CN109970851A - Ccv病毒m蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法 - Google Patents
Ccv病毒m蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法 Download PDFInfo
- Publication number
- CN109970851A CN109970851A CN201910257290.0A CN201910257290A CN109970851A CN 109970851 A CN109970851 A CN 109970851A CN 201910257290 A CN201910257290 A CN 201910257290A CN 109970851 A CN109970851 A CN 109970851A
- Authority
- CN
- China
- Prior art keywords
- ccv
- monoclonal antibody
- protein
- virus
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 238000012360 testing method Methods 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- 241000700605 Viruses Species 0.000 title claims abstract description 37
- 101710085938 Matrix protein Proteins 0.000 title claims abstract description 34
- 101710127721 Membrane protein Proteins 0.000 title claims abstract description 34
- 230000036039 immunity Effects 0.000 title claims abstract description 8
- 241000711506 Canine coronavirus Species 0.000 claims abstract description 86
- 230000014509 gene expression Effects 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 239000000427 antigen Substances 0.000 claims abstract description 16
- 102000036639 antigens Human genes 0.000 claims abstract description 16
- 108091007433 antigens Proteins 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000001262 western blot Methods 0.000 claims abstract description 11
- 102000018120 Recombinases Human genes 0.000 claims abstract description 7
- 108010091086 Recombinases Proteins 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 238000012215 gene cloning Methods 0.000 claims abstract description 5
- 230000003053 immunization Effects 0.000 claims abstract description 5
- 230000002779 inactivation Effects 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 46
- 108090000623 proteins and genes Proteins 0.000 claims description 23
- 210000004408 hybridoma Anatomy 0.000 claims description 15
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 14
- 239000002671 adjuvant Substances 0.000 claims description 11
- 208000015181 infectious disease Diseases 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 239000000020 Nitrocellulose Substances 0.000 claims description 9
- 229920001220 nitrocellulos Polymers 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 9
- 238000001890 transfection Methods 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000001804 emulsifying effect Effects 0.000 claims description 6
- 239000003365 glass fiber Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 230000003612 virological effect Effects 0.000 claims description 6
- 238000011725 BALB/c mouse Methods 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 238000010241 blood sampling Methods 0.000 claims description 5
- 238000002955 isolation Methods 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 208000001528 Coronaviridae Infections Diseases 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000003636 conditioned culture medium Substances 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 230000003902 lesion Effects 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 210000004989 spleen cell Anatomy 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- 244000248349 Citrus limon Species 0.000 claims description 3
- 235000005979 Citrus limon Nutrition 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 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 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 230000007910 cell fusion Effects 0.000 claims description 3
- 210000003608 fece Anatomy 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 238000003119 immunoblot Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000003760 magnetic stirring Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000005266 casting Methods 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 claims description 2
- 230000004544 DNA amplification Effects 0.000 claims 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 229920002678 cellulose Polymers 0.000 claims 1
- 239000001913 cellulose Substances 0.000 claims 1
- 238000007667 floating Methods 0.000 claims 1
- 238000002649 immunization Methods 0.000 claims 1
- 238000009629 microbiological culture Methods 0.000 claims 1
- 229910017604 nitric acid Inorganic materials 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 238000012795 verification Methods 0.000 abstract 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 241000712083 Canine morbillivirus Species 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 241000282472 Canis lupus familiaris Species 0.000 description 7
- 241000282465 Canis Species 0.000 description 6
- 241000711573 Coronaviridae Species 0.000 description 6
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 6
- 241000701931 Canine parvovirus Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000000084 colloidal system Substances 0.000 description 5
- 208000006454 hepatitis Diseases 0.000 description 5
- 231100000283 hepatitis Toxicity 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 238000010276 construction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- JAMAWBXXKFGFGX-KZVJFYERSA-N Ala-Arg-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JAMAWBXXKFGFGX-KZVJFYERSA-N 0.000 description 2
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 2
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 2
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 2
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 2
- QRIYOHQJRDHFKF-UWJYBYFXSA-N Ala-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 QRIYOHQJRDHFKF-UWJYBYFXSA-N 0.000 description 2
- MUGAESARFRGOTQ-IGNZVWTISA-N Ala-Tyr-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N MUGAESARFRGOTQ-IGNZVWTISA-N 0.000 description 2
- DNBMCNQKNOKOSD-DCAQKATOSA-N Arg-Pro-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O DNBMCNQKNOKOSD-DCAQKATOSA-N 0.000 description 2
- RAQMSGVCGSJKCL-FOHZUACHSA-N Asn-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(N)=O RAQMSGVCGSJKCL-FOHZUACHSA-N 0.000 description 2
- ANPFQTJEPONRPL-UGYAYLCHSA-N Asn-Ile-Asp Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O ANPFQTJEPONRPL-UGYAYLCHSA-N 0.000 description 2
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 2
- TZFQICWZWFNIKU-KKUMJFAQSA-N Asn-Leu-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 TZFQICWZWFNIKU-KKUMJFAQSA-N 0.000 description 2
- YUOXLJYVSZYPBJ-CIUDSAMLSA-N Asn-Pro-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O YUOXLJYVSZYPBJ-CIUDSAMLSA-N 0.000 description 2
- GZXOUBTUAUAVHD-ACZMJKKPSA-N Asn-Ser-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GZXOUBTUAUAVHD-ACZMJKKPSA-N 0.000 description 2
- UGKZHCBLMLSANF-CIUDSAMLSA-N Asp-Asn-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UGKZHCBLMLSANF-CIUDSAMLSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- BGIRVSMUAJMGOK-FXQIFTODSA-N Cys-Ala-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CS)N BGIRVSMUAJMGOK-FXQIFTODSA-N 0.000 description 2
- ZEXHDOQQYZKOIB-ACZMJKKPSA-N Cys-Glu-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZEXHDOQQYZKOIB-ACZMJKKPSA-N 0.000 description 2
- WTEACWBAULENKE-SRVKXCTJSA-N Cys-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CS)N WTEACWBAULENKE-SRVKXCTJSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 208000005577 Gastroenteritis Diseases 0.000 description 2
- REJJNXODKSHOKA-ACZMJKKPSA-N Gln-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N REJJNXODKSHOKA-ACZMJKKPSA-N 0.000 description 2
- JXBZEDIQFFCHPZ-PEFMBERDSA-N Gln-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JXBZEDIQFFCHPZ-PEFMBERDSA-N 0.000 description 2
- SGVGIVDZLSHSEN-RYUDHWBXSA-N Gln-Tyr-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O SGVGIVDZLSHSEN-RYUDHWBXSA-N 0.000 description 2
- GCYFUZJHAXJKKE-KKUMJFAQSA-N Glu-Arg-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GCYFUZJHAXJKKE-KKUMJFAQSA-N 0.000 description 2
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 2
- RXJFSLQVMGYQEL-IHRRRGAJSA-N Glu-Tyr-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 RXJFSLQVMGYQEL-IHRRRGAJSA-N 0.000 description 2
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 2
- GWCRIHNSVMOBEQ-BQBZGAKWSA-N Gly-Arg-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O GWCRIHNSVMOBEQ-BQBZGAKWSA-N 0.000 description 2
- UFPXDFOYHVEIPI-BYPYZUCNSA-N Gly-Gly-Asp Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O UFPXDFOYHVEIPI-BYPYZUCNSA-N 0.000 description 2
- QPCVIQJVRGXUSA-LURJTMIESA-N Gly-Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QPCVIQJVRGXUSA-LURJTMIESA-N 0.000 description 2
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 2
- YGHSQRJSHKYUJY-SCZZXKLOSA-N Gly-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN YGHSQRJSHKYUJY-SCZZXKLOSA-N 0.000 description 2
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 2
- TWPSALMCEHCIOY-YTFOTSKYSA-N Ile-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)O)N TWPSALMCEHCIOY-YTFOTSKYSA-N 0.000 description 2
- NUKXXNFEUZGPRO-BJDJZHNGSA-N Ile-Leu-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N NUKXXNFEUZGPRO-BJDJZHNGSA-N 0.000 description 2
- OTSVBELRDMSPKY-PCBIJLKTSA-N Ile-Phe-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OTSVBELRDMSPKY-PCBIJLKTSA-N 0.000 description 2
- WJBOZUVRPOIQNN-KJYZGMDISA-N Ile-Trp-His Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)C1=CN=CN1 WJBOZUVRPOIQNN-KJYZGMDISA-N 0.000 description 2
- QSXSHZIRKTUXNG-STECZYCISA-N Ile-Val-Tyr Chemical compound CC[C@H](C)[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 QSXSHZIRKTUXNG-STECZYCISA-N 0.000 description 2
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 description 2
- MPSBSKHOWJQHBS-IHRRRGAJSA-N Leu-His-Met Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCSC)C(=O)O)N MPSBSKHOWJQHBS-IHRRRGAJSA-N 0.000 description 2
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 2
- RGUXWMDNCPMQFB-YUMQZZPRSA-N Leu-Ser-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RGUXWMDNCPMQFB-YUMQZZPRSA-N 0.000 description 2
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 2
- YLMIDMSLKLRNHX-HSCHXYMDSA-N Leu-Trp-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YLMIDMSLKLRNHX-HSCHXYMDSA-N 0.000 description 2
- SLQJJFAVWSZLBL-BJDJZHNGSA-N Lys-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN SLQJJFAVWSZLBL-BJDJZHNGSA-N 0.000 description 2
- UIJVKVHLCQSPOJ-XIRDDKMYSA-N Lys-Ser-Trp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O UIJVKVHLCQSPOJ-XIRDDKMYSA-N 0.000 description 2
- VVURYEVJJTXWNE-ULQDDVLXSA-N Lys-Tyr-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O VVURYEVJJTXWNE-ULQDDVLXSA-N 0.000 description 2
- ATBJCCFCJXCNGZ-UFYCRDLUSA-N Met-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 ATBJCCFCJXCNGZ-UFYCRDLUSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- MRWOVVNKSXXLRP-IHPCNDPISA-N Phe-Ser-Trp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O MRWOVVNKSXXLRP-IHPCNDPISA-N 0.000 description 2
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 2
- 229940096437 Protein S Drugs 0.000 description 2
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 2
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 2
- YQQKYAZABFEYAF-FXQIFTODSA-N Ser-Glu-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQQKYAZABFEYAF-FXQIFTODSA-N 0.000 description 2
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 2
- ZWSZBWAFDZRBNM-UBHSHLNASA-N Ser-Trp-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O ZWSZBWAFDZRBNM-UBHSHLNASA-N 0.000 description 2
- 101710198474 Spike protein Proteins 0.000 description 2
- 102100021696 Syncytin-1 Human genes 0.000 description 2
- SWIKDOUVROTZCW-GCJQMDKQSA-N Thr-Asn-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C)C(=O)O)N)O SWIKDOUVROTZCW-GCJQMDKQSA-N 0.000 description 2
- RJBFAHKSFNNHAI-XKBZYTNZSA-N Thr-Gln-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N)O RJBFAHKSFNNHAI-XKBZYTNZSA-N 0.000 description 2
- YSXYEJWDHBCTDJ-DVJZZOLTSA-N Thr-Gly-Trp Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O YSXYEJWDHBCTDJ-DVJZZOLTSA-N 0.000 description 2
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 2
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 2
- UTQBQJNSNXJNIH-IHPCNDPISA-N Trp-Asn-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N UTQBQJNSNXJNIH-IHPCNDPISA-N 0.000 description 2
- GBEAUNVBIMLWIB-IHPCNDPISA-N Trp-Ser-Phe Chemical compound C([C@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=CC=C1 GBEAUNVBIMLWIB-IHPCNDPISA-N 0.000 description 2
- WTXQBCCKXIKKHB-JYJNAYRXSA-N Tyr-Arg-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WTXQBCCKXIKKHB-JYJNAYRXSA-N 0.000 description 2
- LUMQYLVYUIRHHU-YJRXYDGGSA-N Tyr-Ser-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LUMQYLVYUIRHHU-YJRXYDGGSA-N 0.000 description 2
- HZWPGKAKGYJWCI-ULQDDVLXSA-N Tyr-Val-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(C)C)C(O)=O HZWPGKAKGYJWCI-ULQDDVLXSA-N 0.000 description 2
- VMRFIKXKOFNMHW-GUBZILKMSA-N Val-Arg-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N VMRFIKXKOFNMHW-GUBZILKMSA-N 0.000 description 2
- JAKHAONCJJZVHT-DCAQKATOSA-N Val-Lys-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N JAKHAONCJJZVHT-DCAQKATOSA-N 0.000 description 2
- FMQGYTMERWBMSI-HJWJTTGWSA-N Val-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N FMQGYTMERWBMSI-HJWJTTGWSA-N 0.000 description 2
- AJNUKMZFHXUBMK-GUBZILKMSA-N Val-Ser-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N AJNUKMZFHXUBMK-GUBZILKMSA-N 0.000 description 2
- VIKZGAUAKQZDOF-NRPADANISA-N Val-Ser-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O VIKZGAUAKQZDOF-NRPADANISA-N 0.000 description 2
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 210000000664 rectum Anatomy 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 108091005703 transmembrane proteins Proteins 0.000 description 2
- 108010045269 tryptophyltryptophan Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000031295 Animal disease Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700003471 Coronavirus 3C Proteases Proteins 0.000 description 1
- 108700002673 Coronavirus M Proteins Proteins 0.000 description 1
- FMDCYTBSPZMPQE-JBDRJPRFSA-N Cys-Ala-Ile Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FMDCYTBSPZMPQE-JBDRJPRFSA-N 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000010772 Dog disease Diseases 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- NHCKESBLOMHIIE-IRXDYDNUSA-N Phe-Gly-Phe Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 NHCKESBLOMHIIE-IRXDYDNUSA-N 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- HHJFMHQYEAAOBM-ZLUOBGJFSA-N Ser-Ser-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O HHJFMHQYEAAOBM-ZLUOBGJFSA-N 0.000 description 1
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- SMUWZUSWMWVOSL-JYJNAYRXSA-N Tyr-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N SMUWZUSWMWVOSL-JYJNAYRXSA-N 0.000 description 1
- PDDJTOSAVNRJRH-UNQGMJICSA-N Val-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](C(C)C)N)O PDDJTOSAVNRJRH-UNQGMJICSA-N 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- -1 White (S Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000000369 enteropathogenic effect Effects 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 201000005111 ocular hyperemia Diseases 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 229920006267 polyester film Polymers 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000012153 swine disease Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—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
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种CCV病毒M蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法,具体涉及CCV病毒M蛋白的单抗,包括CCV M蛋白特异单克隆抗体2H11、2G3。其制备方法;以分离纯化的CCV灭活病毒为免疫抗原,用单克隆抗体技术制备出CCV单抗;经western‑blot试验鉴定单抗针对M蛋白,再用重组酶ExnaseTM II技术对CCV病毒M蛋白的基因克隆进线性化的pCDNA3.1载体,转染293T细胞进行M蛋白表达,IFA试验验证,获得2株CCV病毒M蛋白特异单抗2H11、2G3。本发明还公开了犬冠状病毒胶体金检测试纸条的制备方法。本发明特异性强、敏感性高、操作简单、成本低,适用于临床及田间检测,用于CCV病原学诊断、流行病学调查和动物医院快速诊断。
Description
技术领域
本发明涉及一种CCV病毒M蛋白的单抗及其制备方法、犬冠状病毒胶体金检测试纸条的制备方法,属于生物技术和动物疾病检疫检测领域。
背景技术
犬冠状病毒(CCV)属冠状病毒科冠状病毒属,该病毒形态特征,具有类似冠状的纤突,有囊膜,呈圆形或椭圆形,大小80-120nm。犬冠状病毒主要有4种结构蛋白分别为纤突蛋白(S,Spike Protein,180-220kD)是受体结合位点;小包膜糖蛋白(E,Envelope Protein,9–12kD),此蛋白较小与包膜结合;跨膜蛋白M蛋白(M,Transmembrane Protein,29-32kD)负责营养物质的跨膜运输和新生病毒出芽释放与病毒外包膜的形成;核蛋白N蛋白(N,Nucleocapsid Protein,50–60kD)与病毒的RNA紧密结合成病毒的核心壳。
犬冠状病毒是一种由CCV引起的临床上以呕吐、腹泻、脱水及易复发为特性的高度接触性传染病,一年四季均可发生。该病对幼犬危害尤其严重,病死率较高。病犬和带毒犬是主要传染源,病毒通过直接接触和间接接触,经呼吸道和消化道传染给健康犬及其他易感动物。感染犬可通过粪便排毒2周或更长时间,传染性粪便污染的环境是传染的主要原因。
目前CCV无有效疫苗用于预防,也无特效疗法,因此早期诊断非常重要。CCV的常见临床症状,不易于与其他的肠道病原,如CPV-2,犬轮状病毒和犬腺病毒区分。因此CCV的诊断需要实验室确诊。用于粪便样品中CCV的检测的诊断技术包括电镜(EM),细胞培养分离病毒(VI)和病毒基因检测(RT-PCR)。但这些方法需要特殊设备和熟练的专业技术人员,不便于兽医人员在临床或田间使用。
胶体金免疫层析技术因操作简单,检测灵敏,能稳定保存,成本较低等优点,近年来被临床和基层单位广泛使用。目前国内使用的CCV胶体金试纸条主要依赖进口(韩国)。本发明通过单克隆抗体技术制备了针对CCV病毒M蛋白的单克隆抗体,M蛋白是犬冠状病毒比较保守的蛋白,不同毒株间同源性非常高,用M蛋白作单抗诊断犬冠状病毒感染,可避免因纤突蛋白的变异而造成对犬冠状病毒感染诊断的假阴性,从而更准确地诊断犬冠状病毒感染。CCV M蛋白单抗与胶体金颗粒结合稳定性提高可以特异性的检测出临床及田间的CCV发病动物,能同时检测CCV 1型和2型。
发明内容
本发明的目的在于提供一种CCV病毒M蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法,建立特异性强、敏感性高、操作简单、成本低的适用于检测临床及田间的CCV胶体金免疫层析试纸条,用于CCV病原学诊断、流行病学调查和动物医院快速诊断。
本发明的技术方案包括:一种CCV病毒M蛋白的单克隆抗体,其特征是:包括针对CCV病毒M蛋白的一对杂交瘤细胞株,其中第一个杂交瘤细胞株命名为2H11的保藏编号为CGMCC NO.17287,第二个杂交瘤细胞株命名为2G3的保藏编号为CGMCC NO.17288,2个杂交瘤细胞株均保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址均为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101;CCV病毒M蛋白的单克隆抗体,由所述的2个杂交瘤细胞株2H11和2G3分别产生。
CCV病毒M蛋白的单克隆抗体的制备方法,所述制备方法:以分离纯化的CCV灭活病毒为免疫抗原,用单克隆抗体技术制备出CCV单抗;经western-blot(蛋白免疫印迹)试验鉴定单抗针对M蛋白,再用重组酶ExnaseTM II技术对CCV病毒M蛋白的基因克隆进线性化的pCDNA3.1载体,转染293T细胞进行M蛋白表达,用单抗与表达的M蛋白做IFA鉴定进一步验证;获得2株CCV病毒M蛋白特异单抗2H11、2G3。
所述CCV单抗的制备方法具体为:
(1)CCV病毒的分离与纯化:取临床上发病的犬的新鲜粪便样品,经0.2μm针头滤器过滤后,接种到A-72细胞,进行盲传,出现典型细胞病变后,进行病毒纯化和鉴定;CCV病毒用A-72细胞进行大量培养,用超离法纯化病毒抗原;
(2)针对M蛋白单抗制备:用0.1%(v/v)甲醛灭活的纯化CCV病毒与弗氏佐剂混合乳化,分4次免疫8周龄的BALB/c小鼠;取效价高的小鼠脾脏细胞与瘤细胞SP2/0进行细胞融合;融合后先用感染CCV的细胞板做IFA大量筛选,筛选后的阳性单抗隆抗体细胞上清进行western-blot和转染表达人工重组的M蛋白细胞IFA检测,鉴定、获得针对CCV M蛋白的单克隆抗体。
所述经western-blot(蛋白免疫印迹)试验鉴定单抗针对M蛋白,再用重组酶ExnaseTM II技术对CCV病毒M蛋白的基因克隆进线性化的pCDNA3.1载体,转染293T细胞进行M蛋白表达,具体为:
(1)利用下述引物,以提取犬冠状病毒感染细胞上清的RNA为模板进行M基因的扩增,PCR引物
上游引物:AGCTTGGTACCGAatgaagattttgtta
下游引物:ATATCTGCAGAATTttataccatatgtaat
(2)利用下述引物,以PCDNA3.1+质粒做模板进行线性化载体的扩增;
上游引物:GAATTCTGCAGATATCCAGCACAGTG
下游引物:GCTCGGTACCAAGCTTAAGTTTAAACG
(3)利用重组酶ExnaseTM II对步骤1)和2)得到的PCR产物进行快速重组克隆;阳性克隆经转染293T表达细胞进行M基因的表达。
一种免疫胶体金试纸条的制备方法,所述制备方法包括以下步骤:
(1)胶体金抗体标记:在胶体金上标记权利要求1所述的单克隆抗体2H11;
(2)喷金:将步骤(1)标记好的胶体金喷到玻璃纤维上,制成金标垫;
(3)制作硝酸纤维素膜:将兔抗鼠IgG和权利要求1所述的单克隆抗体2G3共同喷到硝酸纤维素膜上,制成对照线和检测线:
(4)将样品垫、胶体金垫、硝酸纤维素膜、吸水纸自下至上组装在PVC板上,然后切割制成试纸条;所述样品垫为空白的玻璃纤维素膜)。
所述胶体金抗体标记:用柠檬三钠还原法制备粒径约为20nm的胶体金,用0.2mol/L K2CO3调到pH的浓度8.0,在磁力搅拌下逐滴加入纯化单抗100μg/ml 2H11,室温搅拌30min,加入10%(w/v)BSA溶液,使其终浓度为1%,室温继续搅拌均匀,然后依次缓慢滴入终浓度0.2%(w/v)PEG-20000,继续搅拌30min后,置于4℃静置2h;100rpm 4℃离心15min弃去底部未结合的杂质成分,取上清10000rpm 4℃离心30min,弃去上清;沉淀用原体积1/20含5%(w/v)蔗糖、1%(w/v)BSA、0.2%(w/v)PEG20000以及0.02%(w/v)叠氮钠的0.01mol/LpH9.2硼酸盐缓冲液悬浮;置于4℃保存备用。
所述兔抗鼠IgG的制备方法是:将小鼠IgG与弗氏完全佐剂混合乳化,背部皮下、皮内多点注射免疫健康家兔,经3次免疫后,检测血清效价,当琼脂凝胶沉淀试验AGP效价≥1:16时,采血,分离血清,加入饱和硫酸铵沉淀,透析后获得兔抗鼠IgG。
通过本发明,CCV病毒的分离与纯化,针对M蛋白的单抗的制备,兔抗鼠血清的制备、胶体金免疫层析试纸条制备及其对直肠棉试、病料悬液、细胞培养等材料样本中的病毒抗原样本灵敏度和特异性测定。
CCV M蛋白的单克隆抗体的制备方法:
1.CCV病毒的分离与纯化:取临床上发病的犬的新鲜粪便样品,经0.2μm针头滤器过滤后,接种到A-72细胞,进行盲传,出现典型细胞病变后,进行病毒纯化和鉴定。CCV病毒用A-72细胞进行大量培养,用超离法纯化病毒抗原。
2.针对M蛋白单抗制备:用0.1%(v/v)甲醛灭活的纯化CCV病毒与弗氏佐剂混合,分4次免疫8周龄的BALB/c小鼠。取效价高的小鼠脾脏细胞与瘤细胞SP2/0进行细胞融合。融合后先用感染CCV的细胞板做IFA大量筛选,筛选后的阳性单抗隆抗体(细胞上清)进行western-blot和转染表达人工重组的M蛋白细胞IFA检测,鉴定、获得针对CCV M蛋白的单克隆抗体。
单抗的特异性鉴定:分别用犬冠状病毒(CCVJS1706,JS1712),猪传染性胃肠炎病毒(TGEV),犬瘟热病毒(CDV),犬腺病毒(CAV-2),犬细小病毒(CPV-2a)感染A-72、Vero、MDCK、FK81细胞,冷甲醇固定,间接免疫荧光法(IFA)检测细胞特异荧光反应。
兔抗鼠血清的制备:将小鼠IgG:与弗氏完全佐剂混合乳化,背部皮下、皮内多点注射免疫健康家兔,经3次免疫后,检测血清效价,当琼脂凝胶沉淀试验(AGP)效价≥1:16时,采血,分离血清,加入饱和硫酸铵沉淀,透析后获得兔抗鼠IgG。
试纸条制备方法:
(1)在胶体金上标记针对M-蛋白的抗犬冠状病毒抗原特异性单克隆抗体2H11;
(2)将胶体金标记的抗体喷金到玻璃纤维上,制成胶体金垫;
(3)将兔抗鼠IgG和抗犬冠状病毒的单克隆抗体2G3分别喷到硝酸纤维素膜上,制成对照线和检测线;
(4)组装样品垫、胶体金垫、硝酸纤维素膜、吸水纸在PVC底板上,然后切割条制成试纸条。
本发明的试纸条能用于检测直肠粪样悬液、细胞培养等材料样本中的CCV病毒抗原。该方法简单、快速,可以用肉眼直接观察,3-10分钟即出结果,本产品特异性强,敏感性高,适用于基层兽医部门,动物医院进行疾病诊断,也可用于冠状病毒病的流行病学调查,实验室犬病毒快速筛选检测等,应用前景广阔。
附图说明
图1.CCV感染A-72细胞与单克隆抗体的IFA图。CCV单抗隆抗体2H11,2G3均与表达CCV感染的A-72细胞反应。
图2.重组CCV M蛋白表达细胞与单抗隆抗体的IFA鉴定。CCV单抗隆抗体2H11,2G3均与表达CCV M蛋白的293T细胞反应。
图3.单抗隆抗体与CCV M蛋白的western-blot反应性。CCV单抗隆抗体2H11,2G3均特异识别CCV M蛋白(30kD左右)。
图4.胶体金电镜图。胶体金颗粒大小20nm左右。
图5.试纸条与韩国进口试纸条比较检测。符合率100%。
具体实施方式
生物材料来源:
犬冠状病毒(JS1706,JS1712株),由扬州大学农业部畜禽传染病学重点开放实验室分离、鉴定。
猪传染性胃肠炎病毒(TGEV),犬瘟热病毒(CDV),犬腺病毒(CAV-2),犬细小病毒(CPV-2a)毒种,由扬州大学农业部畜禽传染病学重点开放实验室保存、提供。
PCDNA3.1(+)、大肠杆菌DH5α,由扬州大学江苏省动物预防医学重点实验室保存、提供。胶体金试纸条制备材料购自上海金标生物科技有限公司。
具体操作步骤如下:
1.CCV病毒的分离与纯化
(1)CCV病毒的分离
取CCV新鲜的病料,用细胞生长液1:1稀释,经0.2μm针头滤器过滤后,接种到A-72细胞,37℃吸附1h后,换1%(v/v)FBS的DMEM维持液培养3-5d,进行盲传3代,出现典型细胞病变后,进行病毒纯化和鉴定。CCV病毒用A-72细胞进行大量培养,用超离法纯化病毒抗原。
(2)CCV病毒的纯化
取CCV病毒的细胞培养物,10000rpm离心20min,取上清液加入10%(w/v)PEG6000,搅拌溶解后,4℃放置过夜,然后溶液经过10000rpm,4℃离心30min。弃掉上清,沉淀物加入NTE缓冲液重新悬浮至原体积1/10,再加入到含30%(w/v)蔗糖-TNE缓冲液上,140000g离心1.5h,沉淀物加TNE缓冲液到原体积的1/50。测定纯化病毒的蛋白浓度,分装,-20℃保存备用。
2.抗CCV单克隆抗体的制备
纯化的CCV抗原(1.6mg/ml),0.1%(v/v)甲醛灭活24h,作为免疫抗原。第1次免疫,抗原+弗氏完全佐剂1:1混合乳化,皮下多点接种免疫8周龄BALB/c小鼠,0.2ml/只。间隔2周分别进行相同抗原第2、3次接种,使用弗氏不完全佐剂。第4次免疫,不使用佐剂,皮下注射抗原0.1ml,3-4d取脾细胞与SP2/0进行细胞融合,融合剂PEG1500(Roche,USA)。融合后8-10d,用CCV感染细胞进行IFA试验筛选阳性杂交瘤。阳性杂交瘤经有限稀释法进行3次克隆化,建立稳定分泌高特异抗犬冠状病毒的单克隆抗体杂交瘤细胞株。筛选的阳性杂交瘤细胞上清经western-blot鉴定病毒蛋白特异性,进一步与表达重组犬冠状病毒M蛋白(步骤1)的细胞用IFA方法鉴定,得到针对于CCV病毒M蛋白的2株杂交瘤细胞株2H11,2G3。杂交瘤细胞腹腔接种BALB/c小鼠制备单克隆抗体腹水,饱和硫酸铵沉淀,用HiTrap Protein G柱(GE)进行纯化。以纯化的、高特异性单克隆抗体为基础,保证了试纸条的敏感性与特异性。
3.单抗的特异性鉴定:分别用犬冠状病毒(CCVJS1706,JS1712),猪传染性胃肠炎病毒(TGEV),犬瘟热病毒(CDV),犬腺病毒(CAV-2),犬细小病毒(CPV-2a)感染A-72、Vero、MDCK、FK81细胞,冷甲醇固定,间接免疫荧光法(IFA)检测细胞特异荧光反应,CCV单抗2H11,2G3仅与冠状病毒反应。
4.真核表达M蛋白的载体构建、细胞表达
(1)引物的设计
设计扩增含犬冠状病毒M蛋白序列的pcdna3.1+线性化表达载体和M蛋白基因片段引物:扩增pcdna3.1+new线性化载体上游引物位于pcdna3.1+质粒952-977位,扩增pcdna3.1+new线性化载体下游引物位于pcdna3.1+质粒900-926位。扩增M基因序列的上游引物包括基因起始密码ATG及其后15个碱基,且在5’端带有16个与pcdna3.1+线性化载体下游引物反向互补碱基;扩增M基因序列的下游引物包括M全部基因,1383-1399位16个碱基,且在5’端带有15个与pcdna3.1+线性化载体上游引物反向互补碱基。具体引物序列见附表1,由南京金斯瑞生物科技有限公司合成。
(2)M基因和线性化真核表达载体的扩增
由表1所述引物为引物进行PCR扩增。反应体系为:18μl水,25μl 2倍缓冲液,1μl10mM dNTP,2μl 10μM上游引物,2μl 10μM下游引物,1μl商品化的Phanta Super-FidelityDNA聚合酶。模板分别用1μl10ng/μl的pcdna3.1+用来制备线性化PCDNA3.1+载体,M基因目的片段的扩增用细胞上清提取的基因。PCR扩增反应循环参数为:94℃预变性3分钟,随后进行30个循环(94℃变性30秒,55℃退火1分钟,72℃延伸2分钟),最后72℃延伸10分钟。PCR结束后,PCR产物在1%的琼脂糖凝胶中进行电泳。
(3)M基因的重组质粒构建及表达
将以上纯化的线性化载体pcdna3.1以及CCV病毒M基因片段PCR产物在商品化重组酶ExnaseTM II的作用下进行重组克隆。具体重组反应体系如下:纯化的CCV病毒M基因片段片段产物50-100ng,纯化的pcdna3.1线性化表达载体50ng,2μl商品化的ExnaseTM II酶,4μl 5倍的缓冲液,其它补加水至20μl。反应物于37℃作用30分钟后,置冰上5分钟。随后将20μl反应物转化到常规感受态细菌,涂LB板。次日挑取细菌克隆进行质粒制备,阳性克隆鉴定。
将阳性克隆质粒转染到293T细胞中,换5%(v/v)FBS的DMEM细胞培养基,37℃,5%(v/v)CO2培养细胞48h后,将细胞用甲醇4℃固定过夜,用CCV单抗2H11、2G3检测,产生特异免疫荧光反应。
5.Western blot对单抗的分析及鉴定
用纯化的犬细小病毒蛋白裂解过后进行SDS-PAGE,然后电转印到硝酸纤维膜(NC)上,5%(w/v)脱脂乳封闭过后,用一抗单克隆抗体(杂交瘤培养上清),二抗HRP-羊抗鼠稀释液依次进行孵育,最后将NC膜用吸水纸吸干,把增强型ECLPlus化学发光试剂(Solarbio)均匀涂抹在NC膜上,避光曝光,成像***(Tanon 5200)扫描,M蛋白大小29-32kD。
6.兔抗鼠IgG的制备
将小鼠IgG(4.85mg/ml)与弗氏完全佐剂1:1(v/v)混合乳化,背部皮下、皮内多点注射免疫健康家兔(2kg左右),2ml/只。间隔3周分别进行第2、3次接种,第2次接种用弗氏不完全佐剂,接种方式同第1次。第3次接种,耳静脉注射2ml/只,接种后3周,采血制备血清,当琼脂凝胶沉淀试验(AGP)效价≥1:16时,采血,分离血清,加入饱和硫酸铵沉淀,透析后获得兔抗鼠IgG,测定蛋白浓度,分装,-20℃冻存备用。
7.胶体金试纸条的制备
(1)胶体金制备及标记用柠檬三钠还原法制备粒径约为20nm的胶体金,用0.2mol/L K2CO3调到pH的浓度8.0,在磁力搅拌下逐滴加入纯化单抗2H11(100μg/ml),室温搅拌30min,加入10%(w/v)BSA溶液,使其终浓度为1%(w/v),室温继续搅拌均匀,然后依次缓慢滴入终浓度0.2%(w/v)PEG-20000,继续搅拌30min后,置于4℃静置2h。100rpm 4℃离心15min弃去底部未结合的杂质成分,取上清10000rpm 4℃离心30min,弃去上清。沉淀用原体积1/20含5%(w/v)蔗糖、1%(w/v)BSA、0.2%(w/v)PEG20000以及0.02%(w/v)叠氮钠的0.01mol/L pH9.2硼酸盐缓冲液悬浮。置于4℃保存备用。
(2)胶体金标垫的制备用Biodot XYZ3060三维喷点***将胶体金标记抗体2ul/cm的喷量喷在玻璃纤维棉上(公司)制成金标垫。自然干燥,抽真空备用。
(3)NC膜的制备用Biodot XYZ3060三维喷点***以1ul/cm的喷量,分别把单克隆抗体2G3(1mg/ml)和兔抗鼠IgG(1mg/ml)两种抗体条状喷在NC(硝酸纤维素膜)膜上,分别作为检测线(T线)和质控线(C线),两者线之间隔5mm。
(4)试纸条的组装:按照从下到上的顺序依次放置:底盖PVC板→NC膜→金标垫(重叠NC膜T线左端0.25cm处压平放置)→聚酯膜(重叠金标垫左端0.2cm处压平放置)→吸水纸(NC膜C线右端0.2cm处压平)。调整组装好的试纸条长度,用BIO-DOT机切割试纸条宽约3到5mm,最后对应安装与底盖嵌合的面盖(面盖上观察窗对应的T线、C线、加样孔位置分别于试纸条组装顺序一致)
8.试纸条检测
(1)试纸条结果的判定
取待检样品200ul直接加到样品孔里,等待3到10min后,肉眼可见NC膜上出现红色条带,若C、T线同时出现,说明样品中含有待检抗原,判定为阳性;若C线出现而T线未出现,说明样品中不含待检抗原,判定为阴性;若C、T线均未出现或T线出现C线未出现,检测无效。
(2)特异性的检测
分别用试纸条检测犬冠状病毒(CCVJS1706,JS1712),猪传染性胃肠炎病毒(TGEV),犬瘟热病毒(CDV),犬腺病毒(CAV-2),犬细小病毒(CPV-2a),同时用PBS缓冲液做阴性对照。结果表明试纸条只与CCV抗原有反应,证明试纸条就有良好的特异性。
分别用试纸条检测犬冠状病毒2型(CCVJS1706,JS1712)细胞培养物,犬冠状病毒1型(YZ1601)临床样品,能同时检出CCV 1和CCV2。
(3)灵敏性的检测
把培养的CCV病毒进行1:10稀释,用本发明试纸条(见图5下方检测条)进行检测,同时用韩国进口试纸条(见图5上方检测条)检测,样本检测的敏感性相同,符合率100%,见表2。
CCV(JS1706)M基因核苷酸序列
ATGAAGATTT TGTTAATGCT TGCGTGCGTC TTTGCATGCG TTTATGGAGA GCGATATTGT60
GCTATGCAAG CTGATAGCGG CATTACACAA TGTCGTAATG GCACTAATTC TGAGTGTGAA120
TCTTGCTTCA ATGGTGGCGA TCTTATTTGG CATCTCGCTA ACTGGAACTT CAGCTGGTCT180
ATAATATTGA TTGTATTTAT AACTGTGTTA CAATATGGTA GACCTCAATT TAGCTGGTTC240
GTGTATGGCA TTAAAATGCT TATTATGTGG CTACTATGGC CCATTGTGTT AGCTCTTACG300
ATATTTAATG CATACTCGGA ATACGAAGTT TCCAGATATG TAATGTTCGG CTTTAGTGTT360
GCATGTGCAA TTGTTACTTT CATACTTTGG ATTATGTATT TTGTTAGATC CATTCAGTTA420
TACAGAAGGA CTAAGTCTTG GTGGTCTTTC AACCCTGAAA CTAACGCAAT TCTTTGCGTT480
AGTGCTTTAG GAAGAAGCTA CGTGCTTCCT CTTGAAGGTG TACCAACTGG TGTCACTTTA540
ACATTGCTTT CAGGGAATTT GTATGCTGAA GGATTCAAAA TTGCTGGTGG TATGAACATC600
GACAATTTAC CAAAATACGT AATGGTTGCA TTACCTAGCA GGACCATCGT CTACACACTT660
GTTGGCAAGA AATTGAAAGC AAGTAGCGCA ACTGGATGGG CTTACTATGT AAAATCTAAA720
GCCGGTGATT ACTCAACAGA CGCAAGAACT GACAATTTGA GTGAGCAAGA AAAATTATTA780
CATATGGTAT AA792
CCV(JS1706)M基因氨基酸序列
CCV(JS1712)M基因核苷酸序列
ATGAAGATTT TGTTAATGCT TGCGTGCGTC TTTGCATGCG TTTATGGAGA GCGATATTGT60
GCTATGCAAG CTGATAGCGG CATTACACAA TGTCGTAATG GCACTAATTC TGAGTGTGAA120
TCTTGCTTCA ATGGTGGCGA TCTTATTTGG CATCTCGCTA ACTGGAACTT CAGCTGGTCT180
ATAATATTGA TTGTATTTAT AACTGTGTTA CAATATGGTA GACCTCAATT TAGCTGGTTC240
GTGTATGGCA TTAAAATGCT TATTATGTGG CTACTATGGC CCATTGTGTT AGCTCTTACG300
ATATTTAATG CATACTCGGA ATACGAAGTT TCCAGATATG TAATGGTCGG CTTTAGTGTT360
GCATGTGCAA TTGTTACTTT CATACTTTGG ATTATGTATT TTGTTAGATC CATTCAGTTA420
TACAGAAGGA CTAAGTCTTG GTGGTCTTTC AACCCTGAAA CTAACGCAAT TCTTTGCGTT480
AGTGCTTTAG GAAGAAGCTA CGTGCTTCCT CTTGAAGGTG TACCAACTGG TGTCACTTTA540
ACATTGCTTT CAGGGAATTT GTATGCTGAA GGATTCAAAA TTGCTGGTGG TATGAACATC600
GACAATTTAC CAAAATACGT AATGGTTGCA TTACCTAGCA GGACCATCGT CTACACACTT660
GTTGGCAAGA AATTGAAAGC AAGTAGCGTA ACTGGATGGG CTTACTATGT AAAATCTAAA720
GCCGGTGATT ACTCAACAGA CGCAAGAACT GACAATTTGA GTGAGCAAGA AAAATTATTA780
CATATGGTAT AA792
CCV(JS1712)M基因氨基酸序列
表1 CCoV-M真核表达载体构建引物序列
表2胶体金试纸条特异性
表2中:+:阳性反应;-:阴性反应。
序列表
<110> 扬州大学
<120> CCV病毒M蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 792
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgaagattt tgttaatgct tgcgtgcgtc tttgcatgcg tttatggaga gcgatattgt 60
gctatgcaag ctgatagcgg cattacacaa tgtcgtaatg gcactaattc tgagtgtgaa 120
tcttgcttca atggtggcga tcttatttgg catctcgcta actggaactt cagctggtct 180
ataatattga ttgtatttat aactgtgtta caatatggta gacctcaatt tagctggttc 240
gtgtatggca ttaaaatgct tattatgtgg ctactatggc ccattgtgtt agctcttacg 300
atatttaatg catactcgga atacgaagtt tccagatatg taatgttcgg ctttagtgtt 360
gcatgtgcaa ttgttacttt catactttgg attatgtatt ttgttagatc cattcagtta 420
tacagaagga ctaagtcttg gtggtctttc aaccctgaaa ctaacgcaat tctttgcgtt 480
agtgctttag gaagaagcta cgtgcttcct cttgaaggtg taccaactgg tgtcacttta 540
acattgcttt cagggaattt gtatgctgaa ggattcaaaa ttgctggtgg tatgaacatc 600
gacaatttac caaaatacgt aatggttgca ttacctagca ggaccatcgt ctacacactt 660
gttggcaaga aattgaaagc aagtagcgca actggatggg cttactatgt aaaatctaaa 720
gccggtgatt actcaacaga cgcaagaact gacaatttga gtgagcaaga aaaattatta 780
catatggtat aa 792
<210> 2
<211> 269
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Lys Ile Leu Leu Met Leu Ala Cys Val Phe Ala Cys Val Tyr Gly
1 5 10 15
Glu Arg Tyr Cys Ala Met Gln Ala Asp Ser Gly Ile Thr Gln Cys Arg
20 25 30
Asn Gly Thr Asn Ser Glu Cys Glu Ser Cys Phe Asn Gly Gly Asp Leu
35 40 45
Ile Trp His Leu Ala Asn Trp Asn Phe Ser Trp Ser Ile Ile Leu Ile
50 55 60
Val Phe Ile Thr Val Leu Gln Tyr Gly Arg Pro Gln Phe Ser Trp Phe
65 70 75 80
Val Tyr Gly Ile Lys Met Leu Ile Met Trp Leu Leu Trp Pro Ile Val
85 90 95
Leu Ala Leu Thr Ile Phe Asn Ala Tyr Ser Glu Tyr Glu Val Ser Arg
100 105 110
Tyr Val Met Phe Gly Phe Ser Val Ala Cys Ala Ile Val Thr Phe Ile
115 120 125
Leu Trp Ile Met Tyr Phe Val Arg Ser Ile Gln Leu Tyr Arg Arg Thr
130 135 140
Lys Ser Trp Trp Ser Phe Asn Pro Glu Thr Asn Ala Ile Leu Cys Val
145 150 155 160
Ser Ala Leu Gly Arg Ser Tyr Val Leu Pro Leu Glu Gly Val Pro Thr
165 170 175
Gly Val Thr Leu Thr Leu Leu Ser Gly Asn Leu Tyr Ala Glu Gly Phe
180 185 190
Lys Ile Ala Gly Gly Met Asn Ile Asp Asn Leu Pro Lys Tyr Val Met
195 200 205
Val Ala Leu Pro Ser Arg Thr Ile Val Tyr Thr Leu Val Gly Lys Lys
210 215 220
Leu Lys Ala Ser Ser Ala Thr Gly Trp Ala Tyr Tyr Val Lys Ser Lys
225 230 235 240
Ala Gly Asp Tyr Ser Thr Asp Ala Arg Thr Asp Asn Leu Ser Glu Gln
245 250 255
Glu Lys Leu Leu His Met Val Ser Glu Gln Ile Asp Asn
260 265
<210> 3
<211> 792
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaagattt tgttaatgct tgcgtgcgtc tttgcatgcg tttatggaga gcgatattgt 60
gctatgcaag ctgatagcgg cattacacaa tgtcgtaatg gcactaattc tgagtgtgaa 120
tcttgcttca atggtggcga tcttatttgg catctcgcta actggaactt cagctggtct 180
ataatattga ttgtatttat aactgtgtta caatatggta gacctcaatt tagctggttc 240
gtgtatggca ttaaaatgct tattatgtgg ctactatggc ccattgtgtt agctcttacg 300
atatttaatg catactcgga atacgaagtt tccagatatg taatggtcgg ctttagtgtt 360
gcatgtgcaa ttgttacttt catactttgg attatgtatt ttgttagatc cattcagtta 420
tacagaagga ctaagtcttg gtggtctttc aaccctgaaa ctaacgcaat tctttgcgtt 480
agtgctttag gaagaagcta cgtgcttcct cttgaaggtg taccaactgg tgtcacttta 540
acattgcttt cagggaattt gtatgctgaa ggattcaaaa ttgctggtgg tatgaacatc 600
gacaatttac caaaatacgt aatggttgca ttacctagca ggaccatcgt ctacacactt 660
gttggcaaga aattgaaagc aagtagcgta actggatggg cttactatgt aaaatctaaa 720
gccggtgatt actcaacaga cgcaagaact gacaatttga gtgagcaaga aaaattatta 780
catatggtat aa 792
<210> 4
<211> 269
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Lys Ile Leu Leu Met Leu Ala Cys Val Phe Ala Cys Val Tyr Gly
1 5 10 15
Glu Arg Tyr Cys Ala Met Gln Ala Asp Ser Gly Ile Thr Gln Cys Arg
20 25 30
Asn Gly Thr Asn Ser Glu Cys Glu Ser Cys Phe Asn Gly Gly Asp Leu
35 40 45
Ile Trp His Leu Ala Asn Trp Asn Phe Ser Trp Ser Ile Ile Leu Ile
50 55 60
Val Phe Ile Thr Val Leu Gln Tyr Gly Arg Pro Gln Phe Ser Trp Phe
65 70 75 80
Val Tyr Gly Ile Lys Met Leu Ile Met Trp Leu Leu Trp Pro Ile Val
85 90 95
Leu Ala Leu Thr Ile Phe Asn Ala Tyr Ser Glu Tyr Glu Val Ser Arg
100 105 110
Tyr Val Met Val Gly Phe Ser Val Ala Cys Ala Ile Val Thr Phe Ile
115 120 125
Leu Trp Ile Met Tyr Phe Val Arg Ser Ile Gln Leu Tyr Arg Arg Thr
130 135 140
Lys Ser Trp Trp Ser Phe Asn Pro Glu Thr Asn Ala Ile Leu Cys Val
145 150 155 160
Ser Ala Leu Gly Arg Ser Tyr Val Leu Pro Leu Glu Gly Val Pro Thr
165 170 175
Gly Val Thr Leu Thr Leu Leu Ser Gly Asn Leu Tyr Ala Glu Gly Phe
180 185 190
Lys Ile Ala Gly Gly Met Asn Ile Asp Asn Leu Pro Lys Tyr Val Met
195 200 205
Val Ala Leu Pro Ser Arg Thr Ile Val Tyr Thr Leu Val Gly Lys Lys
210 215 220
Leu Lys Ala Ser Ser Val Thr Gly Trp Ala Tyr Tyr Val Lys Ser Lys
225 230 235 240
Ala Gly Asp Tyr Ser Thr Asp Ala Arg Thr Asp Asn Leu Ser Glu Gln
245 250 255
Glu Lys Leu Leu His Met Val Ser Glu Gln Ile Asp Asn
260 265
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agcttggtac cgaatgaaga ttttgtta 28
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atatctgcag aattttatac catatgtaat 30
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gttccagggg cccatgttag aagacagtag c 31
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gttttcaccg tcattacggg ttgtatctgt 30
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgtttaaact taagcttggt accgagc 27
<210> 10
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cactgtgctg gatatctgca gaattc 26
Claims (7)
1.一种CCV病毒M蛋白的单克隆抗体,其特征是:包括针对CCV病毒M蛋白的一对杂交瘤细胞株,其中第一个杂交瘤细胞株命名为2H11的保藏编号为CGMCC NO.17287,第二个杂交瘤细胞株命名为2G3的保藏编号为CGMCC NO.17288,2个杂交瘤细胞株均保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址均为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101;CCV病毒M蛋白的单克隆抗体,由所述的2个杂交瘤细胞株2H11和2G3分别产生。
2.CCV病毒M蛋白的单克隆抗体的制备方法,其特征在于,所述制备方法:以分离纯化的CCV灭活病毒为免疫抗原,用单克隆抗体技术制备出CCV单抗;经western-blot(蛋白免疫印迹)试验鉴定单抗针对M蛋白,再用重组酶ExnaseTM II技术对CCV病毒M蛋白的基因克隆进线性化的pCDNA3.1载体,转染293T细胞进行M蛋白表达,用单抗与表达的M蛋白做IFA鉴定进一步验证;获得2株CCV病毒M蛋白特异单抗2H11、2G3。
3.根据权利要求1所述的CCV病毒M蛋白的单克隆抗体的制备方法,其特征是,所述CCV单抗的制备方法具体为:
(1)CCV病毒的分离与纯化:取临床上发病的犬的新鲜粪便样品,经0.2μm针头滤器过滤后,接种到A-72细胞,进行盲传,出现典型细胞病变后,进行病毒纯化和鉴定;CCV病毒用A-72细胞进行大量培养,用超离法纯化病毒抗原;
(2)针对M蛋白单抗制备:用0.1%(v/v)甲醛灭活的纯化CCV病毒与弗氏佐剂混合乳化,分4次免疫8周龄的BALB/c小鼠;取效价高的小鼠脾脏细胞与瘤细胞SP2/0进行细胞融合;融合后先用感染CCV的细胞板做IFA大量筛选,筛选后的阳性单抗隆抗体细胞上清进行western-blot和转染表达人工重组的M蛋白细胞IFA检测,鉴定、获得针对CCV M蛋白的单克隆抗体。
4.根据权利要求1所述的CCV病毒M蛋白的单克隆抗体的制备方法,其特征是,所述经western-blot(蛋白免疫印迹)试验鉴定单抗针对M蛋白,再用重组酶ExnaseTM II技术对CCV病毒M蛋白的基因克隆进线性化的pCDNA3.1载体,转染293T细胞进行M蛋白表达,具体为:
(1)利用下述引物,以提取犬冠状病毒感染细胞上清的RNA为模板进行M基因的扩增,PCR引物
上游引物:AGCTTGGTACCGAatgaagattttgtta
下游引物:ATATCTGCAGAATTttataccatatgtaat
(2)利用下述引物,以PCDNA3.1+质粒做模板进行线性化载体的扩增;
上游引物:GAATTCTGCAGATATCCAGCACAGTG
下游引物:GCTCGGTACCAAGCTTAAGTTTAAACG
(3)利用重组酶ExnaseTM II对步骤1)和2)得到的PCR产物进行快速重组克隆;阳性克隆经转染293T表达细胞进行M基因的表达。
5.一种免疫胶体金试纸条的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)胶体金抗体标记:在胶体金上标记权利要求1所述的单克隆抗体2H11;
(2)喷金:将步骤(1)标记好的胶体金喷到玻璃纤维上,制成金标垫;
(3)制作硝酸纤维素膜:将兔抗鼠IgG和权利要求1所述的单克隆抗体2G3共同喷到硝酸纤维素膜上,制成对照线和检测线:
(4)将样品垫、胶体金垫、硝酸纤维素膜、吸水纸自下至上组装在PVC板上,然后切割制成试纸条;所述样品垫为空白的玻璃纤维素膜。
6.根据权利要求4所述的一种免疫胶体金试纸条的制备方法,其特征在于,所述胶体金抗体标记:用柠檬三钠还原法制备粒径约为20nm的胶体金,用0.2mol/L K2CO3调到pH的浓度8.0,在磁力搅拌下逐滴加入纯化单抗100μg/ml 2H11,室温搅拌30min,加入10%(w/v)BSA溶液,使其终浓度为1%,室温继续搅拌均匀,然后依次缓慢滴入终浓度0.2%(w/v)PEG-20000,继续搅拌30min后,置于4℃静置2h;100rpm 4℃离心15min弃去底部未结合的杂质成分,取上清10000rpm 4℃离心30min,弃去上清;沉淀用原体积1/20含5%(w/v)蔗糖、1%(w/v)BSA、0.2%(w/v)PEG20000以及0.02%(w/v)叠氮钠的0.01mol/L pH9.2硼酸盐缓冲液悬浮;置于4℃保存备用。
7.根据权利要求4所述的一种免疫胶体金试纸条的制备方法,其特征在于,所述兔抗鼠IgG的制备方法是:将小鼠IgG与弗氏完全佐剂混合乳化,背部皮下、皮内多点注射免疫健康家兔,经3次免疫后,检测血清效价,当琼脂凝胶沉淀试验AGP效价≥1:16时,采血,分离血清,加入饱和硫酸铵沉淀,透析后获得兔抗鼠IgG。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910257290.0A CN109970851B (zh) | 2019-04-01 | 2019-04-01 | Ccv病毒m蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910257290.0A CN109970851B (zh) | 2019-04-01 | 2019-04-01 | Ccv病毒m蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109970851A true CN109970851A (zh) | 2019-07-05 |
CN109970851B CN109970851B (zh) | 2022-12-06 |
Family
ID=67082100
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910257290.0A Active CN109970851B (zh) | 2019-04-01 | 2019-04-01 | Ccv病毒m蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109970851B (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111171138A (zh) * | 2020-01-14 | 2020-05-19 | 大连深蓝肽科技研发有限公司 | 用于检测刺参低聚肽的肽段、单克隆抗体、胶体金试纸条及检测方法 |
CN111239392A (zh) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | 一种新型冠状病毒肺炎(covid-19)血清学诊断试剂盒 |
CN111366734A (zh) * | 2020-03-20 | 2020-07-03 | 广州市康润生物科技有限公司 | 双指标筛选新冠病毒及预判重型肺炎的方法 |
CN115925827A (zh) * | 2022-11-30 | 2023-04-07 | 扬州大学 | 一种应用于检测犬冠状病毒抗体的重组m蛋白及其制备方法与应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103687615A (zh) * | 2011-06-28 | 2014-03-26 | 尼古拉·德卡罗 | 犬冠状病毒疫苗 |
-
2019
- 2019-04-01 CN CN201910257290.0A patent/CN109970851B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103687615A (zh) * | 2011-06-28 | 2014-03-26 | 尼古拉·德卡罗 | 犬冠状病毒疫苗 |
Non-Patent Citations (5)
Title |
---|
GABRIELLAELIA ET AL.: ""Recombinant M protein-based ELISA test for detection of antibodies to canine coronavirus"", 《JOURNAL OF VIROLOGICAL METHODS》 * |
乔军等: "犬冠状病毒基因重组抗原间接ELISA检测方法的研究", 《吉林农业大学学报》 * |
唐利: ""犬冠状病毒及其受体单克隆抗体的研制与免疫过氧化物酶噬斑染色检测方法的建立"", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑 (月刊)》 * |
施洋洋等: "新型环形病毒AGV2的VP3基因的克隆表达及多抗制备", 《中国兽医科学》 * |
甘燕等: "重组SARS病毒M蛋白的免疫效应研究", 《中国病原生物学杂志》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111171138A (zh) * | 2020-01-14 | 2020-05-19 | 大连深蓝肽科技研发有限公司 | 用于检测刺参低聚肽的肽段、单克隆抗体、胶体金试纸条及检测方法 |
CN111239392A (zh) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | 一种新型冠状病毒肺炎(covid-19)血清学诊断试剂盒 |
CN111366734A (zh) * | 2020-03-20 | 2020-07-03 | 广州市康润生物科技有限公司 | 双指标筛选新冠病毒及预判重型肺炎的方法 |
CN111366734B (zh) * | 2020-03-20 | 2021-07-13 | 广州市康润生物科技有限公司 | 双指标筛选新冠病毒及预判重型肺炎的方法 |
CN115925827A (zh) * | 2022-11-30 | 2023-04-07 | 扬州大学 | 一种应用于检测犬冠状病毒抗体的重组m蛋白及其制备方法与应用 |
CN115925827B (zh) * | 2022-11-30 | 2023-11-21 | 扬州大学 | 一种应用于检测犬冠状病毒抗体的重组m蛋白及其制备方法与应用 |
Also Published As
Publication number | Publication date |
---|---|
CN109970851B (zh) | 2022-12-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109970851A (zh) | Ccv病毒m蛋白的单抗及其制备方法、免疫胶体金试纸条的制备方法 | |
CN104198703B (zh) | 人肺炎支原体金标银染免疫层析检测试剂盒及其制备方法和应用 | |
CN105527437B (zh) | 一种检测试剂盒及其应用 | |
ES2344150T3 (es) | Procedimientos y reactivos para diagnosticar la infeccion por hantavirus. | |
CN105198986B (zh) | 抗禽白血病病毒p27蛋白单克隆抗体,包含该单克隆抗体的金标试纸条及应用 | |
CN109254155A (zh) | 一种检测非洲猪瘟病毒抗原胶体金免疫层析试纸及制备方法和应用 | |
CN101881770B (zh) | 猪圆环病毒2型胶体金抗体快速检测试纸条的制备方法 | |
CN102967710A (zh) | 小反刍兽疫抗体检测的竞争elisa试剂盒及其制备方法 | |
CN107656066A (zh) | 一种鸭坦布苏病毒e蛋白截短蛋白及应用 | |
CN110105436A (zh) | 猪圆环病毒3型抗体的elisa检测试剂盒及其制备方法与应用 | |
CN110058019A (zh) | 一种猪瘟抗体阻断检测试纸 | |
CN110068686A (zh) | 一种伪狂犬病抗体阻断检测试纸 | |
CN101363859B (zh) | 布鲁氏菌抗体快速检测试纸条 | |
CN109212239A (zh) | 一种大熊猫促黄体素胶体金免疫层析试纸条、其制备方法及应用 | |
CN109293747A (zh) | ***病毒非结构蛋白3b抗原表位肽及其应用 | |
CN106701687A (zh) | 一种杂交瘤细胞株及其产生的狂犬病毒磷蛋白单克隆抗体 | |
CN109374886A (zh) | 牛传染性鼻气管炎病毒抗体检测试剂盒及其应用 | |
CN101363865B (zh) | 一种猪蓝耳病毒胶体金检测试纸条、其制备方法及其应用 | |
CN108866008A (zh) | 抗锦鲤疱疹病毒的单克隆抗体及其细胞株和应用 | |
CN106399347A (zh) | 一种GyV5新型环形病毒VP3蛋白制备方法 | |
CN114249819A (zh) | 猫泛白细胞减少症病毒抗体、含有猫泛白细胞减少症病毒抗体的试剂盒及应用 | |
CN105968197B (zh) | 一种抗3型解脲支原体mb蛋白抗体及应用该抗体的免疫层析试剂盒 | |
CN110144329A (zh) | 杂交瘤细胞株6b1及其分泌的抗***a型病毒的单克隆抗体与应用 | |
CN109897830A (zh) | 马泰勒虫ema1单克隆抗体及其应用 | |
CN109593122A (zh) | 抗猪戊型肝炎病毒orf2蛋白单克隆抗体及其制备与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |