CN115124616A - Nano antibody with specificity aiming at SARS-CoV-2RBD - Google Patents
Nano antibody with specificity aiming at SARS-CoV-2RBD Download PDFInfo
- Publication number
- CN115124616A CN115124616A CN202210626397.XA CN202210626397A CN115124616A CN 115124616 A CN115124616 A CN 115124616A CN 202210626397 A CN202210626397 A CN 202210626397A CN 115124616 A CN115124616 A CN 115124616A
- Authority
- CN
- China
- Prior art keywords
- antibody
- cov
- sars
- cell
- nucleic acid
- 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.)
- Pending
Links
- 108091005634 SARS-CoV-2 receptor-binding domains Proteins 0.000 title claims abstract description 18
- 238000000746 purification Methods 0.000 claims abstract description 20
- 210000004027 cell Anatomy 0.000 claims description 23
- 108020004707 nucleic acids Proteins 0.000 claims description 21
- 102000039446 nucleic acids Human genes 0.000 claims description 21
- 150000007523 nucleic acids Chemical class 0.000 claims description 21
- 239000012620 biological material Substances 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 20
- 230000014509 gene expression Effects 0.000 claims description 18
- 108010003723 Single-Domain Antibodies Proteins 0.000 claims description 12
- 238000001042 affinity chromatography Methods 0.000 claims description 11
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 230000009261 transgenic effect Effects 0.000 claims description 8
- 241001678559 COVID-19 virus Species 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 5
- 210000004962 mammalian cell Anatomy 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 208000025721 COVID-19 Diseases 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 27
- 102000004169 proteins and genes Human genes 0.000 abstract description 23
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000000427 antigen Substances 0.000 description 26
- 102000036639 antigens Human genes 0.000 description 26
- 108091007433 antigens Proteins 0.000 description 26
- 102100036790 Tubulin beta-3 chain Human genes 0.000 description 23
- 239000000243 solution Substances 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 101100366710 Arabidopsis thaliana SSL12 gene Proteins 0.000 description 14
- 101150007842 SS1 gene Proteins 0.000 description 14
- 241001416177 Vicugna pacos Species 0.000 description 13
- 230000027455 binding Effects 0.000 description 12
- 238000009739 binding Methods 0.000 description 12
- 230000003053 immunization Effects 0.000 description 11
- 238000002649 immunization Methods 0.000 description 11
- 229920002684 Sepharose Polymers 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 8
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 238000002823 phage display Methods 0.000 description 5
- 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 4
- 241000894006 Bacteria Species 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 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 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 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 4
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 4
- 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 4
- 241000700605 Viruses Species 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 108010008355 arginyl-glutamine Proteins 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000945 filler Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000009465 prokaryotic expression Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- HNAUFGBKJLTWQE-IFFSRLJSSA-N Gln-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)N)N)O HNAUFGBKJLTWQE-IFFSRLJSSA-N 0.000 description 3
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 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 3
- 239000007983 Tris buffer Substances 0.000 description 3
- UIDJDMVRDUANDL-BVSLBCMMSA-N Trp-Tyr-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UIDJDMVRDUANDL-BVSLBCMMSA-N 0.000 description 3
- ANHVRCNNGJMJNG-BZSNNMDCSA-N Tyr-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CS)C(=O)O)N)O ANHVRCNNGJMJNG-BZSNNMDCSA-N 0.000 description 3
- 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 3
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 3
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 3
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 108010029020 prolylglycine Proteins 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 108010009962 valyltyrosine Proteins 0.000 description 3
- PQWTZSNVWSOFFK-FXQIFTODSA-N Arg-Asp-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)CN=C(N)N PQWTZSNVWSOFFK-FXQIFTODSA-N 0.000 description 2
- NKBQZKVMKJJDLX-SRVKXCTJSA-N Arg-Glu-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NKBQZKVMKJJDLX-SRVKXCTJSA-N 0.000 description 2
- YVXRYLVELQYAEQ-SRVKXCTJSA-N Asn-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N YVXRYLVELQYAEQ-SRVKXCTJSA-N 0.000 description 2
- MRQQMVZUHXUPEV-IHRRRGAJSA-N Asp-Arg-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MRQQMVZUHXUPEV-IHRRRGAJSA-N 0.000 description 2
- HHWQMFIGMMOVFK-WDSKDSINSA-N Gln-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O HHWQMFIGMMOVFK-WDSKDSINSA-N 0.000 description 2
- JXFLPKSDLDEOQK-JHEQGTHGSA-N Gln-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O JXFLPKSDLDEOQK-JHEQGTHGSA-N 0.000 description 2
- MSHXWFKYXJTLEZ-CIUDSAMLSA-N Gln-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MSHXWFKYXJTLEZ-CIUDSAMLSA-N 0.000 description 2
- VYOILACOFPPNQH-UMNHJUIQSA-N Gln-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N VYOILACOFPPNQH-UMNHJUIQSA-N 0.000 description 2
- FHQRLHFYVZAQHU-IUCAKERBSA-N Gly-Lys-Gln Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O FHQRLHFYVZAQHU-IUCAKERBSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 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 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 2
- XSLXHSYIVPGEER-KZVJFYERSA-N Thr-Ala-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XSLXHSYIVPGEER-KZVJFYERSA-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
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007481 next generation sequencing Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 229940023143 protein vaccine Drugs 0.000 description 2
- 210000003370 receptor cell Anatomy 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229940031626 subunit vaccine Drugs 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- SBGXWWCLHIOABR-UHFFFAOYSA-N Ala Ala Gly Ala Chemical compound CC(N)C(=O)NC(C)C(=O)NCC(=O)NC(C)C(O)=O SBGXWWCLHIOABR-UHFFFAOYSA-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
- JPGBXANAQYHTLA-DRZSPHRISA-N Ala-Gln-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JPGBXANAQYHTLA-DRZSPHRISA-N 0.000 description 1
- AJBVYEYZVYPFCF-CIUDSAMLSA-N Ala-Lys-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O AJBVYEYZVYPFCF-CIUDSAMLSA-N 0.000 description 1
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 1
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 1
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- SBVJJNJLFWSJOV-UBHSHLNASA-N Arg-Ala-Phe Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SBVJJNJLFWSJOV-UBHSHLNASA-N 0.000 description 1
- BVBKBQRPOJFCQM-DCAQKATOSA-N Arg-Asn-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BVBKBQRPOJFCQM-DCAQKATOSA-N 0.000 description 1
- OTCJMMRQBVDQRK-DCAQKATOSA-N Arg-Asp-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OTCJMMRQBVDQRK-DCAQKATOSA-N 0.000 description 1
- PRLPSDIHSRITSF-UNQGMJICSA-N Arg-Phe-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PRLPSDIHSRITSF-UNQGMJICSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- HFPXZWPUVFVNLL-GUBZILKMSA-N Asn-Leu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFPXZWPUVFVNLL-GUBZILKMSA-N 0.000 description 1
- DAYDURRBMDCCFL-AAEUAGOBSA-N Asn-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC(=O)N)N DAYDURRBMDCCFL-AAEUAGOBSA-N 0.000 description 1
- QNMKWNONJGKJJC-NHCYSSNCSA-N Asp-Leu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O QNMKWNONJGKJJC-NHCYSSNCSA-N 0.000 description 1
- XWSIYTYNLKCLJB-CIUDSAMLSA-N Asp-Lys-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O XWSIYTYNLKCLJB-CIUDSAMLSA-N 0.000 description 1
- QSFHZPQUAAQHAQ-CIUDSAMLSA-N Asp-Ser-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O QSFHZPQUAAQHAQ-CIUDSAMLSA-N 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 101100112922 Candida albicans CDR3 gene Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102100031673 Corneodesmosin Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements 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
- VLOLPWWCNKWRNB-LOKLDPHHSA-N Gln-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VLOLPWWCNKWRNB-LOKLDPHHSA-N 0.000 description 1
- QGWXAMDECCKGRU-XVKPBYJWSA-N Gln-Val-Gly Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(N)=O)C(=O)NCC(O)=O QGWXAMDECCKGRU-XVKPBYJWSA-N 0.000 description 1
- NPMSEUWUMOSEFM-CIUDSAMLSA-N Glu-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N NPMSEUWUMOSEFM-CIUDSAMLSA-N 0.000 description 1
- TZXOPHFCAATANZ-QEJZJMRPSA-N Glu-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N TZXOPHFCAATANZ-QEJZJMRPSA-N 0.000 description 1
- YMUFWNJHVPQNQD-ZKWXMUAHSA-N Gly-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN YMUFWNJHVPQNQD-ZKWXMUAHSA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 1
- KRRMJKMGWWXWDW-STQMWFEESA-N Gly-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KRRMJKMGWWXWDW-STQMWFEESA-N 0.000 description 1
- UXJHNZODTMHWRD-WHFBIAKZSA-N Gly-Asn-Ala Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O UXJHNZODTMHWRD-WHFBIAKZSA-N 0.000 description 1
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 1
- XQHSBNVACKQWAV-WHFBIAKZSA-N Gly-Asp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XQHSBNVACKQWAV-WHFBIAKZSA-N 0.000 description 1
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 description 1
- XLFHCWHXKSFVIB-BQBZGAKWSA-N Gly-Gln-Gln Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLFHCWHXKSFVIB-BQBZGAKWSA-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
- HMHRTKOWRUPPNU-RCOVLWMOSA-N Gly-Ile-Gly Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O HMHRTKOWRUPPNU-RCOVLWMOSA-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
- HHRODZSXDXMUHS-LURJTMIESA-N Gly-Met-Gly Chemical compound CSCC[C@H](NC(=O)C[NH3+])C(=O)NCC([O-])=O HHRODZSXDXMUHS-LURJTMIESA-N 0.000 description 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
- 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
- MREVELMMFOLESM-HOCLYGCPSA-N Gly-Trp-Val Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O MREVELMMFOLESM-HOCLYGCPSA-N 0.000 description 1
- RYAOJUMWLWUGNW-QMMMGPOBSA-N Gly-Val-Gly Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O RYAOJUMWLWUGNW-QMMMGPOBSA-N 0.000 description 1
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 1
- PROLDOGUBQJNPG-RWMBFGLXSA-N His-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O PROLDOGUBQJNPG-RWMBFGLXSA-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
- 101000713575 Homo sapiens Tubulin beta-3 chain Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- KVRKAGGMEWNURO-CIUDSAMLSA-N Leu-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(C)C)N KVRKAGGMEWNURO-CIUDSAMLSA-N 0.000 description 1
- UCOCBWDBHCUPQP-DCAQKATOSA-N Leu-Arg-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O UCOCBWDBHCUPQP-DCAQKATOSA-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
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 1
- OIQSIMFSVLLWBX-VOAKCMCISA-N Lys-Leu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OIQSIMFSVLLWBX-VOAKCMCISA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 1
- KIPIKSXPPLABPN-CIUDSAMLSA-N Pro-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 KIPIKSXPPLABPN-CIUDSAMLSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- XVAUJOAYHWWNQF-ZLUOBGJFSA-N Ser-Asn-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O XVAUJOAYHWWNQF-ZLUOBGJFSA-N 0.000 description 1
- SNVIOQXAHVORQM-WDSKDSINSA-N Ser-Gly-Gln Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O SNVIOQXAHVORQM-WDSKDSINSA-N 0.000 description 1
- XKFJENWJGHMDLI-QWRGUYRKSA-N Ser-Phe-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O XKFJENWJGHMDLI-QWRGUYRKSA-N 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- PZVGOVRNGKEFCB-KKHAAJSZSA-N Thr-Asn-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N)O PZVGOVRNGKEFCB-KKHAAJSZSA-N 0.000 description 1
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 1
- JKGGPMOUIAAJAA-YEPSODPASA-N Thr-Gly-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O JKGGPMOUIAAJAA-YEPSODPASA-N 0.000 description 1
- UYTYTDMCDBPDSC-URLPEUOOSA-N Thr-Ile-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N UYTYTDMCDBPDSC-URLPEUOOSA-N 0.000 description 1
- IHAPJUHCZXBPHR-WZLNRYEVSA-N Thr-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N IHAPJUHCZXBPHR-WZLNRYEVSA-N 0.000 description 1
- 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
- BKIOKSLLAAZYTC-KKHAAJSZSA-N Thr-Val-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O BKIOKSLLAAZYTC-KKHAAJSZSA-N 0.000 description 1
- 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 1
- BPGDJSUFQKWUBK-KJEVXHAQSA-N Thr-Val-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BPGDJSUFQKWUBK-KJEVXHAQSA-N 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- AVYVKJMBNLPWRX-WFBYXXMGSA-N Trp-Ala-Ser Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 AVYVKJMBNLPWRX-WFBYXXMGSA-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
- SLOYNOMYOAOUCX-BVSLBCMMSA-N Trp-Phe-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SLOYNOMYOAOUCX-BVSLBCMMSA-N 0.000 description 1
- NUQZCPSZHGIYTA-HKUYNNGSSA-N Tyr-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N NUQZCPSZHGIYTA-HKUYNNGSSA-N 0.000 description 1
- BQASAMYRHNCKQE-IHRRRGAJSA-N Tyr-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N BQASAMYRHNCKQE-IHRRRGAJSA-N 0.000 description 1
- FEXILLGKGGTLRI-NHCYSSNCSA-N Val-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N FEXILLGKGGTLRI-NHCYSSNCSA-N 0.000 description 1
- PGQUDQYHWICSAB-NAKRPEOUSA-N Val-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N PGQUDQYHWICSAB-NAKRPEOUSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229940027570 adenoviral vector vaccine Drugs 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010079413 glycyl-prolyl-glutamic acid Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108700021021 mRNA Vaccine Proteins 0.000 description 1
- 229940126582 mRNA vaccine Drugs 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 108010018625 phenylalanylarginine Proteins 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 229940051173 recombinant immunotoxin Drugs 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Analytical Chemistry (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Communicable Diseases (AREA)
- Food Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Plant Pathology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
Abstract
The invention provides a nano antibody with specificity aiming at SARS-CoV-2RBD, relating to the technical field of antibodies. The specific nano antibody for SARS-CoV-2RBD provided by the invention is any one of SEQ ID No. 1-4. The constructed antibody library is screened to obtain the nano antibody of the invention, the nano antibody has good affinity with SARS-CoV-2RBD, and the nano antibody is applied to RBD purification, which shows high specificity, has high purity of purified protein, and realizes 'one-step' separation and purification.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to a nano antibody specifically aiming at SARS-CoV-2 RBD.
Background
In 2019, pneumonia caused by a novel coronavirus (SARS-CoV-2) spread all over the world, and has posed a serious threat to public life safety. Currently, four types of vaccines are approved for emergency use, including inactivated virus vaccines, recombinant adenoviral vector vaccines, DNA/mRNA vaccines and recombinant subunit protein vaccines. The recombinant subunit vaccine is easy for large-scale production and transportation, and has the advantage of high safety, so that the recombinant subunit vaccine can be used for preventing and controlling epidemic spread. The Receptor Binding Domain (RBD) located on the S protein of SARS-CoV-2 contains most of the neutralizing epitope of antibody, so the subunit protein vaccine of SARS-CoV-2 virus mainly uses the recombinant protein containing RBD region as immunogen to prevent the virus from binding with the ACE2 receptor of host by stimulating the immune response of organism to RBD structure domain.
Various recombinant protein drugs enter clinical research and are produced on the market all over the world, but the separation and purification of recombinant proteins are always key links in the development of pharmaceutical technology and cost control. Since the introduction of the concept of affinity chromatography by professor Cuatrecasas in 1968, affinity chromatography has developed into a powerful tool in biomedical research and biotechnology. Many recombinant Protein drugs currently use antibody Fc fragments as tags and can be purified using Protein a or Protein G chromatography columns. However, in some recombinant proteins not using an Fc tag, if 6 XHis is used as the tag, metal ions remain in the protein after purification by a metal ion affinity column. Therefore, designing and screening specific ligands with low cost, high stability and high specific binding with target protein to prepare affinity chromatography columns is a key step in drug production.
Nanobodies are the antigen-binding regions of heavy chain antibodies (IgG2 and IgG3) in camelids, also known as VHH fragments. Due to its small molecular weight and high stability, it is easy to mass-produce in bacterial expression systems and can be an important source of affinity ligands. Currently, several groups have used anti-Fc VHH antibodies to prepare affinity resins for purification of immunoglobulin g (igg) with a purification efficiency superior to that of classical Protein a purification. The Timothy Pabst group uses camel VHH antibodies as immunoaffinity ligands for "one-step" isolation and purification of biologically active drugs (e.g., recombinant immunotoxins and coagulation factors) to obtain biomolecules with high purity and activity. These applications suggest that nanobody-based affinity ligands can simplify the purification process of biomolecules while ensuring purity and activity.
The phage display technology is a molecular biology technology for displaying various biological macromolecules, including proteins, polypeptides, antibodies, TCR and the like, on the surface of a phage. If the phage library displaying billions of biomolecule variants interacts with the target, biomolecule monoclonals specifically binding to the target can be enriched after 3-5 rounds of biopanning. Based on different panning strategies, various targets such as antigen recombinant proteins, bacteria, viruses, cells, small molecules and the like can be selected to screen the phage library. For example, Rafique a cohort screens VHH from Fab-immunized alpacas for anti-Fab fragments and affinity maturation, ultimately designing VHH affinity ligands that are alkali resistant, easily eluted under acidic conditions, and have high affinity for Fabs.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The present inventors have made extensive efforts to screen and obtain antibodies specific for SARS-CoV-2 RBD. Therefore, the invention provides the following technical scheme.
In a first aspect, the present invention provides an antibody specific for SARS-CoV-2RBD comprising the 3 CDRs in SS1, 3 CDRs in SS3, 3 CDRs in SS4, or 3 CDRs in S12 as shown in table 1, preferably the antibody is a VHH antibody, a heavy chain antibody or a nanobody, characterized in that the amino acid sequence of the VHH antibody, heavy chain antibody or nanobody comprises any one of the amino acid sequences shown in SEQ ID nos. 1 to 4.
In a second aspect, the present invention provides a biomaterial related to the antibody of the first aspect, the biomaterial being any one of:
(a) a nucleic acid molecule encoding the antibody of claim 1;
(b) an expression cassette comprising the nucleic acid molecule of (a);
(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);
(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);
(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).
Preferably, the recombinant vector in (c) is a plasmid, such as pET-25 b.
Further preferably, the prokaryotic cell in (e) is escherichia coli, such as escherichia coli Rosetta 2.
In a third aspect, the invention provides a pharmaceutical composition comprising an antibody according to the first aspect.
In a fourth aspect, the present invention provides a method for producing the antibody of the first aspect, comprising introducing a nucleic acid molecule encoding the antibody of the first aspect into a recipient cell to obtain a transgenic cell, and culturing the transgenic cell to obtain the antibody.
Preferably, the recipient cell is a microbial cell or a mammalian cell.
The fifth aspect of the invention provides the use of the antibody of the first aspect or the biological material of the second aspect in the preparation of a product for detecting SARS-CoV-2.
According to a sixth aspect of the invention, there is provided the use of an antibody according to the first aspect or a biomaterial according to the second aspect for affinity chromatography purification of SARS-CoV-2 RBD.
In a seventh aspect, the invention provides the use of an antibody according to the first aspect or a biomaterial according to the second aspect in the manufacture of a medicament for the treatment of COVID-19.
More specifically, it is an object of the present invention to provide a nanobody specific for SARS-CoV-2RBD, which solves at least one of the technical problems of the prior art.
The second object of the present invention is to provide the above-mentioned nanobody-related biomaterial.
The third purpose of the present invention is to provide a heavy chain antibody containing the nanobody.
The fourth object of the present invention is to provide a method for preparing the above nanobody.
The fifth purpose of the present invention is to provide the application of the above-mentioned nano antibody or biomaterial.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a nano antibody specifically aiming at SARS-CoV-2RBD is disclosed, wherein the amino acid sequence of the nano antibody is any one of SEQ ID NO. 1-4.
The biological material related to the nano antibody is any one of the following materials:
(a) nucleic acid molecules encoding the nanobodies;
(b) an expression cassette comprising the nucleic acid molecule of (a);
(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);
(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);
(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).
Further, the backbone vector in (c) is pET-25b plasmid.
Further, the prokaryotic cell in (e) is Escherichia coli Rosetta 2.
Heavy chain antibody containing the nano antibody.
The preparation method of the nano antibody comprises the steps of introducing nucleic acid molecules for coding the nano antibody into receptor cells to obtain transgenic cells, and culturing the transgenic cells to obtain the nano antibody.
Further, the recipient cell is a microbial cell or a mammalian cell.
The application of the nano antibody or the biological material in preparing SARS-CoV-2 products.
The application of the nano antibody or the biological material in the affinity chromatography purification of SARS-CoV-2 RBD.
The application of the nano antibody or the biological material in preparing a medicament for treating COVID-19.
Compared with the prior art, the invention has the following organic effects:
the specific SARS-CoV-2RBD specific nano antibody provided by the invention is any one of SEQ ID No. 1-4. The constructed antibody library is screened to obtain the nano antibody of the invention, the nano antibody has good affinity with SARS-CoV-2RBD, and the nano antibody is applied to RBD purification, which shows high specificity, has high purity of purified protein, and realizes 'one-step' separation and purification.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is 4 groups with differences in CDRH3 regions obtained by NGS sequencing provided in example 4 of the present invention;
FIG. 2 is an SDS-PAGE (12%) electrophoresis chart of Anti-RBD nanobody provided in example 5 of the present invention;
FIG. 3 shows the detection of the affinity of the nano-antibody VHHs and the antigen (100ng rRBD, human FC tag) provided in example 6 of the present invention;
FIG. 4 shows that the Octet RED384 detection antibodies (SS1, SS4, S12) provided in example 7 of the present invention competitively bind to rRBD;
FIG. 5 shows that the Octet RED384 detection antibodies (SS3, SS4) provided in example 7 of the present invention bind to rRBD non-competitively;
FIG. 6 is a graph showing the results of SS4-Sepharose purified rRBD provided in example 8 of the present invention, wherein lane 1: an RBD positive control; lane 2: after SS4-Sepharose 4FF binds rRBD, 20 microliter of filler is taken and added into 20 microliter of 6 XProtein Loading Buffer for mixing, and the mixture is treated for 20min at 98 ℃; lane 3: eluting;
FIG. 7 is a graph showing the results of SS3-Sepharose 4FF purification of rRBD provided in example 8 of the present invention, wherein lane 1: rRBD eluted from the packing; lane 2: the filler after elution;
FIG. 8 is a rRBD affinity assay recovered after purification of rRBD from SS3-Sepharose 4FF provided in example 8 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The inventor utilizes S1 recombinant protein to immunize alpaca, constructs a VHH phage library, and biologically screens to obtain nano antibody clones combined with RBD, specifically four nano antibodies shown in SEQ ID NO. 1-4.
The invention also provides the biological material related to the nano antibody, which can be nucleic acid molecules, expression cassettes, recombinant vectors, recombinant eukaryotic cells and recombinant prokaryotic cells.
A "nucleotide molecule" may be DNA, such as cDNA or recombinant DNA, or RNA, such as mRNA or hnRNA, etc.
An "expression cassette" comprises a polynucleotide sequence encoding the polypeptide to be expressed (single domain antibody) and sequences controlling its expression such as a promoter and optionally enhancer sequences, including any combination of cis-acting transcriptional control units. Sequences that control the expression of a gene (i.e., its transcription and translation of the transcription product) are often referred to as regulatory units. Most of the regulatory units are located upstream of and operably linked to the coding sequence of the gene. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site.
The vector of the "recombinant vector" may be a plasmid, a phage or a virus.
The host cell of the "recombinant eukaryotic cell" may be a yeast or mammalian cell, etc.
The host cell of the "recombinant prokaryotic cell" may be a bacterium, an alga or the like.
In a preferred embodiment, the recombinant vector is a backbone vector, such as pET-25b plasmid, as a vector of nucleic acid molecules encoding the nanobody of the present invention. The host cell in the recombinant prokaryotic cell may be, for example, Escherichia coli Rosetta 2.
The invention also protects heavy chain antibodies containing the nanobody of the invention.
The invention relates to a preparation method of a nano antibody, which comprises the steps of introducing nucleic acid molecules for coding the nano antibody into receptor cells to obtain transgenic cells, and culturing the transgenic cells to obtain the nano antibody. Among them, the recipient cell may be a microbial cell or a mammalian cell, preferably escherichia coli Rosetta 2.
The nano antibody and the related biological material thereof provided by the invention can be used for preparing products for detecting SARS-CoV-2, such as immunochromatographic test paper, ELISA detection kit and the like; can also be used for affinity chromatography purification of SARS-CoV-2 RBD; can also be used for preparing medicine for treating COVID-19.
The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.
Example 1: antigen preparation and alpaca immunization
Alpaca (Alpaca) immunization: injecting SARS-COV-2 Spike S1 recombinant protein into the neck and back of alpaca subcutaneously and intramuscularly at multiple points to form multiple masses, and tracking and observing the absorption condition of the subcutaneous injection masses to confirm the correctness of immunity. For the first immunization, 0.5mg of antigen is mixed with Freund's complete adjuvant 1:1, emulsified and injected, and the volume is 1mL per alpaca; and (3) second immunization: 3 weeks after the first immunization, 0.25mg of antigen is mixed with Freund's incomplete adjuvant 1:1, emulsified and injected, and the injection volume is 1 mL/alpaca; and (3) third immunization: 3 weeks after the second immunization, 0.25mg of antigen was mixed with Freund's incomplete adjuvant 1:1, emulsified and injected into a volume of 1 mL/alpaca; and (3) fourth immunization: 3 weeks after the third immunization, 0.25mg of antigen was mixed with Freund's incomplete adjuvant 1:1, emulsified and injected in a volume of 1 mL/alpaca.
Serum treatment and titer detection: one week after the fourth immunization, 50mL of peripheral blood was collected, and serum and lymphocytes were separated. The RBD-his antigen was coated in an ELISA 96-well plate, and antibody titer in serum was measured by ELISA. ELISA results showed alpaca quadruplicate immune serum titers > 1: 32000 and meets the library building standard.
Example 2: construction and screening of phage display immune antibody library
Since the fourth serum titer is > 1: 32000, indicating the presence of high affinity antibodies against rRBD in the serum. And further constructing a phage display immune antibody library, and obtaining a nano antibody positive monoclonal of the anti-human rRBD through biological screening.
Collecting blood after fourth immunization of alpaca, and separating lymphocyte PBMC; taking 2X 10 7 Extracting total RNA by using an RNA extraction kit; an appropriate amount of RNA (e.g., 3-5. mu.g) is taken and cDNA is obtained by RT-PCR reverse transcription kit.
IgG2 and IgG3 heavy chain variable region sequences (heavy chain variable region VHH of nanobody) were obtained stepwise by nested PCR, the experimental procedure was as follows:
1) designing two pairs of specific primers to amplify the alpaca heavy chain antibody gene segment, wherein the nested lateral primers are positioned in an antibody heavy chain signal peptide region and a highly conserved region of a CH2 structural domain, and the pair of primers are used for amplifying a VH-CH1-CH2 segment with the size of 900bp and a VH-CH2 segment with the size of 700bp respectively; the nested inside primers were used to amplify a heavy chain antibody variable region VHH fragment of about 400bp from a VH-CH2 fragment of 700 bp.
2) Using cDNA as template, using nested lateral primer to make first round PCR amplification, separating product DNA gel electrophoresis, cutting gel and recovering 700bpPCR product. 3) And amplifying the 700bp product obtained in the first round by using nested inner side primer PCR to obtain a target gene VHH fragment, and purifying and recovering by using a PCR product purification kit.
Inserting the heavy chain variable region sequence into a linearized phagemid vector VHH-libTemplate subjected to enzyme digestion treatment in a homologous recombination or enzyme digestion connection mode to obtain a recombinant vector; after purification and recovery, super competent SS320 cells (containing helper phage M13K07) are transformed; resuspending and activating the transformed bacterial liquid for 1 hour by using an SOC culture medium; taking a small amount of bacterial liquid to perform gradient dilution by 10 times, selecting proper dilution titer, coating the plate on LB/tet10 and LB/Carb50 culture plates, placing the plates in a biochemical incubator at 37 ℃ overnight, and using the next day for calculating the storage capacity; transferring the residual bacteria liquid into a large-volume 2YT/Carb50/Kan25 liquid culture medium, placing the liquid culture medium in a shaking table at 37 ℃, culturing overnight, harvesting supernate the next day, adding 1/4 times of volume of PEG/NaCl solution, precipitating phages, taking a proper amount of PBT solution, re-suspending and diluting to the required concentration to obtain a phage display immune antibody library, and storing the phage display immune antibody library at (-80 ℃ for later use).
The number of clones on LB/Carb50 plates was counted, and the library volume was calculated to be 7.8X 10 9 . 10 single clones were randomly picked from each plate and sequenced with an insertion efficiency of 90%.
Example 3: screening of antibody libraries
Repeating the operation for 3-5 times until the phage enrichment occurs. Successful enrichment was considered if the number of colonies on LB/Carb50 plates from antigen-binding wells was more than 10 times greater than the number of colonies from negative control wells. In this experiment, after the second round of screening, the number of colonies in the antigen binding wells was 1000 times that in the negative control wells, indicating successful enrichment, the Phage ELISA was performed to select high affinity positive clones.
Example 4: high throughput sequencing
Designing primers in constant regions on two sides of the VHH, amplifying a variable region of a phage library, carrying out E-gel detection on a PCR product, and then purifying and recovering PCR library fragments by using a kit for NGS sequencing. The data obtained were statistically analyzed using SPSS 2.0 and Microsoft Excel 2019, and the high throughput sequencing data were used to count and map the sequencing results using Geneius and Clustal Omega. The sequences with the DNA frequency of the first 200 are divided into 4 groups according to the CDRH3 difference of the sequences, and VHHs (SS1, SS3, SS4, S12) with the highest DNA frequency are selected from each group to carry out the next experiment (as shown in figure 1). The amino acid sequences of SS1, SS3, SS4 and S12 are shown in Table 1 below.
TABLE 1 amino acid sequence of anti-RBD Nanobody cloning
Wherein the sequences in italics and in abscissa are the CDR1, CDR2 and CDR3 sequences in that order.
Example 5: prokaryotic expression of Nanobodies
Prokaryotic expression was performed on the sequences in Table 1 of example 4, sequences SS1, SS3, SS4, S12, with the following experimental steps:
1) cloning the VHH fragment amplified by PCR into pET-25b plasmid through enzyme cutting sites BamH I and Xho I, electrically transforming Escherichia coli Rosetta 2, screening ampicillin, sequencing single clone to obtain correct recombinant plasmid pET-25 b-VHH-His;
2) selecting a monoclonal containing a recombinant plasmid, carrying out shake culture at 37 ℃ until OD600 is 0.6-0.8, adding 0.5mM IPTG, and carrying out shake culture at 25 ℃ overnight; collecting thalli the next day, carrying out ultrasonic disruption on the thalli, collecting supernatant, and purifying by nickel affinity chromatography to obtain the VHH-His fusion protein. Prokaryotic expression information is shown in Table 2 below, and the results of SDS-PAGE (12%) electrophorogram are shown in FIG. 2.
TABLE 2 prokaryotic expression of anti-RBD Nanobody clones
Example 6 evaluation of the affinity of VHH by Elisa method
Coating RBD-his antigen (2 mu g/mL) in a 96-well plate, incubating overnight at 4 ℃, removing the antigen supernatant of the 96-well plate the next day, adding 200 mu L of 1% PVA to each hole for sealing, adding 200 mu L of 1% PVA to a blank hole as a negative control hole, and placing the blank hole in a 3D rotary oscillator at room temperature for 2 h; then, remove the supernatant of the protein and control wells, wash 3 times with 200 μ L PT, pat dry, add 100 μ L of gradient diluted primary antibody (SS1, SS3, SS4, S12), incubate for 2h at room temperature, remove the supernatant of the protein and control wells, wash with 200 μ L PT; detecting the combined nano antibody by using an anti-His secondary antibody connected with HRP, detecting for 1h at room temperature, washing, adding 100 mu L of TMB for color development, and measuring the OD value at 450 nm. The analysis results show that SS1, SS3, SS4 and S12 all show good antigen binding activity (as shown in FIG. 3).
Example 7 detection of Epitope Difference in recognition of VHHs epitopes by Epitope Binning method
Firstly, diluting an antigen with a PBST solution to prepare the antigen with a final concentration of 5 mu g/mL; preparing an antibody: diluting the antibody with PBST solution, wherein the final concentration of SS1, SS4 and S12 is 1 μ M, and the final concentration of SS3 is 60 μ M; then, detecting the difference of VHHs epitope by Octet, taking Protein A Sensor solidified antigen (5 mu g/mL), and solidifying the antigen to 3.0 nm; as a preliminary experiment, an antigen was immobilized on a protein a sensor through an Fc region, and signals of binding of nanobodies SS1, SS3, SS4, S12 to the antigen for a short time were detected, and the antibodies SS1, SS3, SS4, S12 bound well to the antigen;
then, antibody SS4 was immobilized on the protein a sensor and saturated with rbd, and then the complex was incubated with each of the indicated antibodies (SS1, SS3, S12), respectively; when the antibody on the sensor competes with the antibody in solution for the same epitope, no additional binding to the antigen is observed. After SS4 bound the antigen, SS1 and S12 did not exhibit significant binding reactions (positions shown as arrows in fig. 4), indicating that the SS1 and S12 binding epitopes are highly overlapping with the SS4 binding epitope. In contrast, SS3 was able to bind antigen again after saturation of SS4 bound antigen (position shown by arrow in fig. 5), indicating that the epitope bound by SS3 was different from the epitope bound by SS 4.
Example 8 purification of rRBD from SS4-Sepharose 4FF and SS3-Sepharose 4FF
The CNBr-4FF medium was resuspended in 5 column volumes of buffer A (0.001M HCl, 0.5M NaCl, pH 3.0), the solvent was completely drained after 5min, and the procedure was repeated 5 times. Selecting nanometer antibodies (SS4, SS3) binding different epitopes as affinity ligands according to example 7 to couple with affinity filler Sepharose 4 FF: displacement of the coupled sample to 0.2M NaHCO 3 0.5M NaCl, pH 8.3 buffer, and concentrated the sample using an ultrafiltration tube. And (3) mixing the washed medium and the concentrated sample in equal volume, gently mixing uniformly for 3 hours at room temperature, then extracting the sample solution, and determining the concentration of the coupled sample extract. ③ resuspend the coupled medium with 5 volumes of buffer B (0.02M Tris-HCl solution, pH 8.3), completely dry the solvent, and repeat this step 3 times. Then, 5 times of buffer solution B is used for resuspension, and after the mixture is gently mixed for 2 hours at room temperature, the solution is drained. Fourthly, the sealed medium is resuspended by 5 times volume of buffer solution C (0.05M Tris-HCl, 0.5M NaCl, pH value is 8.0), and the solvent is drained after 5 min; the medium was then resuspended in 5 volumes of buffer D (0.05M Glycine, 0.5M NaCl, pH3.5) and after 5min the solution was drained. This procedure was repeated 3 times and stored with 20% ethanol. Determination of coupling amount: the Nanodrop determines the content of the antibody (280nm) before and after the reaction, and calculates the coupling amount according to the formula (2-1):
5 column volumes of PBS buffer were added, the solution was run off and the packing settled to the bottom. The sample to be purified (rRBD) was added and allowed to flow out slowly, typically at a flow rate of 1 mL/min. Collecting effluent, and repeating or circulating the loading if special needs exist. The wash was performed with PBS at a flow rate of about 1mL/min, and the protein concentration was monitored using a UV detector until the A280 value stabilized. After washing, adding a conventional elution buffer solution comprising 1)0.1M glycine-HCl, pH 2.5; 2)0.1M citric acid, 0.5M NaCl, pH 2.5; 3)0.1M glycine-NaOH, pH 10; 4)45mM citric acid, 70mM arginine, pH 3.5; 5)20mM Tris, 2M MgCl 2 The pH value is 7.4; 6)50mM Tris, 0.2% SDS, 0.1% Tween-20, pH 8.0. The flow rate of the eluent is about 1mL/min, and the eluent is monitored by an ultraviolet detector until the A280 value is stable. The eluate was collected to calculate the protein content and the protein recovery. Adding 20% ethanol into the chromatographic column, and storing at 4 deg.C. Immediately after elution, the protein was replaced into a stock solution of 10mm Sodium acetate, 270mm sucrose, 0.01% Tween-20 at pH 6.8 and stored at-80 ℃.
The activated coupling amounts of SS3 and SS4 to CNBr were 4.6mg/mL and 8.3mg/mL, respectively. SS3 and SS4 show good coupling performance under the primary amino immobilization mode. The filler was washed with 0.05M Tris-HCl, 0.5M NaCl, pH8.0 buffer and 0.05M Glycine, 0.5M NaCl, pH3.5 buffer, and the nanobody was hardly lost, indicating that SS3 and SS4 could be stably coupled to the column. Compared with the positive control, the rRBD band adsorbed from the affinity chromatography column has almost no foreign protein, which indicates that SS3 and SS4 have high specificity. The affinity chromatography column is used for purifying rRBD protein, loading buffer solution is added for washing, and the hybrid protein which is not adsorbed by the immunoaffinity column flows out of the column. Compared with the positive control, the rRBD protein band adsorbed by the affinity chromatography column has almost no foreign protein, which indicates that SS3 and SS4 have high specificity to the rRBD. The conventional eluent was added for 10min, and the adsorbed rRBD of SS4-Sepharose 4FF could not be eluted (FIG. 6). In contrast, SS3-Sepharose 4 FF-adsorbed rRBD eluted under 50mM Tris, 0.2% SDS, 0.1% Tween-20 (pH8.0) buffer conditions with a protein recovery of 50% and a purity of 90% (FIG. 7) and an EC50 of 0.1nM (FIG. 8).
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Chengdu flourishing age Junlian Biotechnology Co., Ltd
<120> Nanobody specific for SARS-CoV-2RBD
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 111
<212> PRT
<213> Artificial sequence
<220>
<223> SS1 antibody
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly
1 5 10 15
Ser Leu Thr Leu Ala Cys Thr Ala Ser Gly Ser Gly Val Ser Ile Arg
20 25 30
Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Glu Leu Val
35 40 45
Ala Arg Leu Thr Ala Val Leu Ala Thr Met Ile His Asn Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn
85 90 95
Ser Gly Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 2
<211> 118
<212> PRT
<213> Artificial sequence
<220>
<223> SS3 antibody
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Gly
1 5 10 15
Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Arg Asn Met Arg
20 25 30
Gly Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Phe Thr Ala Ala Gly Ala Thr Leu Ser Ser Asn Ala Val Glu
50 55 60
Gly Arg Phe Thr Leu Ser Ala Asp Arg Ala Lys Asn Thr Val Asp Leu
65 70 75 80
Val Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
His Arg Pro Ser Phe Gly Pro Glu Asn Glu Ser Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 3
<211> 111
<212> PRT
<213> Artificial sequence
<220>
<223> SS4 antibody
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Pro Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Thr Ala Ser Gly Asn Ile Arg Asn Leu Asn
20 25 30
Gly Val Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Asp Leu Val
35 40 45
Ala Gln Phe Thr Asn Val Gly Ala Thr Leu Ser Lys Asp Ser Leu Glu
50 55 60
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Leu Arg Ser Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 4
<211> 116
<212> PRT
<213> Artificial sequence
<220>
<223> S12 antibody
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Ala Ile Ser Ser Phe Asp
20 25 30
Ala Val Thr Trp Tyr Arg Gln Val Pro Gly Asn Ala Arg Ala Phe Ile
35 40 45
Ala Ala Ile Ser Gly Gly Asp Val Arg Ser Tyr Ala Arg Ser Ala Lys
50 55 60
Asp Arg Phe Thr Ile Phe Arg Asp Asn Asp Lys Asn Thr Val Asn Leu
65 70 75 80
Glu Met Asn Lys Leu Thr Pro Glu Asp Thr Gly Thr Tyr Val Cys His
85 90 95
Trp Ala Ser His Ser Gly Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser
115
Claims (10)
1. An antibody specific for SARS-CoV-2RBD comprising 3 CDRs in SS1, 3 CDRs in SS3, 3 CDRs in SS4, or 3 CDRs in S12 as set forth in Table 1, preferably the antibody is a VHH antibody, a heavy chain antibody or a nanobody, characterized in that the amino acid sequence of the VHH antibody, heavy chain antibody or nanobody comprises any one of the amino acid sequences set forth as SEQ ID No. 1-4.
2. The antibody-related biomaterial according to claim 1, wherein the biomaterial is any one of:
(a) a nucleic acid molecule encoding the antibody of claim 1;
(b) an expression cassette comprising the nucleic acid molecule of (a);
(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);
(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);
(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).
3. The biomaterial according to claim 2, wherein the recombinant vector in (c) is a plasmid, such as pET-25 b.
4. The biomaterial according to claim 2, wherein the prokaryotic cell in (e) is E.coli, such as E.coli Rosetta 2.
5. A pharmaceutical composition comprising the antibody of claim 1.
6. The method for producing an antibody according to claim 1, wherein the antibody is obtained by introducing a nucleic acid molecule encoding the antibody according to claim 1 into a recipient cell to obtain a transgenic cell, and culturing the transgenic cell.
7. The method according to claim 6, wherein the recipient cell is a microbial cell or a mammalian cell.
8. Use of the antibody of claim 1 or the biological material of any one of claims 2-4 in the preparation of a product for detecting SARS-CoV-2.
9. Use of the antibody of claim 1 or the biological material of any one of claims 2-4 for affinity chromatography purification of SARS-CoV-2 RBD.
10. Use of an antibody according to claim 1 or a biomaterial according to any one of claims 2 to 4 in the manufacture of a medicament for the treatment of COVID-19.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210626397.XA CN115124616A (en) | 2022-06-02 | 2022-06-02 | Nano antibody with specificity aiming at SARS-CoV-2RBD |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210626397.XA CN115124616A (en) | 2022-06-02 | 2022-06-02 | Nano antibody with specificity aiming at SARS-CoV-2RBD |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115124616A true CN115124616A (en) | 2022-09-30 |
Family
ID=83377631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210626397.XA Pending CN115124616A (en) | 2022-06-02 | 2022-06-02 | Nano antibody with specificity aiming at SARS-CoV-2RBD |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115124616A (en) |
-
2022
- 2022-06-02 CN CN202210626397.XA patent/CN115124616A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111995676B (en) | Monoclonal antibody aiming at non-RBD (radial basis function) region of new coronavirus spike protein and application thereof | |
| CN108251431B (en) | Double-carrier system and application thereof | |
| CN110655574B (en) | Nano antibody aiming at green fluorescent protein, application and GFP immunoaffinity adsorption material | |
| CN113045647B (en) | Neutralizing antibody of novel coronavirus SARS-CoV-2 and application thereof | |
| CN109970858B (en) | CD22 single domain antibody, nucleotide sequence and kit | |
| CN111138537B (en) | Anti-human serum albumin antibody fragment, preparation method and application | |
| CN108892723B (en) | Single-domain heavy chain antibody for detecting porcine epidemic diarrhea virus, preparation method and application | |
| WO2023216623A1 (en) | CAMEL-DERIVED HIGH-AFFINITY NANOANTIBODY AGAINST SARS-COV-2 α, γ, δ AND ο MUTANTS | |
| WO2023125520A1 (en) | CAMEL-DERIVED NANOBODY WITH HIGH-AFFINITY FOR α, β, γ AND δ MUTANT STRAINS OF SARS-COV-2 | |
| CN110862455B (en) | Polypeptide capable of binding CD47 and application thereof | |
| CN111138536B (en) | Preparation and application of anti-human serum albumin single-domain antibody | |
| CN110372793B (en) | Nano antibody of PD-L1 and clinical application thereof | |
| CN110423277B (en) | Nano antibody of PD-1 and clinical application thereof | |
| Urushibata et al. | Generation of Fab fragment-like molecular recognition proteins against staphylococcal enterotoxin B by phage display technology | |
| CN108659124B (en) | Single-chain antibody for resisting porcine epidemic diarrhea virus and application thereof | |
| EP3632924A1 (en) | Binders for inhibiting formation of multimeric proteins | |
| CN111138532B (en) | Use of single domain antibodies against hepatitis a virus | |
| CN114805559B (en) | Fully human anti-novel coronavirus receptor binding domain single-chain antibody No4 and application thereof | |
| CN110423274B (en) | Anti-pseudomonas aeruginosa exotoxin A nano antibody and application thereof | |
| CN110885375A (en) | Single-domain antibody specifically aiming at ZnMc structural domain of MMP9 protein, product and application | |
| WO2022037031A1 (en) | Il-5 binding molecule, preparation method therefor, and use thereof | |
| CN115124616A (en) | Nano antibody with specificity aiming at SARS-CoV-2RBD | |
| CN113461816A (en) | Nano antibody aiming at green fluorescent protein GFP and application thereof | |
| CN114478761A (en) | Green fluorescent protein shark source nano antibody, preparation method and application thereof | |
| CN113980127A (en) | Nano antibody for resisting methicillin-resistant staphylococcus and preparation method and application thereof |
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 |