CN110878309B - 结核分枝杆菌分泌蛋白esat-6单抗的重链和轻链可变区基因和编码的多肽及其应用 - Google Patents
结核分枝杆菌分泌蛋白esat-6单抗的重链和轻链可变区基因和编码的多肽及其应用 Download PDFInfo
- Publication number
- CN110878309B CN110878309B CN201911231047.8A CN201911231047A CN110878309B CN 110878309 B CN110878309 B CN 110878309B CN 201911231047 A CN201911231047 A CN 201911231047A CN 110878309 B CN110878309 B CN 110878309B
- Authority
- CN
- China
- Prior art keywords
- esat
- monoclonal antibody
- variable region
- chain variable
- light chain
- 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
- 101710166488 6 kDa early secretory antigenic target Proteins 0.000 title claims abstract description 86
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 86
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 17
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 17
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 17
- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 16
- 102000040739 Secretory proteins Human genes 0.000 title claims abstract description 4
- 108091058545 Secretory proteins Proteins 0.000 title claims abstract description 4
- 201000008827 tuberculosis Diseases 0.000 claims abstract description 29
- 229960005486 vaccine Drugs 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims description 9
- 230000001355 anti-mycobacterial effect Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 20
- 241000699670 Mus sp. Species 0.000 abstract description 15
- 210000004408 hybridoma Anatomy 0.000 abstract description 12
- 230000003053 immunization Effects 0.000 abstract description 10
- 230000003248 secreting effect Effects 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 6
- 206010035226 Plasma cell myeloma Diseases 0.000 abstract description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 5
- 201000000050 myeloid neoplasm Diseases 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000009465 prokaryotic expression Effects 0.000 abstract description 3
- 210000004989 spleen cell Anatomy 0.000 abstract description 2
- 241001646725 Mycobacterium tuberculosis H37Rv Species 0.000 abstract 2
- 108010077805 Bacterial Proteins Proteins 0.000 abstract 1
- 230000037029 cross reaction Effects 0.000 abstract 1
- 150000001413 amino acids Chemical class 0.000 description 24
- 238000001514 detection method Methods 0.000 description 23
- 239000000427 antigen Substances 0.000 description 21
- 102000036639 antigens Human genes 0.000 description 21
- 108091007433 antigens Proteins 0.000 description 21
- 208000015181 infectious disease Diseases 0.000 description 20
- 102000004169 proteins and genes Human genes 0.000 description 20
- 238000000034 method Methods 0.000 description 15
- 241000588724 Escherichia coli Species 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 238000003745 diagnosis Methods 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 238000002649 immunization Methods 0.000 description 7
- 230000001018 virulence Effects 0.000 description 7
- 108010074328 Interferon-gamma Proteins 0.000 description 6
- 101001057048 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) ESAT-6-like protein EsxB Proteins 0.000 description 6
- 108700020164 Mycobacterium tuberculosis ESAT-6 Proteins 0.000 description 6
- 210000004602 germ cell Anatomy 0.000 description 6
- 210000004698 lymphocyte Anatomy 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 241000193998 Streptococcus pneumoniae Species 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000002238 attenuated effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 108020001507 fusion proteins Proteins 0.000 description 5
- 102000037865 fusion proteins Human genes 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 241000187480 Mycobacterium smegmatis Species 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000028996 humoral immune response Effects 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 238000011998 interferon-gamma release assay Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 229960001005 tuberculin Drugs 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 241000186366 Mycobacterium bovis Species 0.000 description 3
- 101100333823 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) esxA gene Proteins 0.000 description 3
- 102100035107 Neurotrimin Human genes 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 3
- 101150079015 esxB gene Proteins 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000000405 serological effect Effects 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- SYZZMPFLOLSMHL-XHNCKOQMSA-N Gln-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)C(=O)O SYZZMPFLOLSMHL-XHNCKOQMSA-N 0.000 description 2
- 206010065048 Latent tuberculosis Diseases 0.000 description 2
- 241000186359 Mycobacterium Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 206010036790 Productive cough Diseases 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
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 208000033353 latent tuberculosis infection Diseases 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000002796 luminescence method Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 210000000680 phagosome Anatomy 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 101150076401 16 gene Proteins 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- KUDREHRZRIVKHS-UWJYBYFXSA-N Ala-Asp-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KUDREHRZRIVKHS-UWJYBYFXSA-N 0.000 description 1
- KXEVYGKATAMXJJ-ACZMJKKPSA-N Ala-Glu-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O KXEVYGKATAMXJJ-ACZMJKKPSA-N 0.000 description 1
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 1
- NTAZNGWBXRVEDJ-FXQIFTODSA-N Arg-Asp-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NTAZNGWBXRVEDJ-FXQIFTODSA-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
- NLRJGXZWTKXRHP-DCAQKATOSA-N Asn-Leu-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NLRJGXZWTKXRHP-DCAQKATOSA-N 0.000 description 1
- LTDGPJKGJDIBQD-LAEOZQHASA-N Asn-Val-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LTDGPJKGJDIBQD-LAEOZQHASA-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
- KYQNAIMCTRZLNP-QSFUFRPTSA-N Asp-Ile-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O KYQNAIMCTRZLNP-QSFUFRPTSA-N 0.000 description 1
- HTSSXFASOUSJQG-IHPCNDPISA-N Asp-Tyr-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HTSSXFASOUSJQG-IHPCNDPISA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- OZHXXYOHPLLLMI-CIUDSAMLSA-N Cys-Lys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OZHXXYOHPLLLMI-CIUDSAMLSA-N 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 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 1
- 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 1
- FQCILXROGNOZON-YUMQZZPRSA-N Gln-Pro-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O FQCILXROGNOZON-YUMQZZPRSA-N 0.000 description 1
- AKDOUBMVLRCHBD-SIUGBPQLSA-N Gln-Tyr-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AKDOUBMVLRCHBD-SIUGBPQLSA-N 0.000 description 1
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 1
- OCJRHJZKGGSPRW-IUCAKERBSA-N Glu-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O OCJRHJZKGGSPRW-IUCAKERBSA-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
- 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
- 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 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
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 1
- 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
- MDOBWSFNSNPENN-PMVVWTBXSA-N His-Thr-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O MDOBWSFNSNPENN-PMVVWTBXSA-N 0.000 description 1
- DLTCGJZBNFOWFL-LKTVYLICSA-N His-Tyr-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N DLTCGJZBNFOWFL-LKTVYLICSA-N 0.000 description 1
- 101001008255 Homo sapiens Immunoglobulin kappa variable 1D-8 Proteins 0.000 description 1
- 101001047628 Homo sapiens Immunoglobulin kappa variable 2-29 Proteins 0.000 description 1
- 101001008321 Homo sapiens Immunoglobulin kappa variable 2D-26 Proteins 0.000 description 1
- 101001047619 Homo sapiens Immunoglobulin kappa variable 3-20 Proteins 0.000 description 1
- 101001008263 Homo sapiens Immunoglobulin kappa variable 3D-15 Proteins 0.000 description 1
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 1
- NZGTYCMLUGYMCV-XUXIUFHCSA-N Ile-Lys-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N NZGTYCMLUGYMCV-XUXIUFHCSA-N 0.000 description 1
- DZMWFIRHFFVBHS-ZEWNOJEFSA-N Ile-Tyr-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N DZMWFIRHFFVBHS-ZEWNOJEFSA-N 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 241000209027 Ilex aquifolium Species 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 102100022949 Immunoglobulin kappa variable 2-29 Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- 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
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 1
- ZXFRGTAIIZHNHG-AJNGGQMLSA-N Lys-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CCCCN)N ZXFRGTAIIZHNHG-AJNGGQMLSA-N 0.000 description 1
- RBEATVHTWHTHTJ-KKUMJFAQSA-N Lys-Leu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O RBEATVHTWHTHTJ-KKUMJFAQSA-N 0.000 description 1
- 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 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
- SPSSJSICDYYTQN-HJGDQZAQSA-N Met-Thr-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O SPSSJSICDYYTQN-HJGDQZAQSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010062207 Mycobacterial infection Diseases 0.000 description 1
- 108700003823 Mycobacterium MPB64 Proteins 0.000 description 1
- 241001467552 Mycobacterium bovis BCG Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- FSPGBMWPNMRWDB-AVGNSLFASA-N Phe-Cys-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N FSPGBMWPNMRWDB-AVGNSLFASA-N 0.000 description 1
- FQUUYTNBMIBOHS-IHRRRGAJSA-N Phe-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N FQUUYTNBMIBOHS-IHRRRGAJSA-N 0.000 description 1
- YMIZSYUAZJSOFL-SRVKXCTJSA-N Phe-Ser-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O YMIZSYUAZJSOFL-SRVKXCTJSA-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
- BAONJAHBAUDJKA-BZSNNMDCSA-N Phe-Tyr-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=CC=C1 BAONJAHBAUDJKA-BZSNNMDCSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- OOKCGAYXSNJBGQ-ZLUOBGJFSA-N Ser-Asn-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OOKCGAYXSNJBGQ-ZLUOBGJFSA-N 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- 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
- ZOPISOXXPQNOCO-SVSWQMSJSA-N Ser-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CO)N ZOPISOXXPQNOCO-SVSWQMSJSA-N 0.000 description 1
- FPCGZYMRFFIYIH-CIUDSAMLSA-N Ser-Lys-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O FPCGZYMRFFIYIH-CIUDSAMLSA-N 0.000 description 1
- IFLVBVIYADZIQO-DCAQKATOSA-N Ser-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N IFLVBVIYADZIQO-DCAQKATOSA-N 0.000 description 1
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 1
- RXUOAOOZIWABBW-XGEHTFHBSA-N Ser-Thr-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RXUOAOOZIWABBW-XGEHTFHBSA-N 0.000 description 1
- KIEIJCFVGZCUAS-MELADBBJSA-N Ser-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CO)N)C(=O)O KIEIJCFVGZCUAS-MELADBBJSA-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
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 1
- VRUFCJZQDACGLH-UVOCVTCTSA-N Thr-Leu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VRUFCJZQDACGLH-UVOCVTCTSA-N 0.000 description 1
- 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
- IEZVHOULSUULHD-XGEHTFHBSA-N Thr-Ser-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O IEZVHOULSUULHD-XGEHTFHBSA-N 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
- RCMHSGRBJCMFLR-BPUTZDHNSA-N Trp-Met-Asn Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(O)=O)=CNC2=C1 RCMHSGRBJCMFLR-BPUTZDHNSA-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
- 108010050970 Type VII Secretion Systems Proteins 0.000 description 1
- YGKVNUAKYPGORG-AVGNSLFASA-N Tyr-Asp-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YGKVNUAKYPGORG-AVGNSLFASA-N 0.000 description 1
- UMXSDHPSMROQRB-YJRXYDGGSA-N Tyr-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O UMXSDHPSMROQRB-YJRXYDGGSA-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
- ZYVAAYAOTVJBSS-GMVOTWDCSA-N Tyr-Trp-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O ZYVAAYAOTVJBSS-GMVOTWDCSA-N 0.000 description 1
- ABSXSJZNRAQDDI-KJEVXHAQSA-N Tyr-Val-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ABSXSJZNRAQDDI-KJEVXHAQSA-N 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- VJOWWOGRNXRQMF-UVBJJODRSA-N Val-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 VJOWWOGRNXRQMF-UVBJJODRSA-N 0.000 description 1
- OVLIFGQSBSNGHY-KKHAAJSZSA-N Val-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N)O OVLIFGQSBSNGHY-KKHAAJSZSA-N 0.000 description 1
- ZXYPHBKIZLAQTL-QXEWZRGKSA-N Val-Pro-Asp Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N ZXYPHBKIZLAQTL-QXEWZRGKSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- JXWGBRRVTRAZQA-ULQDDVLXSA-N Val-Tyr-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N JXWGBRRVTRAZQA-ULQDDVLXSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000003452 antibody preparation method Methods 0.000 description 1
- 210000000776 antibody secreting cell Anatomy 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 230000008349 antigen-specific humoral response Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000000091 biomarker candidate Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 101150016690 esxA gene Proteins 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001759 immunoprophylactic effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 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
- 208000019423 liver disease Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 101150006328 mpb83 gene Proteins 0.000 description 1
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 1
- 230000007110 pathogen host interaction Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229940021993 prophylactic vaccine Drugs 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- -1 vaccines Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
-
- 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
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—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
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- 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
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及结核分枝杆菌分泌蛋白ESAT‑6单抗的重链和轻链可变区基因和编码的多肽及其应用。采用原核表达系统获得结核分枝杆菌毒株H37Rv Rv3875编码的ESAT‑6重组蛋白;重组蛋白免疫小鼠,将免疫小鼠的脾细胞与骨髓瘤细胞SP2/0融合,筛选并获得可持续分泌抗ESAT‑6抗体的杂交瘤细胞系,命名为B1C8。单克隆抗体B1C8可特异识别结核分枝杆菌毒株H37Rv的ESAT‑6,而与其他细菌蛋白无交叉反应。因此,所述的单克隆抗体B1C8重链和轻链可变区基因及编码的多肽可用于结核分枝杆菌感染的诊断、试剂、疫苗和药物的研制,具有比较好的应用前景。
Description
技术领域
本发明属于生物医药技术领域,涉及免疫学和分子生物学相关领域。特别涉及一种结核分枝杆菌ESAT-6蛋白单克隆抗体的重链和轻链可变区基因及其编码的多肽,以及所述基因和编码的多肽作为制备结核分枝杆菌感染的诊断、试剂、疫苗和药物的应用。
背景技术
结核病(tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染引起的一种慢性传染性疾病。WHO报告显示,2018年全球约有1 000万新发病例,死亡约145万,其中HIV阳性者死亡约25万。结核病是全球尤其是发展中国家危害最为严重的慢性传染病之一(WHO Global tuberculosis report 2019.https://www.who.int/tb/publications/global_report/en/)。开发检测速度快、特异性强、灵敏度高的诊断方法是TB疫情控制的关键步骤。卡介苗(BCG)是TB惟一预防疫苗,但其保护率并不完善。临床辅助诊断Mtb的结核菌素皮肤试验(tuberculin skin test,TST)使用结核菌素纯蛋白衍生物(purified protein derivative tuberculin,PPD)混合物为诊断试剂,不能区分Mtb感染和BCG,且检测结果受环境中存在的非结核分枝杆菌(nontuberculosis mycobacteria,NTM)影响。Mtb耐药性问题日益严重,TB合并艾滋病病例不断增加。因此,加强对TB诊断、预防、治疗制剂的研究对于控制TB疫情具有重要意义。
比较Mtb和BCG基因组发现,BCG在减毒的过程中丢失了16个基因差异区(regionof deletion,RD)。早期分泌靶抗原6-kDa(early secretory antigenic target 6kDa,ESAT-6)和CFP-10(culture filtrate protein 10kDa,CFP-10)由RD1区基因编码,在BCG减毒过程中最早丢失。ESAT-6由Mtb RD1区Rv3875(esxA)基因编码,由288个碱基编码的95个氨基酸组成,是一种非信号肽依赖的分泌型小分子抗原。ESAT-6和CFP-10(Rv3874,esxB)受相同的启动子和操纵子调控,一般情况下这两种分泌蛋白形成1:1的异源二聚体发挥功能(Renshaw PS,et al.Structure and function of the complex formed by thetuberculosis virulence factors CFP-10 and ESAT-6.EMBO J.2005 Jul 20;24(14):2491-8.)。ESAT-6存在于Mtb中,而不存在于BCG及绝大多数NTM中(Bennekov T,etal.Alteration of epitope recognition pattern in Ag85B and ESAT-6 has aprofound influence on vaccine-induced protection against Mycobacteriumtuberculosis.Eur J Immunol.2006Dec;36(12):3346-55.),因此被认为是最有应用潜能的诊断抗原。
ESAT-6有多个T细胞和B细胞表位,在Mtb感染早期被机体免疫系统识别,诱导机体产生抗原特异性的体液免疫和细胞免疫应答(Marei A,et al.Superior T cellactivation by ESAT-6 as compared with the ESAT-6-CFP-10 complex.IntImmunol.2005 Nov;17(11):1439-46.)。ESAT-6抗原用于Mtb感染的血清学诊断具有较高的特异性。研究者发现CFP10/ESAT-6抗原与MPB83联用检测牛分枝杆菌感染,可显著提高血清学检测的敏感性(Miller MA,et al.Serological reactivity to MPB83 and CFP10/ESAT-6 antigens in three suid hosts of Mycobacterium bovis infection.VetMicrobiol.2019 Aug;235:285-288.)。Mtb感染的机体内存在多种抗原致敏的效应T细胞,这些细胞在体外受到相同抗原的刺激后会分泌大量的IFN-γ,可以通过检测效应T细胞IFN-γ的释放来判断感染情况。研究发现,ESAT-6可刺激Mtb感染小鼠淋巴细胞增殖,并诱导感染小鼠淋巴细胞释放IFN-γ(Brandt L,Oettinger T,Holm A.Key epitopes on theESAT-6 antigen recognized in mice during the recall of protective immunity toMycobacterium tuberculosis.J Immunol.1996 Oct 15;157(8):3527-33.)。基于该原理的γ-干扰素释放试验(iterferon-gamma release assay,IGRA)已被应用于TB临床诊断。基于ESAT-6和CFP-10建立的TB-IGRA诊断方法已经形成了2种商业化的检测技术T-SPOT.TB和QuantiFERON-Gold(Kim CH,et al.Diagnostic performance of the QuantiFERON-TBGold In-Tube assay and factors associated with nonpositive results inpatients with miliary tuberculosis.Clin Infect Dis.2014 Apr;58(7):986-9.)。用ESAT-6作为刺激抗原,ELISPOT方法检测不同程度的Mtb感染者外周血中分泌IFN-γ的T细胞,发现未感染过Mtb的人群呈阴性反应,而TST阳性、TB密切接触者、淋巴结核患者、痰菌阴性及阳性的肺结核患者均呈阳性反应(Pathan AA,et al.Direct ex vivo analysis ofantigen-specific IFN-gamma-secreting CD4 T cells in Mycobacteriumtuberculosis-infected individuals:associations with clinical disease stateand effect of treatment.J Immunol.2001 Nov 1;167(9):5217-25.)。ESAT-6 ELISPOT诊断活动性TB比PPD ELISPOT和TST的敏感性和特异性均高(Lalvani A,Nagvenkar P,Udwadia Z,ey al.Enumeration of T cells specific for RD1-encoded antigenssuggests a high prevalence of latent Mycobacterium tuberculosis infection inhealthy urban Indians.J Infect Dis.2001 Feb 1;183(3):469-77.)。IGRA检测方法灵敏度高,检出率在85%以上,具有高度的特异性。IGRA在Mtb感染检测的实际临床应用中表现出良好的敏感性和特异性,在欧美等TB低水平流行地区被推荐为TST的替代检测方法。
ESAT-6被认为是一种穿孔素(pore-forming toxin,PFT),与Mtb的毒力相关,ESAT-6缺失的Mtb株毒力减退(Clemmensen HS,An attenuated Mycobacteriumtuberculosis clinical strain with a defect in ESX-1 secretion induces minimalhost immune responses and pathology.Sci Rep.2017Apr 24;7:46666.)。研究发现,Mtb的ESAT-6通过VII型分泌系统ESX-1分泌系统被分泌至细菌胞外,在酸性pH条件下ESAT-6插入吞噬体膜上形成跨膜孔,介导Mtb从巨噬细胞中的吞噬体向胞浆转移,从而激活下游的细胞内信号通路,影响病原菌-宿主间的相互作用,促进了Mtb在组织间的播散(Houben D,etal.ESX-1-mediated translocation to the cytosol controls virulence ofmycobacteria.Cell Microbiol.2012 Aug;14(8):1287-98.)。研究表明,比Mtb H37Rv株毒力强的Erdman、CDC1551和HN878菌株产生更多的ESAT-6蛋白(Luis Solans,et al.ASpecific Polymorphism in Mycobacterium tuberculosis H37Rv Causes DifferentialESAT-6 Expression and Identifies WhiB6 as a Novel ESX-1 Component.InfectImmun.2014 Aug;82(8):3446–3456.)。然而,与Mtb ESAT-6氨基酸序列具有高度同源(72%相似性)的非致病性的耻垢分枝杆菌(Mycobacterium smegmatis,Ms)中的ESAT-6同源蛋白MSMEG_0066却并未发现其打孔效应(Peng X,Sun J.Mechanism of ESAT-6 membraneinteraction and its roles in pathogenesis of Mycobacteriumtuberculosis.Toxicon.2016 Jun 15;116:29-34.)。上述研究表明,ESAT-6是影响分枝杆菌毒力的关键因素,其表达量与毒力成正比,因而也成为抗Mtb感染的重要靶点。
ESAT-6诱导的免疫应答对Mtb感染动物有一定的保护作用(Jiang Q,et al.Anovel recombinant DNA vaccine encoding Mycobacterium tuberculosis ESAT-6 andFL protects against Mycobacterium tuberculosis challenge in mice.J BiomedRes.2013Sep;27(5):406-20.)。第四军医大学(现为空军军医大学)前期研究发现,过表达ESAT-6的重组BCG可诱导更强的体液和细胞免疫应答(Wang LM,et al.Expression andimmunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérinstrains secreting the antigen ESAT-6 from Mycobacterium tuberculosis inmice.Chin Med J(Engl).2007 Jul 20;120(14):1220-5.),ESAT-6-MPT64融合蛋白免疫可诱导小鼠高水平的体液免疫应答,淋巴细胞增殖增加以及IFN-γ分泌增加(Bai YL,etal.Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64fusion protein and its immunoprophylactic potential in mouse model.ProteinExpr Purif.2008 Jun;59(2):189-96.),Ag85B-ESAT-6融合蛋白免疫可诱导豚鼠较高水平的特异性体液免疫应答(许承明等.融合蛋白亚单位疫苗对结核分枝杆菌感染豚鼠和小鼠免疫治疗效果的初步评价[D].第四军医大学,2014,11(02):71-76),Ag85B-ESAT-6融合蛋白免疫可诱导Th1型为主的强烈的免疫应答,有助于机体清除Mtb的感染(Wang,P,etal.Immunotherapeutic efficacy of recombinant Mycobacterium smegmatisexpressing Ag85B-ESAT6 fusion protein against persistent tuberculosisinfection in mice.Hum Vaccin Immunother,2014.10(1):150-8.)。由于ESAT-6可诱导机体产生相应的体液免疫和细胞免疫应答,因此也被广泛应用于新型TB疫苗的研究。
宿主细胞内ESAT-6(intracellular ESAT-6,i/c ESAT-6)与机体内有活的Mtb直接相关。采用抗ESAT-6单克隆抗体对Mtb感染宿主细胞内的i/c ESAT-6进行标记,TB患者阳性检出率高于90%,该方法灵敏度高、特异性强,且检测结果不受机体免疫状态的影响,能够区分真正的潜伏性结核感染和宿主细胞对Mtb抗原的免疫应答,被认为是检测Mtb感染、潜伏性结核感染以及复发感染的候选生物标志物(Poulakis N,et al.IntracellularESAT-6:A New Biomarker for Mycobacterium Tuberculosis Infection.Cytometry BClin Cytom.2016 May;90(3):312-4)。基于抗ESAT-6、抗CFP-10单克隆抗体,研究者开发了适用于多种类型临床样本的胶体金免疫层析试纸检测方法,发现该方法对痰液、培养基样品敏感性高(检出率>90%)、特异强(>88%),可用于TB筛查(Wu X,et al.Preparation ofimmunochromatographic strips for rapid detection of early secreted proteinESAT-6 and culture filtrate protein CFP-10 from Mycobacteriumtuberculosis.Medicine(Baltimore).2017 Dec;96(51):e9350.)。国外研究者使用抗ESAT-6单克隆抗体制备了无标记电化学免疫传感器,能够检测三种Mtb致病菌株(CDC1551,H37Rv和H8N8)的细胞培养滤液中分泌的天然ESAT-6抗原,其检出下限低至7ng/mL(DiouaniMF et al.Detection of ESAT-6 by a label free miniature immuno-electrochemicalbiosensor as a diagnostic tool for tuberculosis.Mater Sci Eng C Mater BiolAppl.2017,74(5):465-470.)。因此,抗ESAT-6单克隆抗体具有较大的检测应用潜能。
目前,商业化市售的ESAT-6单克隆抗体(monoclonal antibody,mAb)仅有Abcam公司生产的11G4(ab26246),这一mAb在ESAT-6的相关研究中应用较多。国内多家单位制备了ESAT-6的鼠源单克隆抗体,包括陈薇课题组(结核分枝杆菌ESAT-6抗原鼠单克隆抗体的制备及初步鉴定[J].生物技术通讯,2011,22(02):258-260.)、姚航平课题组(抗结核杆菌ESAT-6单克隆抗体TBEA8及应用[P].浙江:CN101987872A,2011-03-23.)、焦新安课题组(结核分枝杆菌ESAT-6蛋白单克隆抗体的研制与鉴定[D].扬州大学,2014.)。ESAT-6单克隆抗体的成功制备为ESAT-6应用于Mtb检测、诊断、疫苗及机制研究提供了支持。通过检索国际国家专利汇编数据库(http://patentscope2.wipo.int/),发现目前国内外针对ESAT-6单克隆抗体可变区序列及其编码氨基酸的研究尚无报道。
发明内容
本发明的目的在于提供一种抗ESAT-6单克隆抗体的重链和轻链可变区基因及其编码的多肽,以及所述基因和多肽在Mtb感染的诊断、试剂、疫苗和药物中的应用。
本发明是通过以下技术方案来实现的:
采用分子生物学技术,PCR法从Mtb H37Rv基因组中扩增Rv3875(esat-6)基因,克隆入原核表达载体pET28a(+),诱导表达目的蛋白,用Ni亲和层析法纯化并获得重组的ESAT-6蛋白。重组蛋白皮下免疫BALB/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞SP2/0融合,通过间接ELISA法筛选获得杂交瘤细胞系,命名为B1C8。取上述杂交瘤细胞经小鼠腹腔接种制备腹水,中压液相色谱仪纯化获得单克隆抗体,经鉴定该单克隆抗体可与Mtb ESAT-6蛋白特异性结合。
本发明所述分泌单克隆抗体细胞株的重、轻链可变区基因,是通过提取上述杂交瘤细胞B1C8的RNA,经RT-PCR法获得该单克隆抗体的重链和轻链可变区基因。经过序列测定和在NCBI中BLAST比较分析后,确认所述抗体的重链可变区的核苷酸序列具有SEQ ID NO.1的序列,其编码的氨基酸序列具有SEQ ID NO.2的序列;所述抗体的轻链可变区基因核苷酸序列具有SEQ ID NO.3的序列,其编码的氨基酸序列具有SEQ ID NO.4的序列。
对于本发明成功克隆的单克隆抗体B1C8重、轻链可变区基因,分别与已知抗体基因序列数据库(IMGT)和(NCBI)进行同源性及其胚系基因来源比较和分析,结果表明所获得的基因序列来自鼠胚系基因,与现有报道的各种抗体基因序列均不完全一致。
本发明还涉及所述抗体的重、轻链可变区基因及其编码的多肽在制备Mtb感染的诊断、试剂、疫苗和药物中的应用。
以本发明所述的单克隆抗体,可建立夹心法ELISA夹心法等基于抗原-抗体的反应体系,可用于Mtb的酶联免疫检测。
本发明采用RT-PCR方法成功克隆了所述单克隆抗体的重、轻链可变区基因(VH、VL)。基于上述的重、轻链可变区基因,可用于构建和表达成多种形式的小分子基因工程抗体,如ScFv抗体、Fab抗体、F(ab)2抗体、抗体融合蛋白等,用于Mtb感染的诊断、试剂、疫苗和药物等的设计及应用。由于ESAT-6编码基因仅在Mtb毒株和牛分枝杆菌中特异性存在,本研究获得的单克隆抗体的重、轻链可变区基因可为Mtb感染精准诊断试剂、疫苗和药物的研制奠定一定的基础。
与现有技术相比,本发明具有以下有益的技术效果:
1、本发明获得的单克隆抗体B1C8与Mtb毒株ESAT-6的亲和力高,能够特异性识别Mtb标准毒株H37Rv中ESAT-6,但不识别Mtb减毒株H37Ra中的ESAT-6,推测H37Ra中的ESAT-6表达水平很低,故检测不出;单克隆抗体B1C8与疫苗株BCG、NTM耻垢分枝杆菌(Ms)、肺炎链球菌(Spn)以及大肠埃希菌(E.coli)的菌体裂解物无交叉反应,表明单克隆抗体B1C8具有良好的特异性。
2、本发明克隆的单克隆抗体B1C8的重链、轻链可变区基因和氨基酸序列,序列分析证实了该抗体序列的惟一性。
3、分析单克隆抗体B1C8的重链、轻链可变区基因,获得可变区的CDR区,为Mtb感染的诊断、检测试剂的制备提供了支持。
附图说明
图1间接ELISA法检测ESAT-6-B1C8 mAb抗体亚类结果图
图2间接ELISA法检测ESAT-6-B1C8 mAb抗体亲和力检测结果图
图3 Western-blot检测ESAT-6-B1C8 mAb对ESAT-6识别结果图
图4 Western-blot检测ESAT-6-B1C8 mAb对不同细菌菌体蛋白识别结果图
具体实施方式:
本发明采用分子生物学技术,PCR法从Mtb H37Rv基因组中扩增获得Rv3875(esat-6)基因,构建原核表达载体并转化入E.coli,在IPTG诱导下表达ESAT-6蛋白,用Ni亲和层析法纯化并获得重组的ESAT-6蛋白。纯化的ESAT-6蛋白免疫雌性BALB/c小鼠,取免疫小鼠脾淋巴细胞与骨髓瘤细胞SP2/0融合;通过间接ELISA法筛选获得能稳定分泌高亲和力抗ESAT-6单克隆抗体的杂交瘤细胞株,命名为B1C8。取上述杂交瘤细胞经小鼠腹腔接种制备腹水,中压液相色谱仪纯化获得纯化的单克隆抗体,经鉴定该单克隆抗体可与Mtb ESAT-6蛋白特异性结合。提取B1C8细胞株的总RNA,RT-PCR法获得该单克隆抗体基因序列;通过测序确认了该单克隆抗体基因可变区序列,并通过分析确定了相应的蛋白序列的惟一性及其互补决定区(complementarity determining regions,CDR)序列;为该单克隆抗体的重、轻链可变区基因及其编码的多肽用于结核分枝杆菌及其他分枝杆菌感染的试剂、疫苗和药物的研制提供技术支持。
以下将单克隆抗体B1C8的重、轻链可变区基因及其编码的多肽制备方法以及抗体序列惟一性做详细说明,所述是对本发明的解释而不是限定。
本发明具体按以下步骤实施:
1小鼠抗ESAT-6高亲和力抗体的制备
1.1单克隆抗体的制备与纯化
按照单克隆抗体制备方法(实用单克隆抗体技术,徐志凯主编,P9-P11),用E.coli中表达并纯化的ESAT-6重组蛋白免疫雌性BALB/c小鼠(购自空军军医大学实验动物中心),初次皮下免疫抗原50μg/只+弗氏不完全佐剂;间隔两周,皮下免疫两次;第三次皮下免疫抗原25μg/只;第三次免疫完成后一周,尾静脉采血,检测免疫效果。免疫小鼠加强免疫抗原25μg/只,完成后3天,处死小鼠,制备小鼠脾淋巴细胞悬液并计数。
取对数生长期的小鼠骨髓瘤细胞SP2/0并计数,将骨髓瘤细胞与脾淋巴细胞按照5:1的比例进行细胞融合。融合后的细胞悬液加入含有饲养细胞(正常雌性BALB/c小鼠腹腔巨噬细胞)的96孔板,37℃、5%CO2培养,至细胞克隆出现后,取细胞上清以ESAT-6重组蛋白包被的96孔板间接ELISA检测,挑选阳性克隆。对含有阳性克隆的细胞采用有限稀释法进行克隆化,直至获得能够稳定分泌抗ESAT-6抗体的杂交瘤细胞系,命名为B1C8。
取上述杂交瘤细胞系B1C8扩大培养,接种石蜡油预处理的小鼠腹腔,制备腹水,中压液相色谱仪纯化腹水,获得纯化的单克隆抗体。对B1C8分泌的抗体进行亚类测定,结果表明该单克隆抗体为IgG1亚类,κ型轻链(图1)。
1.2单克隆抗体效价和亲和力测定
用间接ELISA的方法检测单克隆抗体B1C8的相对亲和力。包被抗原为纯化的MtbESAT-6蛋白,待测样品为梯度稀释的纯化的单克隆抗体,检测抗体为HRP-山羊抗小鼠IgG,显色底物为TMB,显色终止液为2M H2SO4。ELISA结果表明,单克隆抗体B1C8纯化抗体效价为1:25 600,亲和力为3.9×10-5(图2)。
1.3单克隆抗体对重组ESAT-6蛋白的识别
将蛋白Marker、纯化的ESAT-6蛋白进行SDS-PAGE;电泳结束后转膜、封闭;一抗分别用His mAb(1:1 000)、Mtb感染小鼠多抗血清(1:400)、纯化的单克隆抗体B1C8(1:20000)进行孵育;二抗为HRP-山羊抗小鼠IgG(1:5 000);ECL发光法检测单克隆抗体B1C8对不同抗原的识别作用。Western blot结果显示,单克隆抗体B1C8可特异性识别纯化的ESAT-6蛋白(图3)。
1.4单克隆抗体对天然ESAT-6蛋白的识别
分别制备Mtb标准毒株(H37Rv)、Mtb减毒株(H37Ra)、BCG、耻垢分枝杆菌(Ms)、肺炎链球菌(Spn)以及大肠埃希菌(E.coli)菌体蛋白。将蛋白Marker和待测各蛋白样品依次上样,进行SDS-PAGE;电泳结束后转膜、封闭;一抗为纯化的单克隆抗体B1C8(1:20 000);二抗为HRP-山羊抗小鼠IgG(1:5 000);ECL发光法观察不同细菌中的蛋白表达。Western blot结果显示,单克隆抗体B1C8可特异地识别Mtb标准毒株H37Rv中的ESAT-6蛋白,而不识别H37Ra同源蛋白MRA_3914(100%相似性),也不识别Ms同源蛋白MSMEG_0066(72%相似性),且不与BCG、Spn、E.coli菌体蛋白非特异性反应,表明单克隆抗体B1C8具有良好的特异性(图4)。
2单克隆抗体重链和轻链可变区基因的克隆
2.1单克隆抗体重链和轻链可变区序列的扩增
将上述分泌单克隆抗体B1C8的杂交瘤细胞用含10%胎牛血清的RPMI 1640完全培养基于37℃、5%CO2孵箱中培养至对数生长期。采用TRIzol(Invitrogen公司)裂解法提取杂交瘤细胞总RNA,定量后采用逆转录试剂盒(TaKaRa公司)进行cDNA的合成。以逆转录获得的cDNA为PCR扩增模板,采用PCR扩增试剂盒(TaKaRa公司)进行扩增,重链可变区引物为VHF(上游引物)和VH R(下游引物),轻链可变区引物为VL F(上游引物)和VL R(下游引物)进行扩增,分别获得单克隆抗体B1C8的VH、VL基因片段。
PCR反应体系为50μL,扩增程序为:95℃1min;95℃5s,58℃30s,72℃1min,循环35次;72℃5min。引物序列为(括号内为简并引物):
2.2单克隆抗体重链和轻链可变区序列的克隆
PCR产物经1%琼脂糖糖凝胶电泳,用PCR清洁回收试剂盒(Axygen公司)回收目的基因片段,用DNA连接试剂盒(TaKaRa公司)将目的基因回收片段分别与pMD19-T Vector(TaKaRa公司)载体连接,连接产物转化至E.coli DH5α感受态细胞,涂布于含Amp抗生素的LB平板,37℃过夜培养。
挑取含Amp抗生素LB平板上的克隆作为PCR模板,分别以重链对应的引物VH F、VHR和轻链对应的引物VL F、VL R为引物,PCR鉴定筛选阳性的E.coli DH5α转化子扩大培养,用质粒小量提取试剂盒(Axygen公司)提取质粒,送至生工生物工程(上海)股份有限公司进行基因测序。测序所得重链可变区的基因序列如SEQ ID NO.1所示,轻链可变区的基因序列如SEQ ID NO.3所示。
3单克隆抗体重链和轻链可变区序列的同源性分析
3.1单克隆抗体可变区核苷酸序列的同源性分析
可变区序列测序无误后,对单克隆抗体B1C8的重、轻链可变区基因分别应用NCBI(GenBank+EMBL+DDBJ+PDB)数据库(http://www.ncbi.nlm.nih.gov/blast)和IMGT数据库(http://www.imgt.org)进行核苷酸序列同源性分析,对所得序列与现有已报道的其它各种抗体基因进行同源性比较,并分析其胚系基因来源。
序列比对结果显示,单克隆抗体B1C8的重链可变区基因序列与编号为GenBank:AB050074.1的小鼠Ig重链可变区基因序列同源性最高,为350/368(95%)。单克隆抗体B1C8的轻链可变区基因序列与编号为GenBank:AF045512.1的小鼠Ig轻链可变区基因序列同源性最高,达310/321(97%)。结果如下显示:
(1)单克隆抗体重链<400>1的胚系基因来源
V-GENE:Musmus IGHV6-6*02F
J-GENE:Musmus IGHJ2*01F
D-GENE:Musmus IGHD2-4*01F
通过FR-IMGT and CDR-IMGT分析显示:
CDR1:ggattcactttcagtaactactgg
CDR2:attaaattgaaatctaacaattatgtaaca
CDR3:acgttctatgattacgacgaaatctactttgactac
NCBI中同源比对结果显示:
RID:89J7WFFM01R
Query Length:447
Database Name:All non-redundant GenBank+EMBL+DDBJ+PDB sequences(noEST,STS,GSS or HTGS sequences)
Sequence ID:AB050074.1 Mus musculus VH10G1 mRNA for anti-dsRNA(RDV-RNA)antibody,partial cds
Length:477
Score:575bits(311)
Expect:5e-160
Identities:350/368(95%)
Gaps:5/368(1%)
Strand:Plus/Plus
(2)单克隆抗体轻链<400>3的胚系基因来源:
V-GENE:Musmus IGKV6-23*01 F
J-GENE:Musmus IGKJ2*01 F
通过FR-IMGT and CDR-IMGT分析显示:
CDR1:caggatgtggatactgct
CDR2:tgggcatcc
CDR3:cagcaatatatcagttatccgtacacg
NCBI中同源比对结果显示:
RID:89JRZEXX015
Query Length:420
Database Name:All non-redundant GenBank+EMBL+DDBJ+PDB sequences(noEST,STS,GSS or HTGS sequences)
Sequence ID:AF045512.1 Mus musculus 9E10 monoclonal antibody kappalight chain variable region,(IgK)mRNA,partial cds
Length:381
Score:532bits(288)
Expect:3e-147
Identities:310/321(97%)
Gaps:0/321(0%)
Strand:Plus/Plus
序列同源性分析表明,编码单克隆抗体B1C8的重、轻链可变区的核苷酸序列,来源于鼠胚系基因,但与现有报道的各种单克隆抗体基因序列均不完全一致,表明本发明在基因序列上具有惟一性。
3.2单克隆抗体可变区氨基酸序列的同源性分析
单克隆抗体B1C8重链可变区的氨基酸序列如SEQ ID NO.2,轻链可变区的氨基酸序列如SEQ ID NO.4所示。在non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF蛋白质数据库中,进行氨基酸序列同源性分析(Blastp)。分析结果表明,单克隆抗体B1C8重链氨基酸序列与编号为BAB87186.1的小鼠Ig重链可变区蛋白的同源性最高,为111/122(91%)。单克隆抗体B1C8轻链氨基酸序列与编号为AAT76273.1的小鼠Ig轻链可变区蛋白的同源性最高,达102/108(94%)。重链、轻链氨基酸同源比对结果如下所示:
(1)单克隆抗体重链氨基酸序列<400>2比对结果:
RID:89KB8X7G015
Query ID:lcl|Query_366857
Query Length:121
Database Name:All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples from WGS projects
Sequence ID:BAB87186.1 anti-dsRNA(RDV-RNA)antibody,partial[Musmusculus]
Length:155
Score:227 bits(579)
Expect:2e-74
Identities:111/122(91%)
Positives:115/122(94%)
Gaps:1/122(0%)
(2)单克隆抗体轻链氨基酸序列<400>4同源比对结果:
RID:89M34JR2014
Query ID:lcl|Query_215378
Query Length:108
Database Name:All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples from WGS projects
Sequence ID:AAT76273.1 immunoglobulin light chain variable region,partial[Mus musculus]
Length:139
Score:213bits(514)
Expect:3e-69
Identities:102/108(94%)
Positives:105/108(97%)
Gaps:0/108(0%)
同源性分析表明,单克隆抗体B1C8重、轻链可变区的氨基酸序列,为鼠源性蛋白,虽然与其他蛋白氨基酸序列有同源性,但未发现与本发明完全相同的氨基酸序列,表明本发明在氨基酸序列上也具有惟一性。
3.3确定CDR区
将测序所得单克隆抗体B1C8重链和轻链可变区序列,在VBASE2网站(http://www.vbase2.org/vbase2.php)进行分析,得出其CDR区。
所述单克隆抗体B1C8重链可变区的3个互补决定区(CDR)序列,如SEQ ID NO.2部分所示,具体为:
CDR1:Gly-Phe-Thr-Phe-Ser-Asn-Tyr-Trp
CDR2:Ile-Lys-Leu-Lys-Ser-Asn-Asn-Tyr-Val-Thr
CDR3:Thr-Phe-Tyr-Asp-Tyr-Asp-Glu-Ile-Tyr-Phe-Asp-Tyr
单克隆抗体B1C8轻链可变区的3个互补决定区(CDR)序列,如SEQ ID NO.4所示,具体为:
CDR1:Gln-Asp-Val-Asp-Thr-Ala
CDR2:Trp-Ala-Ser
CDR3:Gln-Gln-Tyr-Ile-Ser-Tyr-Pro-Tyr-Thr
抗Mtb ESAT-6单克隆抗体B1C8重、轻链可变区基因及其编码的多肽产物的应用
对Mtb H37Rv的ESAT-6序列与Mtb减毒株H37Ra、疫苗株BCG、非结核分枝杆菌Ms及常见病原菌(肺炎链球菌、大肠埃希菌)数据库中的蛋白质氨基酸序列进行分析,Mtb H37RaESAT-6与H37Rv ESAT-6的氨基酸序列均完全一致,但单克隆抗体B1C8仅识别毒株H37Rv中的ESAT-6,而不识别H37Ra ESAT-6。由于ESAT-6表达量与细菌毒力成正比,推测减毒株H37Ra毒力低,其ESAT-6表达量低或不表达,因此单克隆抗体检测不出。Ms同源蛋白MSMEG_0066与Mtb ESAT-6的氨基酸序列有72%的相似性,BCG、肺炎链球菌和大肠杆菌中均不含有ESAT-6蛋白,单克隆抗体B1C8与Ms、BCG、肺炎链球菌和大肠杆菌菌体蛋白无反应,表明本发明制备的单克隆抗体B1C8可特异性识别Mtb H37Rv毒株,因此可用于Mtb感染的检测。由于ESAT-6诱导的免疫应答有助于机体抵抗Mtb感染,单克隆抗体B1C8也可用于Mtb疫苗的研究及免疫治疗药物的研制。
本发明制备的单克隆抗体B1C8对Mtb ESAT-6具有良好的特异性,可用于ESAT-6相互作用蛋白的鉴定研究,为Mtb ESAT-6及相互作用抗原的分离、纯化、鉴定以及TB诊断与防治提供了重要支持。本发明成功获得了单克隆抗体B1C8的重、轻链可变区基因(VH、VL),可用于构建和表达成多种形式的小分子基因工程抗体或药物,例如:小分子抗体,主要Fab抗体、单链抗体、Fv片段抗体、单域抗体及由单个CDR构成的最小识别单位等,用于Mtb感染的诊断、试剂、疫苗或药物的研制。
序列表
<110> 中国人民解放军第四军医大学
<120> 结核分枝杆菌ESAT-6蛋白单抗的重链和轻链可变区基因和编码的多肽及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 363
<212> DNA
<213> 人工序列()
<400> 1
gaagtgaaga ttgaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60
tcctgtgttg cctctggatt cactttcagt aactactgga tgaactgggt ccgccagtct 120
ccagagaagg ggcttgagtg ggttgctgaa attaaattga aatctaacaa ttatgtaaca 180
cattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240
gtctacctgc aaatgaacaa cttaagagct gaagacactg gcatttatta ctgtacgttc 300
tatgattacg acgaaatcta ctttgactac tggggccaag gcaccactct cacagtctcc 360
tca 363
<210> 2
<211> 121
<212> PRT
<213> 人工序列()
<220>
<221> V_region
<222> (26)..(.33)
<223> 重链CDR1区
<220>
<221> V_region
<222> (51)..(.60)
<223> 重链CDR2区
<220>
<221> V_region
<222> (99)..(.110)
<223> 重链CDR3区
<400> 2
Glu Val Lys Ile Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Lys Leu Lys Ser Asn Asn Tyr Val Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr
85 90 95
Tyr Cys Thr Phe Tyr Asp Tyr Asp Glu Ile Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 3
<211> 324
<212> DNA
<213> 人工序列()
<400> 3
gacattgtga tgacgcagtc tcccaaattc atgtccacat cagtaggaga cagggtcagc 60
atcacctgca aggccagtca ggatgtggat actgctgtag cctggtatca acagaaacca 120
ggccaatctc ctaaaatact gatttactgg gcatccaccc gacacactgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcagtct 240
gaagacttgg cagattattt ctgtcagcaa tatatcagtt atccgtacac gttcggaggg 300
gggaccaagg tggagatcaa acgt 324
<210> 4
<211> 108
<212> PRT
<213> 人工序列()
<220>
<221> V_region
<222> (27)..(.32)
<223> 轻链CDR1区
<220>
<221> V_region
<222> (50)..(.52)
<223> 轻链CDR2区
<220>
<221> V_region
<222> (89)..(.97)
<223> 轻链CDR3区
<400> 4
Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ile Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ile Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105
Claims (4)
1.一种结核分枝杆菌分泌蛋白ESAT-6单抗的重链和轻链可变区基因,其特征在于,其重链可变区基因如序列SEQ ID NO.1所示,轻链可变区基因如序列SEQ ID NO.3所示。
2.由权利要求1所述的单抗重链和轻链可变区基因编码的多肽,其特征是:其重链可变区基因编码多肽如序列SEQ ID NO.2所示,其轻链可变区基因编码多肽如序列SEQ ID NO.4所示。
3.由权利要求1所述的单抗重链和轻链可变区基因在制备结核分枝杆菌的诊断试剂、疫苗或制备抗结核分枝杆菌药物的应用。
4.由权利要求2所述的单抗重链和轻链可变区基因编码的多肽在制备结核分枝杆菌诊断试剂、疫苗或制备抗结核分枝杆菌药物的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911231047.8A CN110878309B (zh) | 2019-12-05 | 2019-12-05 | 结核分枝杆菌分泌蛋白esat-6单抗的重链和轻链可变区基因和编码的多肽及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911231047.8A CN110878309B (zh) | 2019-12-05 | 2019-12-05 | 结核分枝杆菌分泌蛋白esat-6单抗的重链和轻链可变区基因和编码的多肽及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110878309A CN110878309A (zh) | 2020-03-13 |
CN110878309B true CN110878309B (zh) | 2023-09-26 |
Family
ID=69730023
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911231047.8A Active CN110878309B (zh) | 2019-12-05 | 2019-12-05 | 结核分枝杆菌分泌蛋白esat-6单抗的重链和轻链可变区基因和编码的多肽及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110878309B (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109988770B (zh) * | 2019-01-10 | 2022-11-22 | 中国人民解放军第四军医大学 | 一种c-di-AMP合成酶单克隆抗体的重链和轻链可变区基因和编码的多肽及其应用 |
CN112759644A (zh) * | 2021-01-28 | 2021-05-07 | 中国人民解放军空军军医大学 | 结核分枝杆菌mpt64蛋白单克隆抗体的重链和轻链可变区基因和编码的多肽及其应用 |
CN113584117A (zh) * | 2021-07-15 | 2021-11-02 | 潍坊医学院 | 高通量鉴定病原菌与宿主分子之间的相互作用的方法 |
CN116376845A (zh) * | 2023-02-28 | 2023-07-04 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | 针对豚鼠IFN-γ的单克隆抗体及其用途 |
CN116925212B (zh) * | 2023-08-09 | 2024-08-02 | 中国农业大学 | Hbha单克隆抗体及其在抗牛分枝杆菌感染中的应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1712629A2 (en) * | 1995-09-01 | 2006-10-18 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
CN101985473A (zh) * | 2010-10-12 | 2011-03-16 | 浙江大学 | 抗结核杆菌esat-6单克隆抗体tbef3及应用 |
CN101987872A (zh) * | 2010-10-12 | 2011-03-23 | 浙江大学 | 抗结核杆菌esat-6单克隆抗体tbea8及应用 |
AU2013254904A1 (en) * | 2006-03-02 | 2014-02-20 | The Uab Research Foundation | Mycobacterial disease detection, treatment, and drug discovery |
-
2019
- 2019-12-05 CN CN201911231047.8A patent/CN110878309B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1712629A2 (en) * | 1995-09-01 | 2006-10-18 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
AU2013254904A1 (en) * | 2006-03-02 | 2014-02-20 | The Uab Research Foundation | Mycobacterial disease detection, treatment, and drug discovery |
CN101985473A (zh) * | 2010-10-12 | 2011-03-16 | 浙江大学 | 抗结核杆菌esat-6单克隆抗体tbef3及应用 |
CN101987872A (zh) * | 2010-10-12 | 2011-03-23 | 浙江大学 | 抗结核杆菌esat-6单克隆抗体tbea8及应用 |
Non-Patent Citations (2)
Title |
---|
结核分枝杆菌ESAT-6蛋白单克隆抗体的研制与鉴定;解晓莉;《中国优秀硕士论文全文数据库 农业科技辑》;20150115;第2.3节和第2.4节、讨论部分第5-6段 * |
结核分枝杆菌ESAT-6重组二聚体的表达、纯化及其在血清学诊断中的初步应用;郭建等;《中国生物制品学杂志》;20090220(第02期);第29-32页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110878309A (zh) | 2020-03-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110878309B (zh) | 结核分枝杆菌分泌蛋白esat-6单抗的重链和轻链可变区基因和编码的多肽及其应用 | |
Feng et al. | Novel monoclonal antibodies to ESAT-6 and CFP-10 antigens for ELISA-based diagnosis of pleural tuberculosis | |
Singh et al. | Construction of a single-chain variable-fragment antibody against the superantigen staphylococcal enterotoxin B | |
JP2012515551A5 (zh) | ||
US8501179B2 (en) | Antibody against PcrV | |
US9085611B2 (en) | Humanized PcrV antibody having anti-pseudomonal activity | |
CN116239683A (zh) | 一种抗艰难梭菌毒素b的单克隆抗体及其制备方法和应用 | |
US8475803B2 (en) | Assays for diagnosis of tuberculosis and uses thereof | |
Krishnananthasivam et al. | An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis | |
Bannantine et al. | MAP1272c encodes an NlpC/P60 protein, an antigen detected in cattle with Johne's disease | |
Klimovich et al. | Development of Immunoreagents for Diagnostics of CagA‐Positive Helicobacter pylori Infections | |
CN111378628B (zh) | 一种分泌结核分枝杆菌esat6蛋白特异性抗体的杂交瘤细胞株、其抗体及应用 | |
JP2017531657A (ja) | 組換えボレリアタンパク質およびその使用のための方法 | |
WO1991014448A1 (en) | New proteins, peptides and corresponding dna or rna sequences and probes useful for diagnosing tuberculosis | |
Xue et al. | Evaluation of P1 adhesin epitopes for the serodiagnosis of Mycoplasma pneumoniae infections | |
CN112759644A (zh) | 结核分枝杆菌mpt64蛋白单克隆抗体的重链和轻链可变区基因和编码的多肽及其应用 | |
Zhao et al. | Monoclonal antibodies against a Mycobacterium tuberculosis Ag85B-Hsp16. 3 fusion protein | |
Rementeria et al. | Characterization of a monoclonal antibody directed against Salmonella enterica serovar typhimurium and serovar [4, 5, 12: i:−] | |
Leng et al. | Development of a novel anti ESAT-6 monoclonal antibody for screening of Mycobacterium tuberculosis | |
IE904536A1 (en) | New methods for diagnosis of tuberculosis | |
CN109988770B (zh) | 一种c-di-AMP合成酶单克隆抗体的重链和轻链可变区基因和编码的多肽及其应用 | |
Khalilpour et al. | Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity | |
CN114957474B (zh) | 一种抗猪cd69蛋白单克隆抗体及其制备方法与应用 | |
Li et al. | An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis. | |
CN114702585B (zh) | 一种抗cd22的单克隆抗体4f6及其制备方法和用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
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 |