CN113740536A - 一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用 - Google Patents
一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用 Download PDFInfo
- Publication number
- CN113740536A CN113740536A CN202111137754.8A CN202111137754A CN113740536A CN 113740536 A CN113740536 A CN 113740536A CN 202111137754 A CN202111137754 A CN 202111137754A CN 113740536 A CN113740536 A CN 113740536A
- Authority
- CN
- China
- Prior art keywords
- swine fever
- fever virus
- african swine
- seq
- enzyme
- 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
- 101900228950 African swine fever virus Phosphoprotein p30 Proteins 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000002965 ELISA Methods 0.000 title claims abstract description 31
- 230000000903 blocking effect Effects 0.000 title claims abstract description 26
- 108090000790 Enzymes Proteins 0.000 claims abstract description 44
- 102000004190 Enzymes Human genes 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 241000701386 African swine fever virus Species 0.000 claims abstract description 21
- 208000007407 African swine fever Diseases 0.000 claims abstract description 19
- 101710120319 Photosystem I reaction center subunit IV Proteins 0.000 claims abstract description 17
- 239000000427 antigen Substances 0.000 claims abstract description 9
- 108091007433 antigens Proteins 0.000 claims abstract description 9
- 102000036639 antigens Human genes 0.000 claims abstract description 9
- 241000710777 Classical swine fever virus Species 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 74
- 210000002966 serum Anatomy 0.000 claims description 67
- 150000001413 amino acids Chemical class 0.000 claims description 54
- 239000000758 substrate Substances 0.000 claims description 27
- 241000282898 Sus scrofa Species 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 23
- 239000013642 negative control Substances 0.000 claims description 21
- 239000013641 positive control Substances 0.000 claims description 18
- 239000008055 phosphate buffer solution Substances 0.000 claims description 16
- 230000000295 complement effect Effects 0.000 claims description 10
- 239000003085 diluting agent Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- 101710151911 Phosphoprotein p30 Proteins 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 8
- 238000000576 coating method Methods 0.000 claims description 8
- 101710132601 Capsid protein Proteins 0.000 claims description 7
- 102100037623 Centromere protein V Human genes 0.000 claims description 7
- 101710189818 Non-structural protein 2a Proteins 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000012089 stop solution Substances 0.000 claims description 6
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- 244000052769 pathogen Species 0.000 claims description 5
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 claims description 4
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical group C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 229920002223 polystyrene Polymers 0.000 claims description 4
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 4
- 229960005486 vaccine Drugs 0.000 claims description 4
- 238000008157 ELISA kit Methods 0.000 claims description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 16
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 claims 2
- 229940031567 attenuated vaccine Drugs 0.000 claims 1
- 238000012217 deletion Methods 0.000 claims 1
- 230000037430 deletion Effects 0.000 claims 1
- 229940031551 inactivated vaccine Drugs 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 claims 1
- 238000002255 vaccination Methods 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 31
- 102000004169 proteins and genes Human genes 0.000 abstract description 18
- 241000700605 Viruses Species 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 206010037660 Pyrexia Diseases 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 17
- 210000004408 hybridoma Anatomy 0.000 description 15
- 238000002156 mixing Methods 0.000 description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 238000012216 screening Methods 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- 238000012163 sequencing technique Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 239000012224 working solution Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 230000003053 immunization Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000010839 reverse transcription Methods 0.000 description 7
- 239000013605 shuttle vector Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000011534 wash buffer Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 5
- 241000710202 Foot-and-mouth disease virus - type A Species 0.000 description 5
- 241000710194 Foot-and-mouth disease virus - type O Species 0.000 description 5
- 238000010009 beating Methods 0.000 description 5
- 230000009465 prokaryotic expression Effects 0.000 description 5
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000012620 biological material Substances 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 101150049619 p30 gene Proteins 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 239000012898 sample dilution Substances 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000011888 foil Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- AIRUUHAOKGVJAD-JYJNAYRXSA-N Leu-Phe-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIRUUHAOKGVJAD-JYJNAYRXSA-N 0.000 description 2
- 240000000220 Panda oleosa Species 0.000 description 2
- 235000016496 Panda oleosa Nutrition 0.000 description 2
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 2
- 241000202347 Porcine circovirus Species 0.000 description 2
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000519995 Stachys sylvatica Species 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- LXWYCLOUQZZDBD-LIYNQYRNSA-N csfv Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LXWYCLOUQZZDBD-LIYNQYRNSA-N 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 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 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 229910000406 trisodium phosphate Inorganic materials 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 101100406721 African swine fever virus (strain Badajoz 1971 Vero-adapted) Ba71V-93 gene Proteins 0.000 description 1
- AWAXZRDKUHOPBO-GUBZILKMSA-N Ala-Gln-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O AWAXZRDKUHOPBO-GUBZILKMSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- FQNILRVJOJBFFC-FXQIFTODSA-N Ala-Pro-Asp Chemical compound C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N FQNILRVJOJBFFC-FXQIFTODSA-N 0.000 description 1
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 1
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 1
- XKXAZPSREVUCRT-BPNCWPANSA-N Ala-Tyr-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=C(O)C=C1 XKXAZPSREVUCRT-BPNCWPANSA-N 0.000 description 1
- ZDILXFDENZVOTL-BPNCWPANSA-N Ala-Val-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZDILXFDENZVOTL-BPNCWPANSA-N 0.000 description 1
- NONSEUUPKITYQT-BQBZGAKWSA-N Arg-Asn-Gly Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N)CN=C(N)N NONSEUUPKITYQT-BQBZGAKWSA-N 0.000 description 1
- BEXGZLUHRXTZCC-CIUDSAMLSA-N Arg-Gln-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N BEXGZLUHRXTZCC-CIUDSAMLSA-N 0.000 description 1
- LLUGJARLJCGLAR-CYDGBPFRSA-N Arg-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N LLUGJARLJCGLAR-CYDGBPFRSA-N 0.000 description 1
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- VKCOHFFSTKCXEQ-OLHMAJIHSA-N Asn-Asn-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VKCOHFFSTKCXEQ-OLHMAJIHSA-N 0.000 description 1
- MECFLTFREHAZLH-ACZMJKKPSA-N Asn-Glu-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N MECFLTFREHAZLH-ACZMJKKPSA-N 0.000 description 1
- KMCRKVOLRCOMBG-DJFWLOJKSA-N Asn-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(=O)N)N KMCRKVOLRCOMBG-DJFWLOJKSA-N 0.000 description 1
- KSGAFDTYQPKUAP-GMOBBJLQSA-N Asn-Met-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KSGAFDTYQPKUAP-GMOBBJLQSA-N 0.000 description 1
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 1
- CGYKCTPUGXFPMG-IHPCNDPISA-N Asn-Tyr-Trp Chemical compound [H]N[C@@H](CC(N)=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 CGYKCTPUGXFPMG-IHPCNDPISA-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
- ZELQAFZSJOBEQS-ACZMJKKPSA-N Asp-Asn-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZELQAFZSJOBEQS-ACZMJKKPSA-N 0.000 description 1
- QNFRBNZGVVKBNJ-PEFMBERDSA-N Asp-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N QNFRBNZGVVKBNJ-PEFMBERDSA-N 0.000 description 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
- ALMIMUZAWTUNIO-BZSNNMDCSA-N Asp-Tyr-Tyr 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=CC=C(O)C=C1)C(O)=O ALMIMUZAWTUNIO-BZSNNMDCSA-N 0.000 description 1
- 101001105440 Chlamydomonas reinhardtii Photosystem I reaction center subunit IV, chloroplastic Proteins 0.000 description 1
- 241001533384 Circovirus Species 0.000 description 1
- DIHCYBRLTVEPBW-SRVKXCTJSA-N Cys-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CS)N DIHCYBRLTVEPBW-SRVKXCTJSA-N 0.000 description 1
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 1
- VOLVNCMGXWDDQY-LPEHRKFASA-N Gln-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)C(=O)O VOLVNCMGXWDDQY-LPEHRKFASA-N 0.000 description 1
- YPMDZWPZFOZYFG-GUBZILKMSA-N Gln-Leu-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YPMDZWPZFOZYFG-GUBZILKMSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 1
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 1
- PHONAZGUEGIOEM-GLLZPBPUSA-N Glu-Glu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PHONAZGUEGIOEM-GLLZPBPUSA-N 0.000 description 1
- QYPKJXSMLMREKF-BPUTZDHNSA-N Glu-Glu-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)N QYPKJXSMLMREKF-BPUTZDHNSA-N 0.000 description 1
- ZCOJVESMNGBGLF-GRLWGSQLSA-N Glu-Ile-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZCOJVESMNGBGLF-GRLWGSQLSA-N 0.000 description 1
- DWBBKNPKDHXIAC-SRVKXCTJSA-N Glu-Leu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCC(O)=O DWBBKNPKDHXIAC-SRVKXCTJSA-N 0.000 description 1
- SWRVAQHFBRZVNX-GUBZILKMSA-N Glu-Lys-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O SWRVAQHFBRZVNX-GUBZILKMSA-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
- ADZGCWWDPFDHCY-ZETCQYMHSA-N Gly-His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 ADZGCWWDPFDHCY-ZETCQYMHSA-N 0.000 description 1
- VBOBNHSVQKKTOT-YUMQZZPRSA-N Gly-Lys-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O VBOBNHSVQKKTOT-YUMQZZPRSA-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
- 208000032843 Hemorrhage Diseases 0.000 description 1
- XJQDHFMUUBRCGA-KKUMJFAQSA-N His-Asn-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XJQDHFMUUBRCGA-KKUMJFAQSA-N 0.000 description 1
- UPGJWSUYENXOPV-HGNGGELXSA-N His-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N UPGJWSUYENXOPV-HGNGGELXSA-N 0.000 description 1
- GGXUJBKENKVYNV-ULQDDVLXSA-N His-Val-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N GGXUJBKENKVYNV-ULQDDVLXSA-N 0.000 description 1
- TZCGZYWNIDZZMR-NAKRPEOUSA-N Ile-Arg-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](C)C(=O)O)N TZCGZYWNIDZZMR-NAKRPEOUSA-N 0.000 description 1
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 1
- JRYQSFOFUFXPTB-RWRJDSDZSA-N Ile-Gln-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N JRYQSFOFUFXPTB-RWRJDSDZSA-N 0.000 description 1
- LGMUPVWZEYYUMU-YVNDNENWSA-N Ile-Glu-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N LGMUPVWZEYYUMU-YVNDNENWSA-N 0.000 description 1
- LPFBXFILACZHIB-LAEOZQHASA-N Ile-Gly-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)O)N LPFBXFILACZHIB-LAEOZQHASA-N 0.000 description 1
- TWYOYAKMLHWMOJ-ZPFDUUQYSA-N Ile-Leu-Asn Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O TWYOYAKMLHWMOJ-ZPFDUUQYSA-N 0.000 description 1
- VEPIBPGLTLPBDW-URLPEUOOSA-N Ile-Phe-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N VEPIBPGLTLPBDW-URLPEUOOSA-N 0.000 description 1
- JODPUDMBQBIWCK-GHCJXIJMSA-N Ile-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O JODPUDMBQBIWCK-GHCJXIJMSA-N 0.000 description 1
- FBGXMKUWQFPHFB-JBDRJPRFSA-N Ile-Ser-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N FBGXMKUWQFPHFB-JBDRJPRFSA-N 0.000 description 1
- QQVXERGIFIRCGW-NAKRPEOUSA-N Ile-Ser-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)O)N QQVXERGIFIRCGW-NAKRPEOUSA-N 0.000 description 1
- DTPGSUQHUMELQB-GVARAGBVSA-N Ile-Tyr-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 DTPGSUQHUMELQB-GVARAGBVSA-N 0.000 description 1
- PRTZQMBYUZFSFA-XEGUGMAKSA-N Ile-Tyr-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)NCC(=O)O)N PRTZQMBYUZFSFA-XEGUGMAKSA-N 0.000 description 1
- 102100024067 Inhibitor of growth protein 2 Human genes 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- OGUUKPXUTHOIAV-SDDRHHMPSA-N Leu-Glu-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N OGUUKPXUTHOIAV-SDDRHHMPSA-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
- SGIIOQQGLUUMDQ-IHRRRGAJSA-N Leu-His-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N SGIIOQQGLUUMDQ-IHRRRGAJSA-N 0.000 description 1
- FAELBUXXFQLUAX-AJNGGQMLSA-N Leu-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C FAELBUXXFQLUAX-AJNGGQMLSA-N 0.000 description 1
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- 108010062166 Lys-Asn-Asp Proteins 0.000 description 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 description 1
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- NVGBPTNZLWRQSY-UWVGGRQHSA-N Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN NVGBPTNZLWRQSY-UWVGGRQHSA-N 0.000 description 1
- ZCWWVXAXWUAEPZ-SRVKXCTJSA-N Lys-Met-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZCWWVXAXWUAEPZ-SRVKXCTJSA-N 0.000 description 1
- XFOAWKDQMRMCDN-ULQDDVLXSA-N Lys-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)CC1=CC=CC=C1 XFOAWKDQMRMCDN-ULQDDVLXSA-N 0.000 description 1
- AZOFEHCPMBRNFD-BZSNNMDCSA-N Lys-Phe-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=CC=C1 AZOFEHCPMBRNFD-BZSNNMDCSA-N 0.000 description 1
- WGILOYIKJVQUPT-DCAQKATOSA-N Lys-Pro-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WGILOYIKJVQUPT-DCAQKATOSA-N 0.000 description 1
- 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
- RPWTZTBIFGENIA-VOAKCMCISA-N Lys-Thr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O RPWTZTBIFGENIA-VOAKCMCISA-N 0.000 description 1
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- PJWDQHNOJIBMRY-JYJNAYRXSA-N Met-Arg-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PJWDQHNOJIBMRY-JYJNAYRXSA-N 0.000 description 1
- XOMXAVJBLRROMC-IHRRRGAJSA-N Met-Asp-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XOMXAVJBLRROMC-IHRRRGAJSA-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
- KPVLLNDCBYXKNV-CYDGBPFRSA-N Met-Val-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KPVLLNDCBYXKNV-CYDGBPFRSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- KAHUBGWSIQNZQQ-KKUMJFAQSA-N Phe-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KAHUBGWSIQNZQQ-KKUMJFAQSA-N 0.000 description 1
- DHZOGDVYRQOGAC-BZSNNMDCSA-N Phe-Cys-Tyr Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N DHZOGDVYRQOGAC-BZSNNMDCSA-N 0.000 description 1
- BFYHIHGIHGROAT-HTUGSXCWSA-N Phe-Glu-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BFYHIHGIHGROAT-HTUGSXCWSA-N 0.000 description 1
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 1
- MVIJMIZJPHQGEN-IHRRRGAJSA-N Phe-Ser-Val Chemical compound CC(C)[C@@H](C([O-])=O)NC(=O)[C@H](CO)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 MVIJMIZJPHQGEN-IHRRRGAJSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- ZLXKLMHAMDENIO-DCAQKATOSA-N Pro-Lys-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLXKLMHAMDENIO-DCAQKATOSA-N 0.000 description 1
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 1
- QKWYXRPICJEQAJ-KJEVXHAQSA-N Pro-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@@H]2CCCN2)O QKWYXRPICJEQAJ-KJEVXHAQSA-N 0.000 description 1
- FIXILCYTSAUERA-FXQIFTODSA-N Ser-Ala-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FIXILCYTSAUERA-FXQIFTODSA-N 0.000 description 1
- UGJRQLURDVGULT-LKXGYXEUSA-N Ser-Asn-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UGJRQLURDVGULT-LKXGYXEUSA-N 0.000 description 1
- HVKMTOIAYDOJPL-NRPADANISA-N Ser-Gln-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVKMTOIAYDOJPL-NRPADANISA-N 0.000 description 1
- HJEBZBMOTCQYDN-ACZMJKKPSA-N Ser-Glu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HJEBZBMOTCQYDN-ACZMJKKPSA-N 0.000 description 1
- OHKFXGKHSJKKAL-NRPADANISA-N Ser-Glu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OHKFXGKHSJKKAL-NRPADANISA-N 0.000 description 1
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 1
- SNVIOQXAHVORQM-WDSKDSINSA-N Ser-Gly-Gln Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O SNVIOQXAHVORQM-WDSKDSINSA-N 0.000 description 1
- 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
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 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
- GYDFRTRSSXOZCR-ACZMJKKPSA-N Ser-Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GYDFRTRSSXOZCR-ACZMJKKPSA-N 0.000 description 1
- FLMYSKVSDVHLEW-SVSWQMSJSA-N Ser-Thr-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLMYSKVSDVHLEW-SVSWQMSJSA-N 0.000 description 1
- PCMZJFMUYWIERL-ZKWXMUAHSA-N Ser-Val-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMZJFMUYWIERL-ZKWXMUAHSA-N 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- PXQUBKWZENPDGE-CIQUZCHMSA-N Thr-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)O)N PXQUBKWZENPDGE-CIQUZCHMSA-N 0.000 description 1
- NLSNVZAREYQMGR-HJGDQZAQSA-N Thr-Asp-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NLSNVZAREYQMGR-HJGDQZAQSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- 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
- DEGCBBCMYWNJNA-RHYQMDGZSA-N Thr-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O DEGCBBCMYWNJNA-RHYQMDGZSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 1
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 1
- PELIQFPESHBTMA-WLTAIBSBSA-N Thr-Tyr-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 PELIQFPESHBTMA-WLTAIBSBSA-N 0.000 description 1
- BKIOKSLLAAZYTC-KKHAAJSZSA-N Thr-Val-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O BKIOKSLLAAZYTC-KKHAAJSZSA-N 0.000 description 1
- PWONLXBUSVIZPH-RHYQMDGZSA-N Thr-Val-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O PWONLXBUSVIZPH-RHYQMDGZSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- UOXPLPBMEPLZBW-WDSOQIARSA-N Trp-Val-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 UOXPLPBMEPLZBW-WDSOQIARSA-N 0.000 description 1
- XGEUYEOEZYFHRL-KKXDTOCCSA-N Tyr-Ala-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 XGEUYEOEZYFHRL-KKXDTOCCSA-N 0.000 description 1
- PEVVXUGSAKEPEN-AVGNSLFASA-N Tyr-Asn-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PEVVXUGSAKEPEN-AVGNSLFASA-N 0.000 description 1
- IUQDEKCCHWRHRW-IHPCNDPISA-N Tyr-Asn-Trp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O IUQDEKCCHWRHRW-IHPCNDPISA-N 0.000 description 1
- GHUNBABNQPIETG-MELADBBJSA-N Tyr-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O GHUNBABNQPIETG-MELADBBJSA-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
- JKUZFODWJGEQAP-KBPBESRZSA-N Tyr-Gly-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O JKUZFODWJGEQAP-KBPBESRZSA-N 0.000 description 1
- OFHKXNKJXURPSY-ULQDDVLXSA-N Tyr-Met-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O OFHKXNKJXURPSY-ULQDDVLXSA-N 0.000 description 1
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 1
- PLVVHGFEMSDRET-IHPCNDPISA-N Tyr-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC3=CC=C(C=C3)O)N PLVVHGFEMSDRET-IHPCNDPISA-N 0.000 description 1
- LVFZXRQQQDTBQH-IRIUXVKKSA-N Tyr-Thr-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LVFZXRQQQDTBQH-IRIUXVKKSA-N 0.000 description 1
- JQOMHZMWQHXALX-FHWLQOOXSA-N Tyr-Tyr-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JQOMHZMWQHXALX-FHWLQOOXSA-N 0.000 description 1
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 1
- JYVKKBDANPZIAW-AVGNSLFASA-N Val-Arg-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C(C)C)N JYVKKBDANPZIAW-AVGNSLFASA-N 0.000 description 1
- IWZYXFRGWKEKBJ-GVXVVHGQSA-N Val-Gln-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N IWZYXFRGWKEKBJ-GVXVVHGQSA-N 0.000 description 1
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 1
- APEBUJBRGCMMHP-HJWJTTGWSA-N Val-Ile-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 APEBUJBRGCMMHP-HJWJTTGWSA-N 0.000 description 1
- UOUIMEGEPSBZIV-ULQDDVLXSA-N Val-Lys-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 UOUIMEGEPSBZIV-ULQDDVLXSA-N 0.000 description 1
- UXODSMTVPWXHBT-ULQDDVLXSA-N Val-Phe-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N UXODSMTVPWXHBT-ULQDDVLXSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 108010038850 arginyl-isoleucyl-tyrosine Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010033719 glycyl-histidyl-glycine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010091871 leucylmethionine Proteins 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108010022588 methionyl-lysyl-proline Proteins 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000009251 neurologic dysfunction Effects 0.000 description 1
- 208000015015 neurological dysfunction Diseases 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- 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
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/12011—Asfarviridae
- C12N2710/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用。该试剂盒包括包括以非洲猪瘟病毒p30蛋白为包被抗原的的酶联反应板和酶标抗体；所述酶标抗体为与非洲猪瘟病毒p30蛋白特异性结合的单克隆抗体(6G10)制成的酶标抗体。本试剂盒采用非洲猪瘟病毒p30纯化蛋白和抗非洲猪瘟病毒p30蛋白特异性单克隆抗体制备的阻断法酶联免疫抗体检测试剂盒，灵敏度高、特异性强，可以有效地检测样品中非洲猪瘟病毒抗体的含量，与进口试剂盒符合率达95％以上，具有广阔的市场前景和良好的经济、社会效益。
Description
技术领域
本发明属于生物检测技术领域，更具体地，本发明涉及一种非洲猪瘟p30阻断ELISA抗体检测试剂盒及其应用，适用于非洲猪瘟抗体的特异、快速、准确检测。
背景技术
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fevervirus,ASFV)引起的猪的一种急性、烈性、高度接触传染性疾病。非洲猪瘟在临床症状上与猪瘟极为相似，以高热、高死亡率、全身广泛性出血、呼吸障碍以及神经功能紊乱为主要特征，病死率高达100％。我国将其列为一类动物传染病。非洲猪瘟1921年于肯尼亚发现，2007年传入高加索地区和俄罗斯，2014年已传入东欧大部分国家，流行趋势扩大，2018年，我国首例非洲猪瘟在沈阳确诊并迅速蔓延，给我国养猪业造成巨大经济损失。
非洲猪瘟病毒是一种由囊膜包裹的双股线性DNA病毒。不同毒株基因组大小存在些许差异，长度介于170-190Kb之间，有150多个主要开放阅读框，可编码150～200种蛋白质，其中结构蛋白为54种。其中p30蛋白是ASFV的主要结构蛋白，由CP204L基因编码，也被称为p32蛋白，该蛋白有很强的抗原性，能诱导机体产生中和抗体，通常被用做诊断抗原。非洲猪瘟病毒p30蛋白是病毒感染早期产生的蛋白，以此为基础的抗体检测方法可在病毒感染早期就实现有效检测。
由于对ASFV的生物学特性研究不充分，目前还没有研发出针对非洲猪瘟特别有效的疫苗，对非洲猪瘟的防控主要依赖于动物检疫，但非洲猪瘟与猪瘟临床症状极为相似且作为我国的新发病，国内相应的检测产品尚属于空白状态，因此研发出一款简便、快捷、高效的检测试剂变得尤为重要。
酶联免疫吸附试验(Enzyme-Linked ImmunosorbentAssays,ELISA)是市场上主流的免疫测定技术，已广泛应用于临床检测，快速便捷且灵敏度高，且不需要特殊的仪器设备，可用于非洲猪瘟病毒的抗体检测。
发明内容
本发明获得了具有较好反应原性及免疫原性的可溶性p30蛋白，以该蛋白免疫BALB/c小鼠，通过细胞融合和亚克隆筛选，获得了一株特异性单抗；分析抗体序列，构建并表达p30特异性单抗，以避免抗体生产过程中，杂交瘤细胞抗体分泌不稳定以及腹水制备批次间存在差异的问题，能够保证抗体供应高效、稳定；研发出基于抗非洲猪瘟病毒p30蛋白特异性单克隆抗体的阻断ELISA抗体检测方法，解决了间接ELISA方法特异性较差的问题，使得该试剂盒具有很好的敏感性和特异性。
基于此，本发明的目的在于提供一种敏感性高、特异性强、能够快速、简便地检测非洲猪瘟病毒p30抗体的阻断ELISA试剂盒。
该试剂盒的优点之一是使用了辣根过氧化物酶(HRP)标记的抗非洲猪瘟病毒p30蛋白的单克隆抗体，提高了检测的敏感性和特异性。
基于上述目的，本发明的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，包括以非洲猪瘟病毒p30蛋白为包被抗原的的酶联反应板和酶标抗体；所述酶标抗体为与非洲猪瘟病毒p30蛋白特异性结合的单克隆抗体(6G10)制成的酶标抗体。所述酶标抗体优选为经辣根化物酶标记抗体，所述辣根过氧化物酶可通过戊二醛法或过碘酸法交联在抗体上。
优选的，所述非洲猪瘟p30蛋白的序列为序列表中SEQ ID No.5序列，为密码子优化后序列，构建入原核表达载体pET-28a，该蛋白为利用大肠杆菌原核表达系统获得的可溶性p30纯化蛋白，具有良好的特异性、敏感性及免疫原性。
优选的，与非洲猪瘟病毒p30蛋白特异性结合的单克隆抗体含有重链可变区6G10-VH和轻链可变区6G10-VL；所述重链可变区6G10-VH和轻链可变区6G10-VL均由决定簇互补区和框架区组成；所述6G10-VH和所述6G10-VL的决定簇互补区均由CDR1、CDR2和CDR3组成；所述6G10-VH的CDR1的氨基酸序列如SEQ ID No.1的第26～30位氨基酸所示；所述6G10-VH的CDR2的氨基酸序列如SEQ ID No.1的第48～63位氨基酸所示；所述6G10-VH的CDR3的氨基酸序列如SEQ ID No.1的第102～111位氨基酸所示；所述6G10-VL的CDR1的氨基酸序列如SEQID No.2的第24～40位氨基酸所示；所述6G10-VL的CDR2的氨基酸序列如SEQ ID No.2的第56～62位氨基酸所示；所述6G10-VL的CDR3的氨基酸序列如SEQ ID No.2的第95～102位氨基酸所示。
优选的，所述6G10-VH的氨基酸序列如序列表中SEQ ID No.1的第1～119位所示；其6G10-VL的氨基酸序列如序列表中SEQ ID No.2的第1～113位所示。
所述酶联反应板的最佳包被制备方法及条件是将所述非洲猪瘟病毒p30蛋白溶于100μl的pH 9.6的碳酸盐溶液，然后加到96孔聚苯乙烯酶联反应板，每孔0.1μg～1μg非洲猪瘟病毒p30蛋白，2～8℃下放置8～12小时，使包被抗原与酶联反应板充分结合，然后按照300μl/孔加入含有10mg/ml牛血清白蛋白pH7.4的PBS缓冲液，37℃封闭处理2～3小时，甩干后，待酶联反应板干燥后4℃密封保存。
优选的，所述试剂盒还包括阳性对照血清和阴性对照血清，所述阳性对照血清为用p30重组蛋白免疫后的猪血清；所述阴性对照血清为无特定病原体且无疫苗接种的猪血清。
更进一步的，所述试剂盒还包括样品稀释液、20倍浓缩洗涤液、底物液A、底物液B、终止液。所述酶联反应板为可拆卸96孔酶标板。所述样品稀释液为含有5mg/ml酪蛋白的0.01M、pH值为7.4的磷酸盐缓冲液。所述20倍浓缩洗涤液为含有浓度为0.8％～1.2％(ml/ml)的Tween-20的0.01M，pH值为7.4的磷酸盐缓冲液。所述底物液A为含0.6mg/ml过氧化氢尿素的柠檬酸磷酸盐缓冲液，所述底物液B为0.2mg/ml的四甲基联苯胺溶液，使用时两者以1:1的比例混合。所述终止液为2mol/L的硫酸溶液。
本发明还要求保护单克隆抗体，其可与非洲猪瘟病毒特异性结合，是下述任意一项所述的单克隆抗体：
1)含有重链可变区6G10-VH和轻链可变区6G10-VL；所述重链可变区6G10-VH和轻链可变区6G10-VL均由决定簇互补区和框架区组成；所述6G10-VH和所述6G10-VL的决定簇互补区均由CDR1、CDR2和CDR3组成；所述6G10-VH的CDR1的氨基酸序列如SEQ ID No.1的第26～30位氨基酸所示；所述6G10-VH的CDR2的氨基酸序列如SEQ ID No.1的第48～63位氨基酸所示；所述6G10-VH的CDR3的氨基酸序列如SEQ ID No.1的第102～111位氨基酸所示；所述6G10-VL的CDR1的氨基酸序列如SEQ ID No.2的第24～40位氨基酸所示；所述6G10-VL的CDR2的氨基酸序列如SEQ ID No.2的第56～62位氨基酸所示；所述6G10-VL的CDR3的氨基酸序列如SEQ ID No.2的第95～102位氨基酸所示。
2)含有重链可变区6G10-VH和轻链可变区6G10-VL；所述6G10-VH的氨基酸序列如序列表中SEQ ID No.1的第1～119位所示；其6G10-VL的氨基酸序列如序列表中SEQ ID No.2的第1～113位所示。
通过上述重链可变区和轻链可变区序列，可以与动物源恒定区(如鼠抗体重链和轻链恒定区)连接，制备得到可与非洲猪瘟病毒特异性结合的单克隆抗体。
上述酶联免疫试剂盒在非洲猪瘟病毒p30阻断抗体检测中的应用也属于本发明的保护范围。
上述可以与非洲猪瘟毒特异性结合的单克隆抗体在制备检测非洲猪瘟病毒的试剂盒中的应用也是本发明的保护范围。特别是在制备检测非洲猪瘟病毒抗体的试剂盒中的应用。
上述非洲猪瘟病毒p30蛋白为利用大肠杆菌原核表达系统表达获得的非洲猪瘟病毒p30可溶性纯化蛋白，可包括下述步骤：
1)基因序列的合成、表达载体的构建：根据GenBank报道(MK238347.1)毒株的p30基因序列，加入His标签，并在p30基因的5’和3’分别插入Nde Ⅰ和Xho Ⅰ两个酶切位点，经大肠杆菌密码子优化后合成，合成基因构建在pET-28a(+)载体中，对插入质粒的目的片段进行测序鉴定。
2)质粒转化及阳性菌株筛选：将构建好的质粒转化BL21(DE3)感受态，将1μL质粒加入100μL感受态，轻轻震荡混匀，冰浴30min，45℃热激45s，冰浴3min，加入700μL无抗LB培养基，37℃，200rpm培养1h，取部分培养基涂布于卡纳抗性的LB平板，37℃培养过夜。挑取菌落进行培养，送往测序公司对插入的目的片段进行测序，比对后筛选出测序无误菌株。
3)蛋白的诱导表达：接种阳性菌株于卡纳抗性LB培养基，37℃，200rpm过夜培养作为种子液，第二天以1：100的比例加入卡纳抗性LB培养基，当培养基OD600达到0.6～0.8时，加入终浓度为0.5mM的IPTG，37℃诱导表达4h。
4)菌体收集及破碎：菌液4000rpm离心30min，弃上清，用PBS按照1：10的比例重悬菌体。超声破碎菌体，超声破碎仪参数设置为：功率190W，工作5s，关闭10s，超声15min。离心后收集破碎上清。
5)蛋白纯化：用0.22μm滤膜过滤细菌破碎上清后备用。20mmol/L Tris 150mmol/LNacl PH8.0溶液平衡His柱，然后细菌破碎上清挂柱，以不同浓度的咪唑溶液洗脱，进行亲和层析纯化并鉴定，进而获得纯化的非洲猪瘟病毒p30蛋白。
上述的可以与非洲猪瘟病毒p30蛋白及非洲猪瘟病毒特异性结合的单克隆抗体的获得方法如下：按照本领域已知的常规方法筛选本发明非洲猪瘟病毒特异性单克隆细胞株，再采用基因测序的方法测定特异性单克隆细胞株的基因序列，利用基因合成，构建重组表达载体的方法制备稳定表达的单克隆抗体作为本发明的酶标单克隆抗体。具体来讲，本发明非洲猪瘟病毒的特异性单克隆抗体的获得方法，可包括下述步骤：
1)以大肠杆菌原核表达系统表达并纯化的p30蛋白为免疫原；
2连续免疫4次，每次间隔14天，前3次采用多点皮下免疫方式，第4次采取腹腔注射的免疫方式，每次10μg/只动物；
3)分离免疫动物的脾细胞，将其与骨髓瘤细胞进行融合，用HAT选择性培养基筛选杂交瘤细胞，对杂交瘤细胞上清用间接ELISA方法进行筛选特异性阳性克隆。当被免疫动物的血清抗体水平用间接ELISA进行检测效价超过1:50000时，可分离动物的脾细胞并制备成单细胞悬液，并在适当的融合剂(如聚乙二醇)的诱导下与骨髓瘤细胞(优选为小鼠骨髓瘤细胞SP2/0)融合以形成杂交瘤；经检测，优选单克隆细胞株分泌的6G10能够特异性与非洲猪瘟病毒p30蛋白反应。
4)特异性阳性克隆杂交瘤细胞株总RNA提取：取杂交瘤细胞悬液250μl，加入750μl的Trizol，上下颠倒混匀，加入200μl的氯仿，混匀，4℃12000rpm离心15min。吸取上清至新的1.5ml EP管中，加入600μl的异丙醇，混匀离心10min。将异丙醇弃去，用75％的DEPC乙醇洗涤，离心。将乙醇弃去，烘干，用20μl无RNA酶水溶解RNA。
5)反转录、PCR扩增及基因测序：利用Invitrogen反转录试剂盒按说明书进行反转录，获得杂交瘤细胞的cDNA。针对重链(VH-1：5’-GTGAATTCATGCAGGTGCAGCTGTTGGAGTCTGG-3’；VH-2：5’-ATGTCGACTGAGGAGACGGTGACCAGGGTGCC-3’)和轻链(VL-1：5’-GTGAATTCATGGACATTGTGATGACCCAGTCTCC-3’；VL-2：5’-CAGTCGACTTACGTTTGATCTCCAGCTTGGTCCC-3’)可变区设计通用引物，利用扩增引物对目的片段进行扩增，扩增完后胶回收片段，然后连接载体进行序列测定，获取单克隆抗体重链和轻链可变区序列信息。
6)特异性单克隆抗体的基因序列的合成、穿梭载体的构建、重组Bacmid的筛选与提取、重组杆状病毒的拯救：①基因序列的合成：根据已测得单抗6G10的重链和轻链可变区的序列，将鼠抗体重链和轻链恒定区的序列补充在可变区部分，然后进行基因序列的合成，并进行昆虫细胞密码子优化。②穿梭载体的构建：根据重链和轻链的序列信息以及pFastBacdual载体序列信息，构建穿梭载体，pFastBacdual载体含有两个启动子，即PH启动子和P10启动子，连入载体后进行序列测定确保序列的准确性。③重组Bacmid的筛选与提取：将构建好的穿梭载体转化DH10Bac感受态，然后涂布三抗平板(卡那霉素、庆大霉素、四环霉素)，37℃培养箱培养48h后挑取白斑，进行鉴定，阳性克隆目的片段大小为4600bp，阴性克隆为300bp，选取完全无300bp条带的克隆菌摇菌，12h后采用异丙醇沉淀法进行Bacmid的提取，然后利用Nanodrop进行浓度测定。④重组杆状病毒的拯救：转染前将密度为2×106的SF9细胞铺六孔板，重组Bacmid按5μg和2.5μg的量进行转染，转染试剂用量为8μl，转染后4～6h进行换液，28℃培养，72h后收获扩增P2代病毒，采用同样方法进行P3代病毒扩增。P4代病毒的扩增采取摇瓶扩增，病毒接种比例为1：100。
7)特异性单克隆抗体的表达与纯化：将P4代病毒按1:5的比例接种密度为2×106的Hi5细胞，28℃培养，48h后收获细胞，8000r/min离心1h取上清，然后用0.22μm滤膜过滤备用。用Na3PO4 pH值为7.0溶液平衡ProteinA预装柱，平衡3～5个柱体积，然后将细胞上清结合ProteinA预装柱，样品结合完后用Glycine-HCL pH值为3.0洗脱液进行洗脱，即获得纯化的抗非洲猪瘟病毒p30蛋白特异性单克隆抗体6G10。
本发明试剂盒的检测程序为：
1)平衡：将试剂盒从冷藏环境中取出，置室温平衡30min备用；液体试剂用前混匀。
2)配液：将浓缩洗涤液用蒸馏水或去离子水20倍稀释得到洗涤缓冲液；
3)样品稀释：在血清稀释板中将待检血清用样品稀释液进行2倍稀释，阴、阳性对照血清已稀释，可直接使用。
4)加样：取出所需板条，剩余板条装入铝箔袋中封好，置于2～8℃保存备用。将稀释好的待检血清、阴性对照血清和阳性对照血清加入到抗原包被板中，100μl/孔。每份待检血清设1孔，阴性对照和阳性对照各设2孔，加样过程时间跨度应尽量短。如图1所示加样：N：表示加阴性对照血清；P：表示加阳性对照血清；S1、S2、S3、S4等表示加各待检血清。
5)温育：震荡混匀，置37℃温箱中，反应60min。
6)洗板：弃去反应液，每孔加300μl稀释后的洗涤缓冲液，浸泡15s，甩弃洗液，连续洗板4次后拍干。
7)加酶：各孔加入辣根过氧化物酶标记的单克隆抗体100μl(浓度为0.5ug/ml)。
8)温育：置37℃温箱，反应30min。
9)洗板：弃去反应液，每孔加入稀释后的洗涤缓冲液300μl，浸泡15s，甩弃洗涤液，连续洗板4次后拍干。
10)显色：每孔加入100μl底物工作液(将底物液A和底物液B等量混合即为底物工作液，现用现配)，震荡混匀，置37℃温箱中，避光反应15min。
11)每孔加入显色终止液50μl，振荡混匀终止反应，15分钟内测定结果。
12)试验成立条件：阴性对照OD450nm值均应≥1.0。阳性对照孔S/N值应≤0.5。
13)判定：在酶标仪上测各孔OD450nm值。S/N＝样本OD450nm值/阴性对照OD450nm值。通过计算每个样品的S/N值，判定其抗体的有无。阴性：S/N≥0.7；可疑0.6＜S/N＜0.7；阳性S/N≤0.6。
本发明的积极效果在于：本发明提供了非洲猪瘟病毒抗体检测的酶联免疫检测试剂盒。该试剂盒是采用非洲猪瘟病毒p30纯化蛋白和抗非洲猪瘟病毒p30蛋白特异性单克隆抗体制备的阻断法酶联免疫抗体检测试剂盒，可通过检测酶催化底物产生的信号变化来测定样品中非洲猪瘟病毒特异性抗体的含量，且与目前其他病原如猪口蹄疫病毒O型、猪口蹄疫病毒A型、猪圆环病毒、猪繁殖与呼吸综合征病毒阳性血清均不发生交叉反应。
综上所述，本试剂盒采用非洲猪瘟病毒p30纯化蛋白和抗非洲猪瘟病毒p30蛋白特异性单克隆抗体制备的阻断法酶联免疫抗体检测试剂盒，灵敏度高、特异性强，可以有效地检测样品中非洲猪瘟病毒抗体的含量，与进口试剂盒符合率达95％以上，具有广阔的市场前景和良好的经济、社会效益。
附图说明
图1为本发明试剂盒酶联免疫板加样示意图。
具体实施方式
下述实施例中的方法，如无特别说明，均为常规方法。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和伦理道德可获取的生物材料都可按照实施例中的提示替换使用。
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
实施例1、制备非洲猪瘟病毒p30纯化蛋白
包括下述步骤：
上述非洲猪瘟病毒p30蛋白为利用大肠杆菌原核表达系统表达获得的非洲猪瘟病毒p30可溶性纯化蛋白，可包括下述步骤：
1)根据GenBank报道(MK238347.1)的p30基因序列，加入His标签，并在p30基因的5’和3’分别插入NdeⅠ和XhoⅠ两个酶切位点，经大肠杆菌密码子优化后合成，合成基因构建在pET-28a(+)载体中，对插入质粒的目的片段进行测序鉴定。
2)将构建好的质粒转化BL21(DE3)感受态，将1μL质粒加入100μL感受态，轻轻震荡混匀，冰浴30min，45℃热激45s，冰浴3min，加入700μL无抗LB培养基，37℃，200rpm培养1h，取部分培养基涂布于卡纳抗性的LB平板，37℃培养过夜。挑取菌落进行培养，送往测序公司对插入的目的片段进行测序，比对后筛选出测序无误菌株。
3)接种阳性菌株于卡纳抗性LB培养基，37℃，200rpm过夜培养作为种子液，第二天以1：100的比例加入卡纳抗性LB培养基，当培养基OD600达到0.6～0.8时，加入终浓度为0.5mM的IPTG，37℃诱导表达4h。
4)菌液4000rpm离心30min，弃上清，用PBS按照1：10的比例重悬菌体。超声破碎菌体，超声破碎仪参数设置为：功率190W，工作5s，关闭10s，超声15min。离心后收集破碎上清。
5)用0.22μm滤膜过滤细菌破碎上清后备用。20mmol/L Tris 150mmol/LNaclPH8.0溶液平衡His柱，然后细菌破碎上清挂柱，以不同浓度的咪唑溶液洗脱，进行亲和层析纯化并鉴定；通过分子筛对蛋白进行凝胶过滤层析纯化并鉴定，进而获得纯化的非洲猪瘟病毒p30蛋白(序列5)。
实施例2、非洲猪瘟病毒p30蛋白特异性杂交瘤细胞株的筛选
非洲猪瘟病毒p30蛋白特异性杂交瘤细胞株的筛选，包括以下步骤：
1)以实施例1获得的p30蛋白为免疫原，免疫动物为BALB/c小鼠(购自北京维通利华实验动物技术有限公司)，连续免疫4次，每次间隔14天，前3次采用多点皮下免疫方式，第4次采取腹腔注射的免疫方式，每次每只小鼠注射p30蛋白10μg；
2)末次免疫后7天，取小鼠尾血分离血清后，用间接ELISA进行检测，效价＞1:50000后，分离免疫动物的脾细胞，将其与生长状态良好的骨髓瘤细胞SP2/0进行融合，用HAT选择性培养基筛选获得杂交瘤细胞；
3)对杂交瘤细胞上清用间接ELISA方法进行筛选特异性阳性克隆，经检测，最终获得分泌6G10的单克隆细胞株能够特异性与非洲猪瘟病毒p30蛋白反应。具体操作步骤：非洲猪瘟病毒p30蛋白溶于100μl的pH 9.6的碳酸盐溶液稀释浓度至2μg/ml，然后加到96孔聚苯乙烯酶联反应板，每孔100μl，2～8℃下放置8～12小时，使特异性单克隆抗体与酶联反应板充分结合，然后按照300μl/孔加入含有10mg/ml牛血清白蛋白pH7.4的PBS缓冲液，37℃封闭处理2～3小时，甩干后，待酶联反应板干燥后用铝箔纸进行封袋，置2～8℃保存备用。
取细胞培养上清加入包被有P30纯化蛋白的酶标板中，37℃反应30分钟，用洗涤液(含有浓度为0.8％～1.2％(ml/ml)的Tween-20的0.01M，pH值为7.4的磷酸盐缓冲液，使用时用双蒸水稀释20倍。)洗板4次，拍干后，向每孔加入1:5000稀释的兔抗鼠IgG-HRP标记物(购自美国Sigma公司)，37℃反应30分钟，用洗涤液4次，拍干后，向每孔加入底物液A(为含0.6mg/ml过氧化氢尿素的柠檬酸磷酸盐缓冲液)和底物液B(为0.2mg/ml的四甲基联苯胺溶液)各50μl底物工作液，37℃避光反应15分钟。向每孔加入50μl终止液(2mol/L的硫酸溶液)，振荡混匀终止反应。15分钟内测定每孔的OD450nm值。以吸光度值＞阴性对照(即洗板培养液)×2.1倍为阳性判定标准，测定细胞培养上清中特异性单克隆抗体效价，最后获得1株特异性细胞克隆，与非洲猪瘟病毒p30蛋白有强烈信号反应，将这株编号为6G10。
实施例3、抗非洲猪瘟病毒p30蛋白特异性单克隆抗体重组表达系统的建立及单克隆抗体的纯化
非洲猪瘟病毒p30蛋白的特异性杂交瘤细胞株的基因测序、单克隆抗体重组表达系统的建立及单克隆抗体的纯化，包括以下步骤：
1)特异性阳性克隆杂交瘤细胞株总RNA提取、反转录、PCR及序列测定：
①总RNA提取：取杂交瘤细胞悬液250μl，加入750μl的Trizol，上下颠倒混匀，加入200μl的氯仿，混匀，4℃12000rpm离心15min。吸取上清至新的1.5ml EP管中，加入600μl的异丙醇，混匀离心10min。将异丙醇弃去，用75％的DEPC乙醇洗涤，离心。将乙醇弃去，烘干，用20μl无RNA酶水溶解RNA。
②反转录：利用Invitrogen反转录试剂盒按说明书进行反转录，获得杂交瘤细胞的cDNA。
③PCR反应及其产物的克隆测序：针对重链和轻链可变区设计通用引物，序列信息如下：
表1重链及轻链可变区通用引物
| 名称 | 序列(5’-3’) |
| V<sub>H</sub>-1(正向) | GTGAATTCATGCAGGTGCAGCTGTTGGAGTCTGG |
| V<sub>H</sub>-2(反向) | ATGTCGACTGAGGAGACGGTGACCAGGGTGCC |
| V<sub>L</sub>-1(正向) | GTGAATTCATGGACATTGTGATGACCCAGTCTCC |
| V<sub>L</sub>-2(反向) | CAGTCGACTTACGTTTGATCTCCAGCTTGGTCCC |
利用扩增引物对目的片段进行扩增，扩增完后胶回收片段，然后连接载体进行序列测定，获取单克隆抗体重链和轻链可变区序列信息。
单克隆抗体6G10含有重链可变区6G10-VH、轻链可变区6G10-VL，其6G10-VH的氨基酸序列如序列表中SEQ ID No.1的第1～119位所示；其6G10-VL的氨基酸序列如序列表中SEQ ID No.2的第1～113位所示。
所述重链可变区6G10-VH和轻链可变区6G10-VL均由决定簇互补区和框架区组成；所述6G10-VH和所述6G10-VL的决定簇互补区均由CDR1、CDR2和CDR3组成；所述6G10-VH的CDR1的氨基酸序列如SEQ ID No.1的第26～30位氨基酸所示；所述6G10-VH的CDR2的氨基酸序列如SEQ ID No.1的第48～63位氨基酸所示；所述6G10-VH的CDR3的氨基酸序列如SEQ IDNo.1的第102～111位氨基酸所示；所述6G10-VL的CDR1的氨基酸序列如SEQ ID No.2的第24～40位氨基酸所示；所述6G10-VL的CDR2的氨基酸序列如SEQ ID No.2的第56～62位氨基酸所示；所述6G10-VL的CDR3的氨基酸序列如SEQ ID No.2的第95～102位氨基酸所示。
2)特异性单克隆抗体的基因序列的合成及重组表达系统的建立
①基因序列的合成：根据已测得单抗6G10的重链和轻链可变区的序列，将鼠抗体重链和轻链恒定区的序列补充在可变区部分，然后进行基因序列的合成，并进行昆虫细胞密码子优化，6G10重链可变区编码的核苷酸序列如序列表中SEQ ID No.3所示，轻链可变区编码的核苷酸序列如序列表中SEQ ID No.4所示。
②穿梭载体的构建：根据重链和轻链的序列信息以及pFastBacdual(购自ThermoFisher公司，货号10712024)载体序列信息，构建穿梭载体。
③重组Bacmid的筛选与提取：将构建好的穿梭载体转化DH10Bac感受态，然后涂布三抗平板(卡那霉素、庆大霉素、四环霉素)，37℃培养箱培养48h后挑取白斑，利用M13引物进行鉴定，阳性克隆目的片段大小为4600bp，阴性克隆为300bp，选取完全无300bp条带的克隆菌摇菌，12h后采用异丙醇沉淀法进行Bacmid的提取，然后利用Nanodrop进行浓度测定。
④重组杆状病毒的拯救：转染前将密度为2×106的SF9细胞铺六孔板，重组Bacmid按5μg和2.5μg的量进行转染，转染试剂用量为8μl，转染后4～6h进行换液，28℃培养，72h后收获扩增P2代病毒，采用同样方法进行P3代病毒扩增。P4代病毒的扩增采取摇瓶扩增，病毒接种比例为1：100。
3)特异性单克隆抗体的表达与纯化：将P4代病毒按1:5的比例接种密度为2×106的Hi5细胞，28℃培养，48h后收获细胞，8000r/min离心1h取上清，然后用0.22μm滤膜过滤备用。用Na3PO4 pH值为7.0溶液平衡ProteinA预装柱，平衡3～5个柱体积，然后将细胞上清结合ProteinA预装柱，样品结合完后用Glycine-HCL pH值为3.0洗脱液进行洗脱，即获得纯化的抗非洲猪瘟病毒p30蛋白特异性单克隆抗体6G10。
实施例4、制备非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒
1)用非洲猪瘟病毒p30蛋白制备抗原包被板：将p30蛋白用pH 9.6的碳酸盐溶液稀释成1μg/ml的包被工作液，然后加到96孔聚苯乙烯酶联反应板，100μl/孔，2～8℃下放置8～12小时，使包被抗原与酶联反应板充分结合，然后按照300μl/孔加入含有10mg/ml牛血清白蛋白pH7.4的PBS缓冲液，37℃封闭处理2～3小时，甩干后，待酶联反应板干燥后2～8℃密封保存。
2)制备辣根过氧化物酶标记的抗非洲猪瘟病毒p30蛋白特异性单克隆抗体
将抗非洲猪瘟病毒p30蛋白特异性单克隆抗体用戊二醛氧化法与辣根过氧化物酶(HRP)进行偶联，用pH7.4的PBS缓冲液充分透析，加等量的优质丙三醇，-20℃以下保存。具体步骤如下：
①将5mg HRP溶于0.2ml含有1.25％戊二醛的0.1mol/L pH值6.8的PBS缓冲液中，置室温偶联18个小时，充分透析出去多余戊二醛；
②加生理盐水至1ml，然后加入2.5mg纯化的抗非洲猪瘟病毒p30蛋白特异性单克隆抗体及0.1mlpH值9.6的1mol/L碳酸盐缓冲液，置于2～8℃放置24小时；
③加入0.1ml 0.3mol/L的赖氨酸溶液，室温放置2小时；
④用pH7.4的PBS缓冲液充分透析，通过离心除去沉淀，上清即为酶结合物。用酶标记物稀释液按一定比例稀释后即为酶标记物的工作液(0.5ug/ml)。
3)阳性对照血清：是以非洲猪瘟p30重组蛋白免疫后猪血清，作为试剂盒的阳性对照血清(1管，1.5ml/管)。
4)阴性对照血清：是无特定病原体(SPF)猪血清，作为试剂盒的阴性对照血清(1管，1.5ml/管)。
5)样品稀释液的制备：为含有5mg/ml酪蛋白的0.01M、pH值为7.4的磷酸盐缓冲液，1瓶(24ml/瓶)。
6)底物液A的制备为含0.6mg/ml过氧化氢尿素的柠檬酸磷酸盐缓冲液(1瓶，12ml/瓶)
7)底物液B的制备为0.2mg/ml的四甲基联苯胺(TMB)溶液(1瓶，12ml/瓶)。
8)20倍浓缩洗涤液的制备为含有浓度为1.0％(ml/ml)的Tween-20的0.01M，pH值为7.4的磷酸盐缓冲液(50ml/瓶，2瓶)。
9)终止液的制备2mol/L的硫酸溶液(1瓶，12ml/瓶)。
10)根据需要，试剂盒中还可以有样品稀释板(2块，96孔/块)，用于样品的稀释。
实施例5、非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒的使用方法
1)平衡：将试剂盒从冷藏环境中取出，置室温平衡30min备用；液体试剂用前混匀。
2)配液：将浓缩洗涤液用蒸馏水或去离子水20倍稀释得到洗涤缓冲液；
3)样品稀释：在血清稀释板中将待检血清用样品稀释液进行2倍稀释，阴、阳性对照血清已稀释，可直接使用。
4)加样：取出所需板条，剩余板条装入铝箔袋中封好，置于2～8℃保存备用。将稀释好的待检血清、阴性对照血清和阳性对照血清加入到抗原包被板中，100μl/孔。每份待检血清设1孔，阴性对照和阳性对照各设2孔，加样过程时间跨度应尽量短。如图1所示加样：N：表示加阴性对照血清；P：表示加阳性对照血清；S1、S2、S3、S4等表示加各待检血清。
5)温育：震荡混匀，置37℃温箱中，反应60min。
6)洗板：弃去反应液，每孔加300μl稀释后的洗涤缓冲液，浸泡15s，甩弃洗液，连续洗板4次后拍干。
7)加酶：各孔加入实施例4中制备的酶标记物的工作液100μl。
8)温育：置37℃温箱，反应30min。
9)洗板：弃去反应液，每孔加入稀释后的洗涤缓冲液300μl，浸泡15s，甩弃洗涤液，连续洗板4次后拍干。
10)显色每孔加入100μl底物工作液(将底物液A和底物液B等量混合即为底物工作液，现用现配)，震荡混匀，置37℃温箱中，避光反应15min。
11)每孔加入显色终止液50μl，振荡混匀终止反应，15分钟内测定结果。
12)试验成立条件：阴性对照OD450nm值均应≥1.0。阳性对照孔S/N值应≤0.5。
13)判定：在酶标仪上测各孔OD450nm值。S/N＝样本OD450nm值/阴性对照OD450nm值。通过计算每个样品的S/N值，判定其抗体的有无。阴性：S/N≥0.7；可疑0.6＜S/N＜0.7；阳性S/N≤0.6。
实施例6、敏感性试验
使用按照实施例4的方法制备的3批非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒(批次ZM302101、ZM302102、ZM302103)，按照实施例5的使用方法对非洲猪瘟病毒感染猪血清62份进行检测，实验结果见表2，本发明的试剂盒共检测出62份，结果表明本试剂盒对62份已知阳性血清的敏感性为100.0％。
表2敏感性检测结果
| 试剂盒批号 | 检出率 | 敏感性 |
| ZM302101 | 62/62 | 100.0％ |
| ZM302102 | 62/62 | 100.0％ |
| ZM302103 | 62/62 | 100.0％ |
实施例7、特异性试验
使用按照实施例4的方法制备的3批非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒(批次ZM302101、ZM302102、ZM302103)，按照实施例5的使用方法对100份健康猪血清、2份猪口蹄疫病毒O型(FMD-O)阳性血清、2份猪口蹄疫病毒A型(FMD-A)阳性血清、2份猪圆环病毒阳性血清(PCV2)、2份猪繁殖与呼吸综合征阳性血清(PRRS)，分别进行检测。
试剂盒的特异性检测结果如下表(表3)显示，对100份健康猪血清的检测结果显示，3批试剂盒的特异性均为100.0％。对2份猪口蹄疫病毒O型(FMD-O)阳性血清、2份猪口蹄疫病毒A型(FMD-A)阳性血清、2份猪圆环病毒阳性血清(PCV2)、2份猪繁殖与呼吸综合征阳性血清(PRRS)的检测结果均显示为阴性，因此3批试剂盒对这8份相关病原阳性血清检测的特异性均为100％。
表3非洲猪瘟病毒阻断ELISA抗体检测试剂盒特异性检测结果
实施例8、符合率试验
采用西班牙Ingenasa试剂盒和本发明的试剂盒同时对50份健康猪血清、25份病毒感染血清同时进行检测，比较2种试剂盒检测结果的符合率。
美国进口试剂盒操作方法：
(1)每孔加入50μl稀释液，任选两孔加入50μl的阳性对照血清，任选两孔加入50μl的阴性对照血清，其余每孔加入50μl的待测血清样品，36℃±1℃孵育1h或20～25℃过夜孵育(16～20h)；
(2)倾去孔内液体，洗板4次；
(3)每孔加入100μl的结合物(用稀释液进行1:100倍稀释)，36℃±1℃孵育30min；
(4)倾去孔内液体，洗板5次，每孔加100μl的底物溶液，室温20～25℃避光放置15min；
(5)每孔加入100μl终止液，5min内读取OD450nm值。判定标准：①有效性：阴性对照OD值/阳性对照OD值≥4；②血清阻断率计算％＝(NC-样品OD值)/(NC-PC)×100；③阳性Cut-off值＝NC-[(NC-PC)×0.5]，阴性Cut-off值＝NC-[(NC-PC)×0.4]；④阻断率≥50％，判为阳性，阻断率≤40％，判为阴性，两者之间为可疑，可疑样品重新检测。
本发明试剂盒和西班牙Ingenasa试剂盒对50份健康猪血清、30份病毒感染血清的检测结果见表4，本发明试剂盒和西班牙Ingenasa试剂盒检测为阳性的血清份数均为26份，检测为阴性的血清份数均为52份。80份待检血清中，两种试剂盒检测结果一致的血清份数是78份，符合率为97.5％。
表4符合率试验结果
序列表
<110> 中牧实业股份有限公司
<120> 一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用
<130> WHOI210051
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Asn Ile Ser Cys Lys Ala Ser Tyr Tyr Glu Ser Thr Ile Glu
20 25 30
Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu Thr
35 40 45
Pro Tyr Thr Thr Gly Gly Lys Phe Arg Val Ala Thr Met Arg Tyr Lys
50 55 60
Tyr Asn Glu Lys Phe Lys Gly Lys Ala Ile Phe Thr Val Glu Thr Ser
65 70 75 80
Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
85 90 95
Ala Val Tyr Phe Cys Tyr Asp Tyr Tyr Ser Leu Ser Tyr Ala Phe Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 2
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Leu His Arg Gln Ser Arg Asn Gly Asn
20 25 30
Lys Val Ser Ser Gly Gln Asn Tyr Trp Tyr Gln Gln Lys Pro Asp Gly
35 40 45
Thr Val Lys Leu Leu Ile Tyr Ser Trp Thr Val Asn Thr Tyr Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
65 70 75 80
Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Pro Ala
85 90 95
Asn Asn Thr His Val Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 3
<211> 366
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caggtgcagc ttcagcaaag cggtgcagag cttatgaaac ctggtgcgag tgtgaacatc 60
tcatgcaagg cctcgtatta cgagtccacc atcgaatggg ttaaacagcg cccaggacac 120
ggcctggaat ggatcggaga gactccctac actacgggag gtaagttccg tgtggctacc 180
atgcgctata agtacaacga gaaattcaag ggtaaggcta tcttcacggt ggagaccagt 240
tcaaacacgg cctacatgca gctttctagt ctcacctccg aggacagcgc cgtctacttt 300
tgctacgact actactcact gtcctacgcc ttttggggac agggcactac cgtcaccgta 360
tcatcg 366
<210> 4
<211> 339
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gatattcaga tgacccagac tacctcatcg ctttcagcca gtttgggtga ccgcgtgaca 60
atcagttgtc tccatcgtca gtctcgcaac ggcaacaagg tgagctccgg ccagaactac 120
tggtatcaac aaaaacctga tggtacagtg aagctcctta tatactcatg gactgtcaac 180
acatacggcg tccctagtcg ttttagtgga tcaggatccg gcacagatta ctccctgacc 240
atctcaaacc tcgaacctga agacatcgcc acatactact gtccagccaa caacacccat 300
gtgttcacgt tcggcggcgg taccaagctg gaaatcaaa 339
<210> 5
<211> 194
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Asp Phe Ile Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe Lys
1 5 10 15
Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser Leu
20 25 30
Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val Thr
35 40 45
Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val Lys
50 55 60
Ser Ala Arg Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala Gln
65 70 75 80
Glu Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr Glu
85 90 95
Ser Ser Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp Asn Glu Thr
100 105 110
Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro Ser
115 120 125
Ser Glu Val Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys Thr
130 135 140
Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val
145 150 155 160
Ile Arg Ala His Asn Phe Ile Gln Thr Ile Tyr Gly Thr Pro Leu Lys
165 170 175
Glu Glu Glu Lys Glu Val Val Arg Leu Met Val Ile Lys Leu Leu Lys
180 185 190
Lys Lys
Claims (10)
1.一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，包括酶联反应板和酶标抗体；其中，所述酶联反应板包被有非洲猪瘟病毒p30蛋白，酶标抗体为用酶标记的抗非洲猪瘟病毒p30蛋白的单克隆抗体。
2.根据权利要求1所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，其特征在于：所述非洲猪瘟病毒p30蛋白的序列为序列表中SEQ ID No.5序列。
3.根据权利要求1所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，其特征在于：所述抗非洲猪瘟病毒p30蛋白的单克隆抗体，含有重链可变区6G10-VH和轻链可变区6G10-VL；所述重链可变区6G10-VH和轻链可变区6G10-VL均由决定簇互补区和框架区组成；
所述6G10-VH和所述6G10-VL的决定簇互补区均由CDR1、CDR2和CDR3组成；
所述6G10-VH的CDR1的氨基酸序列如SEQ ID No.1的第26～30位氨基酸所示；
所述6G10-VH的CDR2的氨基酸序列如SEQ ID No.1的第48～63位氨基酸所示；
所述6G10-VH的CDR3的氨基酸序列如SEQ ID No.1的第102～111位氨基酸所示；
所述6G10-VL的CDR1的氨基酸序列如SEQ ID No.2的第24～40位氨基酸所示；
所述6G10-VL的CDR2的氨基酸序列如SEQ ID No.2的第56～62位氨基酸所示；
所述6G10-VL的CDR3的氨基酸序列如SEQ ID No.2的第95～102位氨基酸所示。
4.根据权利要求3所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，其特征在于：所述6G10-VH的氨基酸序列如序列表中SEQ ID No.1的第1～119位所示；其6G10-VL的氨基酸序列如序列表中SEQ ID No.2的第1～113位所示。
5.根据权利要求1-4中任意一项所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，其特征在于：所述酶联反应板的获得方法是将所述非洲猪瘟病毒p30蛋白溶于100μl的pH9.6的碳酸盐溶液，然后加到96孔聚苯乙烯酶联反应板，每孔0.1μg～1μg 非洲猪瘟病毒p30蛋白，2～8℃下放置8～12小时，使包被抗原与酶联反应板充分结合，然后按照300μl/孔加入含有10mg/ml牛血清白蛋白pH7.4的PBS缓冲液，37℃封闭处理2～3小时，甩干后，待酶联反应板干燥后4℃密封保存。
6.根据权利要求1所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，其特征在于：所述试剂盒还包括底物液A、底物液B和终止液；所述底物液A为含0.6mg/ml过氧化氢尿素的柠檬酸磷酸盐缓冲液，所述底物液B为0.2mg/ml的四甲基联苯胺溶液，使用时两者以1:1的比例混合；所述终止液为2mol/L的硫酸溶液。
7.根据权利要求1所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒，其特征在于：所述试剂盒还包括样品稀释液和20倍浓缩洗涤液；样品稀释液为含有5mg/ml酪蛋白的0.01M、pH值为7.4的磷酸盐缓冲液；浓缩洗涤液为含有体积百分浓度为0.8％～1.2％的Tween-20的0.01M，pH值为7.4的磷酸盐缓冲液；
和/或，所述酶联免疫试剂盒还包括阳性对照血清和阴性对照血清；所述阳性对照血清为用p30重组蛋白免疫后的猪血清；所述阴性对照血清为无猪伪狂犬病毒病原体且无疫苗接种的猪血清。
8.根据权利要求1～7所述的非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒在制备检测猪伪狂犬病毒感染或疫苗接种的待测样品的试剂中的应用，其中，待测样品为感染野毒株或应用过普通的弱毒疫苗、灭活疫苗或应用过非gE缺失疫苗猪只血清。
9.一株单克隆抗体，其可与抗非洲猪瘟病毒p30蛋白特异性结合，是下述任意一项所述的单克隆抗体：
1)所述抗非洲猪瘟病毒p30蛋白的单克隆抗体，含有重链可变区6G10-VH和轻链可变区6G10-VL；所述重链可变区6G10-VH和轻链可变区6G10-VL均由决定簇互补区和框架区组成；
所述6G10-VH和所述6G10-VL的决定簇互补区均由CDR1、CDR2和CDR3组成；
所述6G10-VH的CDR1的氨基酸序列如SEQ ID No.1的第26～30位氨基酸所示；
所述6G10-VH的CDR2的氨基酸序列如SEQ ID No.1的第48～63位氨基酸所示；
所述6G10-VH的CDR3的氨基酸序列如SEQ ID No.1的第102～111位氨基酸所示；
所述6G10-VL的CDR1的氨基酸序列如SEQ ID No.2的第24～40位氨基酸所示；
所述6G10-VL的CDR2的氨基酸序列如SEQ ID No.2的第56～62位氨基酸所示；
所述6G10-VL的CDR3的氨基酸序列如SEQ ID No.2的第95～102位氨基酸所示；
2)含有重链可变区6G10-VH和轻链可变区6G10-VL；所述6G10-VH的氨基酸序列如序列表中SEQ ID No.1的第1～119位所示；其6G10-VL的氨基酸序列如序列表中SEQ ID No.2的第1～113位所示。
10.权利要求9所述的单克隆抗体在制备检测非洲猪瘟病毒p30抗体的试剂盒中的应用。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2021110645864 | 2021-09-10 | ||
| CN202111064586 | 2021-09-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113740536A true CN113740536A (zh) | 2021-12-03 |
| CN113740536B CN113740536B (zh) | 2023-10-27 |
Family
ID=78741400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111137754.8A Active CN113740536B (zh) | 2021-09-10 | 2021-09-27 | 一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113740536B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114966013A (zh) * | 2022-05-10 | 2022-08-30 | 中牧实业股份有限公司 | 检测非洲猪瘟病毒抗原的酶联免疫试剂盒及其应用 |
| CN116444653A (zh) * | 2023-03-09 | 2023-07-18 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一株阻断性非洲猪瘟病毒单克隆抗体杂交瘤细胞株的制备与应用 |
| CN116769019A (zh) * | 2023-03-09 | 2023-09-19 | 河北农业大学 | 一种ASFVp30蛋白单克隆抗体及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111912991A (zh) * | 2020-08-12 | 2020-11-10 | 南京农业大学 | 一种检测非洲猪瘟病毒血清抗体的试纸条及其应用 |
| CN112920266A (zh) * | 2021-03-05 | 2021-06-08 | 中国检验检疫科学研究院 | 一种基于非洲猪瘟病毒p30基因的竞争性单克隆抗体、试剂盒及其应用 |
| CN113607952A (zh) * | 2021-08-18 | 2021-11-05 | 杭州恒奥科技有限公司 | 非洲猪瘟病毒阻断elisa抗体检测试剂盒及其制备方法和应用 |
-
2021
- 2021-09-27 CN CN202111137754.8A patent/CN113740536B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111912991A (zh) * | 2020-08-12 | 2020-11-10 | 南京农业大学 | 一种检测非洲猪瘟病毒血清抗体的试纸条及其应用 |
| CN112920266A (zh) * | 2021-03-05 | 2021-06-08 | 中国检验检疫科学研究院 | 一种基于非洲猪瘟病毒p30基因的竞争性单克隆抗体、试剂盒及其应用 |
| CN113607952A (zh) * | 2021-08-18 | 2021-11-05 | 杭州恒奥科技有限公司 | 非洲猪瘟病毒阻断elisa抗体检测试剂盒及其制备方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| FANGFENG YUAN ET AL: "Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against African Swine Fever Virus", 《PATHOGENS》, vol. 10, pages 9 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114966013A (zh) * | 2022-05-10 | 2022-08-30 | 中牧实业股份有限公司 | 检测非洲猪瘟病毒抗原的酶联免疫试剂盒及其应用 |
| CN116444653A (zh) * | 2023-03-09 | 2023-07-18 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一株阻断性非洲猪瘟病毒单克隆抗体杂交瘤细胞株的制备与应用 |
| CN116769019A (zh) * | 2023-03-09 | 2023-09-19 | 河北农业大学 | 一种ASFVp30蛋白单克隆抗体及其应用 |
| CN116769019B (zh) * | 2023-03-09 | 2023-12-15 | 河北农业大学 | 一种ASFVp30蛋白单克隆抗体及其应用 |
| CN116444653B (zh) * | 2023-03-09 | 2024-03-15 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一株阻断性非洲猪瘟病毒单克隆抗体杂交瘤细胞株的制备与应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113740536B (zh) | 2023-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109900902B (zh) | 一种猪伪狂犬病毒gB阻断ELISA抗体检测试剂盒及其应用 | |
| CN113740536B (zh) | 一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用 | |
| CN112920266B (zh) | 一种基于非洲猪瘟病毒p30基因的竞争性单克隆抗体、试剂盒及其应用 | |
| CN113151187B (zh) | 一株非洲猪瘟病毒单克隆抗体杂交瘤细胞及其应用 | |
| CN111848786B (zh) | 一种单克隆抗体、其制备方法及用途 | |
| CN110698543A (zh) | 一种用于非洲猪瘟病毒抗体检测的双抗原间接elisa试剂盒 | |
| CN110261616B (zh) | 一种丙型肝炎病毒检测试剂盒 | |
| CN111999497B (zh) | 检测狂犬病毒糖蛋白抗原的酶联免疫试剂盒及其应用 | |
| CN114874995B (zh) | 猪瘟病毒2型Erns蛋白的单克隆抗体杂交瘤细胞株及应用 | |
| WO2012163263A1 (en) | Reagents and methods for prrsv detection | |
| CN109580945B (zh) | 检测口蹄疫o型广西株抗原的酶联免疫试剂盒及其应用 | |
| CN109900903B (zh) | 一种猪伪狂犬病毒gE阻断ELISA抗体检测试剂盒及其应用 | |
| CN113563461B (zh) | 一种基于非洲猪瘟病毒CD2v蛋白的竞争性单克隆抗体、试剂盒及其应用 | |
| CN113687073B (zh) | 一种非洲猪瘟病毒p54阻断ELISA抗体检测试剂盒及其应用 | |
| KR102086089B1 (ko) | 재조합 n 단백질에 대한 단클론항체를 이용한 아까바네바이러스 블러킹 효소결합면역측정법 | |
| CN110257405B (zh) | 牛支原体乙醇脱氢酶基因及其编码蛋白与应用 | |
| CN113009139B (zh) | 检测猪伪狂犬病毒抗原的酶联免疫试剂盒及其应用 | |
| KR101080071B1 (ko) | 재조합 ｎ 단백질에 대한 단클론 항체를 이용한 리프트계곡열 경합적 효소결합면역측정법 | |
| CN109824775B (zh) | 口蹄疫a型武汉株单克隆抗体及其组合物与应用 | |
| CN110894216B (zh) | 一种猪流行性腹泻病毒抗原表位肽、单克隆抗体及应用 | |
| CN114966013B (zh) | 检测非洲猪瘟病毒抗原的酶联免疫试剂盒及其应用 | |
| CN116718765A (zh) | 一种牛结节性皮肤病阻断elisa抗体检测试剂盒及其应用 | |
| CN109613238B (zh) | 检测口蹄疫a型武汉株抗原的酶联免疫试剂盒及其应用 | |
| CN114685619B (zh) | 一种抗原蛋白、其单克隆抗体或多克隆抗体及应用 | |
| CN114230661B (zh) | 一种用于番茄黄化斑驳相关病毒检测的抗体及其制备方法和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |