln-Gly-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N IKFZXRLDMYWNBU-YUMQZZPRSA-N 0.000 description 1
- ILKYYKRAULNYMS-JYJNAYRXSA-N Gln-Lys-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ILKYYKRAULNYMS-JYJNAYRXSA-N 0.000 description 1
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 1
- SXFPZRRVWSUYII-KBIXCLLPSA-N Gln-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N SXFPZRRVWSUYII-KBIXCLLPSA-N 0.000 description 1
- GHAXJVNBAKGWEJ-AVGNSLFASA-N Gln-Ser-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GHAXJVNBAKGWEJ-AVGNSLFASA-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
- INGJLBQKTRJLFO-UKJIMTQDSA-N Glu-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O INGJLBQKTRJLFO-UKJIMTQDSA-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
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 1
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 1
- OCQUNKSFDYDXBG-QXEWZRGKSA-N Gly-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OCQUNKSFDYDXBG-QXEWZRGKSA-N 0.000 description 1
- 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 1
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-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
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 1
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 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
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-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
- 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
- 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 1
- CCQLQKZTXZBXTN-NHCYSSNCSA-N Leu-Gly-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CCQLQKZTXZBXTN-NHCYSSNCSA-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
- UCRJTSIIAYHOHE-ULQDDVLXSA-N Leu-Tyr-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UCRJTSIIAYHOHE-ULQDDVLXSA-N 0.000 description 1
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 1
- IXHKPDJKKCUKHS-GARJFASQSA-N Lys-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N IXHKPDJKKCUKHS-GARJFASQSA-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
- 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
- MIFFFXHMAHFACR-KATARQTJSA-N Lys-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN MIFFFXHMAHFACR-KATARQTJSA-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
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- FVKRQMQQFGBXHV-QXEWZRGKSA-N Met-Asp-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O FVKRQMQQFGBXHV-QXEWZRGKSA-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
- 208000025370 Middle East respiratory syndrome Diseases 0.000 description 1
- 241001529936 Murinae Species 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
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 description 1
- QPQDWBAJWOGAMJ-IHPCNDPISA-N Phe-Asp-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 QPQDWBAJWOGAMJ-IHPCNDPISA-N 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- XZONQWUEBAFQPO-HJGDQZAQSA-N Pro-Gln-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XZONQWUEBAFQPO-HJGDQZAQSA-N 0.000 description 1
- VZKBJNBZMZHKRC-XUXIUFHCSA-N Pro-Ile-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O VZKBJNBZMZHKRC-XUXIUFHCSA-N 0.000 description 1
- FMDHKPRACUXATF-ACZMJKKPSA-N Ser-Gln-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O FMDHKPRACUXATF-ACZMJKKPSA-N 0.000 description 1
- 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 1
- 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 1
- WEQAYODCJHZSJZ-KKUMJFAQSA-N Ser-His-Tyr Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 WEQAYODCJHZSJZ-KKUMJFAQSA-N 0.000 description 1
- 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 1
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 1
- OLKICIBQRVSQMA-SRVKXCTJSA-N Ser-Ser-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OLKICIBQRVSQMA-SRVKXCTJSA-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
- 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 1
- 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 1
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 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
- 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 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
- 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 1
- 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 1
- MEZCXKYMMQJRDE-PMVMPFDFSA-N Trp-Leu-Tyr Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)CC(C)C)C(O)=O)C1=CC=C(O)C=C1 MEZCXKYMMQJRDE-PMVMPFDFSA-N 0.000 description 1
- MBLJBGZWLHTJBH-SZMVWBNQSA-N Trp-Val-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 MBLJBGZWLHTJBH-SZMVWBNQSA-N 0.000 description 1
- VCXWRWYFJLXITF-AUTRQRHGSA-N Tyr-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VCXWRWYFJLXITF-AUTRQRHGSA-N 0.000 description 1
- NSOMQRHZMJMZIE-GVARAGBVSA-N Tyr-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NSOMQRHZMJMZIE-GVARAGBVSA-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
- 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 1
- OLWFDNLLBWQWCP-STQMWFEESA-N Tyr-Gly-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O OLWFDNLLBWQWCP-STQMWFEESA-N 0.000 description 1
- LUMQYLVYUIRHHU-YJRXYDGGSA-N Tyr-Ser-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LUMQYLVYUIRHHU-YJRXYDGGSA-N 0.000 description 1
- 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 1
- LTTQCQRTSHJPPL-ZKWXMUAHSA-N Val-Ser-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N LTTQCQRTSHJPPL-ZKWXMUAHSA-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
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 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
- 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
- 238000002835 absorbance Methods 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
本发明涉及一种抗SARS‑CoV‑2的单克隆抗体1E10,该抗体的六个CDR区为:(1)重链CDR1(VHCDR1)包含如SEQ ID NO.1所示的氨基酸序列;(2)重链CDR2(VHCDR2)包含如SEQ ID NO.2所示的氨基酸序列;(3)重链CDR3(VHCDR3)包含如SEQ ID NO.3所示的氨基酸序列;(4)轻链CDR1(VLCDR1)包含如SEQ ID NO.4所示的氨基酸序列;(5)轻链CDR2(VLCDR2)包含如SEQ ID NO.5所示的氨基酸序列;(6)轻链CDR3(VLCDR3)包含如SEQ ID NO.6所示的氨基酸序列。
Description
技术领域
本发明属于生物医药技术领域,具体的,涉及一种抗SARS-CoV-2的单克隆抗体1E10。
背景技术
含有高浓度的抗SARS-CoV-2抗体的康复期血浆已在治疗中显示出积极的效果,说明抗SARS-CoV-2特异性抗体能有效阻断病毒与细胞的结合。另外,在SARS、MERS等烈性传染病爆发期间研发了一系列的具有中和活性的单克隆抗体,已被证明在疾病的预防和治疗中具有安全性和有效性。这些都提示在应对SARS-CoV-2病毒时,可考虑制备抗SARS-CoV-2单克隆抗体,尤其是全人源单克隆抗体,该种抗体不仅能通过阻断SARS-CoV-2与受体细胞的结合从而避免病毒的侵入,达到保护的作用,而且相对人源化或人鼠嵌合抗体具有副作用更小的优点,将为COVID-19的特异性预防和治疗提供新的手段。
发明内容
本发明首先涉及针对SARS-CoV-2病毒的单克隆抗体1E10,其特征在于,该抗体的六个CDR区为:
(1)重链CDR1(VHCDR1)包含如SEQ ID NO.1所示的氨基酸序列:GGTFSSYAIS;
(2)重链CDR2(VHCDR2)包含如SEQ ID NO.2所示的氨基酸序列:RIIPILGIANYAQKFQG;
(3)重链CDR3(VHCDR3)包含如SEQ ID NO.3所示的氨基酸序列:EVSDFDWLYRSHYGMDV;
(4)轻链CDR1(VLCDR1)包含如SEQ ID NO.4所示的氨基酸序列:RASQSISSYLN;
(5)轻链CDR2(VLCDR2)包含如SEQ ID NO.5所示的氨基酸序列:AASSLQS;
(6)轻链CDR3(VLCDR3)包含如SEQ ID NO.6所示的氨基酸序列:QQSYSTPQT。
进一步的,
所述的单克隆抗体1E10的重链可变区全长包含如SEQ ID NO.7所示的氨基酸序列: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYM ELSSLRSEDTAVYYCAREVSDFDWLYRSHYGMDVWGQGTTVTVSS;
所述的单克隆抗体1E10的轻链可变区全长包含如SEQ ID NO.8所示的氨基酸序列: EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQSYSTPQTFGGGTKVEIK。
进一步的,所述的单克隆抗体1E10为人IgG型抗体。
进一步的,所述的单克隆抗体1E10结合的抗原为SARS-CoV-2病毒的刺突蛋白S1,具体的,所述的单克隆抗体1E10结合的抗原结构区域为SARS-CoV-2病毒的刺突蛋白S1中的RBD结构域。
最优选的,所述的单克隆抗体1E10的轻链可变区和重链可变区分别是SEQ IDNO.8和SEQ ID NO.7所示的氨基酸序列。
本发明还涉及编码所述单克隆抗体1E10的核酸片段。
本发明还涉及一种抗体,所述的抗体的轻链为:
(1)SEQ ID NO.8所示的氨基酸序列经替换、缺失或添加一个或几个氨基酸后,形成的具有同等功能的序列;
或(2)与SEQ ID NO.8所示的氨基酸序列具有95%以上同源性的氨基酸序列;
所述的抗体的重链为:
(1)SEQ ID NO.7所示的氨基酸序列经替换、缺失或添加一个或几个氨基酸后,形成的具有同等功能的序列;
或(2)与SEQ ID NO.7所示的氨基酸序列具有95%以上同源性的氨基酸序列。
本发明还包括所述的单克隆抗体1E10在制备检测SARS-CoV-2病毒的试剂中的应用。
本发明还包括所述的单克隆抗体1E10在制备抑制SARS-CoV-2病毒的试剂中的应用。
本发明还包括单克隆抗体1E10在制备药物中的应用,所述的药物为预防和/或治疗因SARS-CoV-2病毒感染导致的疾病的药物。
本发明的有益效果:由于目前针对SARS-CoV-2病毒引起的疾病仍无上市的疫苗和特异药物,虽然康复期血浆治疗已被证明是一种有希望的治疗方法,但是由于大规模制备受限,且该方法主要针对重症和危重症患者,而单克隆抗体则具有纯度高,靶向性强、副作用小、可大量制备等优点。本发明所涉及的1E10抗体的实验结果表明,该单克隆抗体1E10中和活性和阻断活性均较高。因此,本发明获得的人源单克隆抗体 1E10为COVID-19的特异性预防和治疗提供了新的候选药物。
附图说明
图1、单克隆抗体1E10的SDS-PAGE电泳检测结果:泳道1为非还原SDS-PAGE电泳,泳道2为还原 SDS-PAGE电泳。
图2、单克隆抗体1E10的SEC-HPLC检测结果。
图3、单克隆抗体1E10结合活性检测结果。A为针对SARS-CoV-2刺突蛋白S1的结合活性;B为针对 SARS-CoV-2刺突蛋白S1的RBD结构域的结合活性。
图4、单克隆抗体1E10的活病毒中和实验检测结果。
具体实施方式
若未特别说明,以下实施例中所用的技术手段均为本领域技术人员所熟知的常规手段,所有试剂耗材均为市售商品。
实施例1、单克隆抗体1E10表达载体构建、的表达与纯化
将含1E10抗体重链和轻链可变区的编码基因片段分别整合进含人IgG1抗体重轻链恒定区序列的 pcDNA3.4表达载体中,得到分别表达目标抗体重链和轻链的重组表达载体。
细胞的转染、抗体表达和纯化
1、转染:采用Gibco公司转染试剂盒并按照说明书进行转染操作,步骤简述为:
将两种重组表达载体总DNA与转染试剂混合以形成DNA ExpiFectamineTM 293复合物;
随后加入含40mL、2.94x106个/mL的Expi293细胞培养物中;
最后置于37℃,125rpm,8%CO2培养。
2、纯化:
培养5天后,4000rpm,25℃离心10分钟收集上清,随后用MabSelect Sure亲和层析柱进行纯化。纯化步骤简述为:
用pH7.0的0.1M Tris缓冲液平衡;
上样后,用pH7.0的0.1M Tris缓冲液淋洗;
随后用pH8.0的1.0M Tris缓冲液进行洗脱。
收集洗脱液进一步于PBS缓冲液中透析。取纯化后的抗体进行SDS-PAGE和HPLC-SEC检测分析。
3、结果分析:SDS-PAGE结果见图1,显示在非还原条件下,人源SARS-CoV-2抗体呈现分子量约为150kDa 的条带;在还原条件下呈现分子量约为50kDa和25kDa的两条带,分别对应抗体的重链和轻链。SEC-HPLC结果见图2,显示纯化后的单克隆抗体纯度达到95%以上。纯化后的单克隆抗体经肽图分析后得到的氨基酸序列与预期的氨基酸序列一致。具体的,轻链可变区和重链可变区的序列分别与SEQ ID NO.8、SEQ ID NO.7相同。
实施例2、抗体的功能分析
1、人源抗SARS-CoV-2抗体1E10与抗原的结合活性检测
采用ELISA法测定抗体与SARS-CoV-2(2019-nCoV)病毒的刺突蛋白S1和RBD结构域结合能力。步骤简述为:
(1)以SARS-CoV-2刺突S1-His重组蛋白(Sino公司,货号:40591-V08H)或RBD结构域重组蛋白(Sino 公司,货号40592-V05H)为包被抗原,用重碳酸盐缓冲液将1.0μg/mL抗原包被于酶标板,4℃过夜;
(2)用酪蛋白缓冲液于37℃封闭1h;加入系列稀释后的待检抗体,37℃1h;
(3)加入1:10000稀释后的羊抗人IgG-HRP(Bethyl公司,货号:A80-304P),37℃1h;
(4)用显色液显色后,2M HCl终止反应;酶标仪检测吸光度A450和A540值。
结果:
抗体1E10与抗原结合活性检测结果见图3,可知单克隆抗体1E10针对S1蛋白的结合活性(图3A)为 EC50=0.029nM;针对RBD结构域的结合活性(图3B)为EC50=0.044nM。
2、人源抗SARS-CoV-2抗体活病毒中和试验
病毒蚀斑减少中和试验采用SARS-CoV-2病毒BetaCoV/IVDC-HB-envF13/2020株进行。步骤简述为:将定量的SARS-CoV-2病毒和系列倍比稀释的单克隆抗体混合后孵育,然后加入到预先准备好的含Vero细胞的检测平板中,培养后观察记录病毒蚀斑数,并计算病毒中和活性(以IC50表示)。
结果:
抗体1E10的活病毒中和实验检测结果见图4,显示该单克隆抗体能较好的中和SARS-CoV-2病毒 (IC50<48.8ng/mL)。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
SEQUENCE LISTING
<110> 武汉生物制品研究所有限责任公司
<120> 一种抗SARS-CoV-2的单克隆抗体1E10
<210> 1
<211> 10
<212> PRT
<213> 人工序列
<400> 1
Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser
1 5 10
<210> 2
<211> 17
<212> PRT
<213> 人工序列
<400> 2
Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 3
<211> 17
<212> PRT
<213> 人工序列
<400> 3
Glu Val Ser Asp Phe Asp Trp Leu Tyr Arg Ser His Tyr Gly Met Asp
1 5 10 15
Val
<210> 4
<211> 11
<212> PRT
<213> 人工序列
<400> 4
Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
1 5 10
<210> 5
<211> 7
<212> PRT
<213> 人工序列
<400> 5
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列
<400> 6
Gln Gln Ser Tyr Ser Thr Pro Gln Thr
1 5
<210> 7
<211> 126
<212> PRT
<213> 人工序列
<400> 7
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Val Ser Asp Phe Asp Trp Leu Tyr Arg Ser His Tyr Gly
100 105 110
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 8
<211> 107
<212> PRT
<213> 人工序列
<400> 8
Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Gln
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
Claims (9)
1.一种抗SARS-CoV-2病毒的单克隆抗体1E10,其特征在于,该抗体的六个CDR区为:
(1)重链CDR1(VHCDR1)为如SEQ ID NO.1所示的氨基酸序列:GGTFSSYAIS;
(2)重链CDR2(VHCDR2)为如SEQ ID NO.2所示的氨基酸序列:RIIPILGIANYAQKFQG;
(3)重链CDR3(VHCDR3)为如SEQ ID NO.3所示的氨基酸序列:EVSDFDWLYRSHYGMDV;
(4)轻链CDR1(VLCDR1)为如SEQ ID NO.4所示的氨基酸序列:RASQSISSYLN;
(5)轻链CDR2(VLCDR2)为如SEQ ID NO.5所示的氨基酸序列:AASSLQS;
(6)轻链CDR3(VLCDR3)为如SEQ ID NO.6所示的氨基酸序列:QQSYSTPQT。
2.根据权利要求1所述的抗体,其特征在于,
所述的单克隆抗体1E10的重链可变区全长如SEQ ID NO.7所示的氨基酸序列:
所述的单克隆抗体1E10的轻链可变区全长如SEQ ID NO.8所示的氨基酸序列。
3.根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体1E10为人IgG型抗体。
4.根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体1E10结合的抗原为SARS-CoV-2病毒的刺突蛋白S1。
5.根据权利要求4所述的抗体,其特征在于,所述的单克隆抗体1E10结合的抗原结构区域为SARS-CoV-2病毒的刺突蛋白S1中的RBD结构域。
6.编码权利要求1-5任一所述的抗体的核酸片段。
7.权利要求1-5任一所述的单克隆抗体1E10在制备检测SARS-CoV-2病毒的试剂中的应用。
8.权利要求1-5任一所述的单克隆抗体1E10在制备抑制SARS-CoV-2病毒的试剂中的应用。
9.权利要求1-5任一所述的单克隆抗体1E10在制备药物中的应用,所述的药物为预防和/或治疗因SARS-CoV-2病毒感染导致的疾病的药物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010566786.9A CN111732654B (zh) | 2020-06-19 | 2020-06-19 | 一种抗SARS-CoV-2的单克隆抗体1E10 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010566786.9A CN111732654B (zh) | 2020-06-19 | 2020-06-19 | 一种抗SARS-CoV-2的单克隆抗体1E10 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111732654A CN111732654A (zh) | 2020-10-02 |
CN111732654B true CN111732654B (zh) | 2021-05-25 |
Family
ID=72651819
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010566786.9A Active CN111732654B (zh) | 2020-06-19 | 2020-06-19 | 一种抗SARS-CoV-2的单克隆抗体1E10 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111732654B (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG11202103404PA (en) | 2020-04-02 | 2021-04-29 | Regeneron Pharma | Anti-sars-cov-2-spike glycoprotein antibodies and antigen-binding fragments |
CN112326962B (zh) * | 2020-11-03 | 2021-10-12 | 山西康健恩生物科技有限公司 | 一种新型冠状病毒抗原胶体金快速诊断试剂盒及其制备方法 |
WO2022095045A1 (zh) * | 2020-11-09 | 2022-05-12 | 上海济煜医药科技有限公司 | SARS-CoV-2抗体及其应用 |
WO2022179561A1 (en) * | 2021-02-24 | 2022-09-01 | The University Of Hong Kong | Neutralizing antibodies against covid-19 and methods of use thereof |
CN114031685B (zh) * | 2022-01-10 | 2022-03-25 | 中国人民解放军军事科学院军事医学研究院 | 一种全人源抗新冠病毒中和抗体zw2g10及应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITTO20120570A1 (it) * | 2012-06-27 | 2013-12-28 | Pomona Ricerca Srl | Anticorpo monoclonale diretto contro il virus jc |
CN107056938B (zh) * | 2017-05-23 | 2020-09-25 | 深圳普兰达科技有限公司 | 人源抗h7n9禽流感病毒高亲和力抗体10k及其应用 |
CN107226861B (zh) * | 2017-05-26 | 2020-11-10 | 深圳市第三人民医院 | 人源抗h7n9禽流感病毒中和性抗体1f7l及其应用 |
CN111217917B (zh) * | 2020-02-26 | 2020-10-23 | 康希诺生物股份公司 | 一种新型冠状病毒SARS-CoV-2疫苗及其制备方法 |
-
2020
- 2020-06-19 CN CN202010566786.9A patent/CN111732654B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN111732654A (zh) | 2020-10-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111690059B (zh) | 一种抗SARS-CoV-2的单克隆抗体1D7 | |
CN111718411B (zh) | 一种抗SARS-CoV-2的单克隆抗体1F2 | |
CN111732654B (zh) | 一种抗SARS-CoV-2的单克隆抗体1E10 | |
CN112521494B (zh) | 一种抗SARS-CoV-2的单克隆抗体2B11 | |
CN106687479B (zh) | 抗ctla4的单克隆抗体或其抗原结合片段、药物组合物及用途 | |
CN112166121B (zh) | 神经生长因子的单克隆抗体及其编码基因和应用 | |
CN110016079B (zh) | 抗呼吸道合胞病毒的中和抗体及其应用 | |
HUE025329T2 (en) | Monoclonal antibodies capable of responding to more than one influenza A subtype | |
CN113354733B (zh) | 一种抗SARS-CoV-2流行突变株的单克隆抗体20D8 | |
KR20100021577A (ko) | 에스.아우레우스 ORF0657n을 표적화하는 항원 결합 단백질 | |
US20120269822A1 (en) | Anti-Botulinum Neurotoxin a Single Domain Antibody Antibodies | |
US20170158753A1 (en) | Monoclonal antibodies directed against envelope glycoproteins from multiple filovirus species | |
KR102132604B1 (ko) | 비형 간염 표면 항원에 대한 항체 및 그의 용도 | |
WO2020186687A1 (zh) | 一种特异性结合四种血清型登革病毒的人源抗体 | |
CN108690134A (zh) | 用于治疗乙肝感染及相关疾病的抗体 | |
CN111153988B (zh) | 广谱抗肠道病毒d68型的中和性单克隆抗体 | |
CN115093477A (zh) | 一种抗新型冠状病毒核蛋白n端区的单克隆抗体及其应用 | |
WO2019128119A1 (zh) | 一种针对破伤风毒素的全人源单克隆中和抗体及其应用 | |
CN113817051B (zh) | 一种抗SARS-CoV-2的单克隆抗体1B6 | |
EP4353744A1 (en) | Antibody against respiratory syncytial virus and use thereof | |
CN107207583A (zh) | 对呼吸道合胞体病毒(rsv)的f蛋白具有特异性的人单克隆抗体 | |
CN113817050B (zh) | 一种抗SARS-CoV-2的单克隆抗体1H8 | |
CN115260306A (zh) | 靶向SARS-CoV-2受体结合基序的单克隆抗体及其识别抗原表位和应用 | |
US7622111B2 (en) | Fusion protein of human IgG1 heavy chain constant region and scFv antibody against equine encephalitis virus | |
CN116715757A (zh) | 全人源抗狂犬病毒中和抗体及其用途 |
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 | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20211228 Address after: 100024 2F, building 2, yard 2, Shuangqiao Road B, Chaoyang District, Beijing Patentee after: China Biotechnology Co.,Ltd. Patentee after: Wuhan Biological Products Research Institute Co., Ltd Address before: 430207 No.1, Huangjin Industrial Park Road, Zhengdian Town, Jiangxia District, Wuhan City, Hubei Province Patentee before: WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS Co.,Ltd. |