CN113122551B - 一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 - Google Patents
一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 Download PDFInfo
- Publication number
- CN113122551B CN113122551B CN202110338067.6A CN202110338067A CN113122551B CN 113122551 B CN113122551 B CN 113122551B CN 202110338067 A CN202110338067 A CN 202110338067A CN 113122551 B CN113122551 B CN 113122551B
- Authority
- CN
- China
- Prior art keywords
- group
- gene
- coxsackie virus
- type
- recombinant expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 67
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 48
- 241000988559 Enterovirus A Species 0.000 title claims abstract description 29
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 25
- 101150024766 VP1 gene Proteins 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 210000003141 lower extremity Anatomy 0.000 claims description 14
- 230000003053 immunization Effects 0.000 claims description 7
- 210000002363 skeletal muscle cell Anatomy 0.000 claims description 7
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 241000709687 Coxsackievirus Species 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 10
- 230000002163 immunogen Effects 0.000 abstract description 2
- 230000009465 prokaryotic expression Effects 0.000 abstract description 2
- 241000146305 Coxsackievirus A19 Species 0.000 description 31
- 241000699666 Mus <mouse, genus> Species 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 241000709661 Enterovirus Species 0.000 description 6
- 238000011999 immunoperoxidase monolayer assay Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 210000003608 fece Anatomy 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 4
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 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 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- 208000020061 Hand, Foot and Mouth Disease Diseases 0.000 description 2
- 208000025713 Hand-foot-and-mouth disease Diseases 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- RPZFUIQVAPZLRH-GHCJXIJMSA-N Ile-Asp-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)O)N RPZFUIQVAPZLRH-GHCJXIJMSA-N 0.000 description 2
- 241000709664 Picornaviridae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- KLQPIEVIKOQRAW-IZPVPAKOSA-N Tyr-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O KLQPIEVIKOQRAW-IZPVPAKOSA-N 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 210000001519 tissue Anatomy 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
- 238000012795 verification Methods 0.000 description 2
- 102100040023 Adhesion G-protein coupled receptor G6 Human genes 0.000 description 1
- SSSROGPPPVTHLX-FXQIFTODSA-N Ala-Arg-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSSROGPPPVTHLX-FXQIFTODSA-N 0.000 description 1
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 1
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 1
- FSXDWQGEWZQBPJ-HERUPUMHSA-N Ala-Trp-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(=O)O)C(=O)O)N FSXDWQGEWZQBPJ-HERUPUMHSA-N 0.000 description 1
- AOAKQKVICDWCLB-UWJYBYFXSA-N Ala-Tyr-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N AOAKQKVICDWCLB-UWJYBYFXSA-N 0.000 description 1
- 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 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- HJVGMOYJDDXLMI-AVGNSLFASA-N Arg-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCCNC(N)=N HJVGMOYJDDXLMI-AVGNSLFASA-N 0.000 description 1
- WVNFNPGXYADPPO-BQBZGAKWSA-N Arg-Gly-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O WVNFNPGXYADPPO-BQBZGAKWSA-N 0.000 description 1
- DGFXIWKPTDKBLF-AVGNSLFASA-N Arg-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCCN=C(N)N)N DGFXIWKPTDKBLF-AVGNSLFASA-N 0.000 description 1
- YCYXHLZRUSJITQ-SRVKXCTJSA-N Arg-Pro-Pro Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 YCYXHLZRUSJITQ-SRVKXCTJSA-N 0.000 description 1
- VLIJAPRTSXSGFY-STQMWFEESA-N Arg-Tyr-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 VLIJAPRTSXSGFY-STQMWFEESA-N 0.000 description 1
- 208000006740 Aseptic Meningitis Diseases 0.000 description 1
- HAJWYALLJIATCX-FXQIFTODSA-N Asn-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N HAJWYALLJIATCX-FXQIFTODSA-N 0.000 description 1
- XVAPVJNJGLWGCS-ACZMJKKPSA-N Asn-Glu-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XVAPVJNJGLWGCS-ACZMJKKPSA-N 0.000 description 1
- YNQIDCRRTWGHJD-ZLUOBGJFSA-N Asp-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(O)=O YNQIDCRRTWGHJD-ZLUOBGJFSA-N 0.000 description 1
- RDRMWJBLOSRRAW-BYULHYEWSA-N Asp-Asn-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O RDRMWJBLOSRRAW-BYULHYEWSA-N 0.000 description 1
- NHSDEZURHWEZPN-SXTJYALSSA-N Asp-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CC(=O)O)N NHSDEZURHWEZPN-SXTJYALSSA-N 0.000 description 1
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 description 1
- QOCFFCUFZGDHTP-NUMRIWBASA-N Asp-Thr-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QOCFFCUFZGDHTP-NUMRIWBASA-N 0.000 description 1
- BPAUXFVCSYQDQX-JRQIVUDYSA-N Asp-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](CC(=O)O)N)O BPAUXFVCSYQDQX-JRQIVUDYSA-N 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- ZOKPRHVIFAUJPV-GUBZILKMSA-N Cys-Pro-Arg Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CS)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O ZOKPRHVIFAUJPV-GUBZILKMSA-N 0.000 description 1
- 108010090461 DFG peptide Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241001466953 Echovirus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- FYAULIGIFPPOAA-ZPFDUUQYSA-N Gln-Ile-Met Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(O)=O FYAULIGIFPPOAA-ZPFDUUQYSA-N 0.000 description 1
- BJPPYOMRAVLXBY-YUMQZZPRSA-N Gln-Met-Gly Chemical compound CSCC[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N BJPPYOMRAVLXBY-YUMQZZPRSA-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
- LTUVYLVIZHJCOQ-KKUMJFAQSA-N Glu-Arg-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LTUVYLVIZHJCOQ-KKUMJFAQSA-N 0.000 description 1
- UENPHLAAKDPZQY-XKBZYTNZSA-N Glu-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCC(=O)O)N)O UENPHLAAKDPZQY-XKBZYTNZSA-N 0.000 description 1
- GPSHCSTUYOQPAI-JHEQGTHGSA-N Glu-Thr-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O GPSHCSTUYOQPAI-JHEQGTHGSA-N 0.000 description 1
- HHSKZJZWQFPSKN-AVGNSLFASA-N Glu-Tyr-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O HHSKZJZWQFPSKN-AVGNSLFASA-N 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 1
- UXJHNZODTMHWRD-WHFBIAKZSA-N Gly-Asn-Ala Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O UXJHNZODTMHWRD-WHFBIAKZSA-N 0.000 description 1
- QPTNELDXWKRIFX-YFKPBYRVSA-N Gly-Gly-Gln Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O QPTNELDXWKRIFX-YFKPBYRVSA-N 0.000 description 1
- UTYGDAHJBBDPBA-BYULHYEWSA-N Gly-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN UTYGDAHJBBDPBA-BYULHYEWSA-N 0.000 description 1
- GGAPHLIUUTVYMX-QWRGUYRKSA-N Gly-Phe-Ser Chemical compound OC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)C[NH3+])CC1=CC=CC=C1 GGAPHLIUUTVYMX-QWRGUYRKSA-N 0.000 description 1
- GLACUWHUYFBSPJ-FJXKBIBVSA-N Gly-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GLACUWHUYFBSPJ-FJXKBIBVSA-N 0.000 description 1
- YABRDIBSPZONIY-BQBZGAKWSA-N Gly-Ser-Met Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O YABRDIBSPZONIY-BQBZGAKWSA-N 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- BZKDJRSZWLPJNI-SRVKXCTJSA-N His-His-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O BZKDJRSZWLPJNI-SRVKXCTJSA-N 0.000 description 1
- YVCGJPIKRMGNPA-LSJOCFKGSA-N His-Met-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O YVCGJPIKRMGNPA-LSJOCFKGSA-N 0.000 description 1
- WHKLDLQHSYAVGU-ACRUOGEOSA-N His-Phe-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WHKLDLQHSYAVGU-ACRUOGEOSA-N 0.000 description 1
- 101100378639 Homo sapiens ADGRG6 gene Proteins 0.000 description 1
- HZMLFETXHFHGBB-UGYAYLCHSA-N Ile-Asn-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HZMLFETXHFHGBB-UGYAYLCHSA-N 0.000 description 1
- SPQWWEZBHXHUJN-KBIXCLLPSA-N Ile-Glu-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O SPQWWEZBHXHUJN-KBIXCLLPSA-N 0.000 description 1
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 1
- NZOCIWKZUVUNDW-ZKWXMUAHSA-N Ile-Gly-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O NZOCIWKZUVUNDW-ZKWXMUAHSA-N 0.000 description 1
- NYEYYMLUABXDMC-NHCYSSNCSA-N Ile-Gly-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)O)N NYEYYMLUABXDMC-NHCYSSNCSA-N 0.000 description 1
- UDBPXJNOEWDBDF-XUXIUFHCSA-N Ile-Lys-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)O)N UDBPXJNOEWDBDF-XUXIUFHCSA-N 0.000 description 1
- BATWGBRIZANGPN-ZPFDUUQYSA-N Ile-Pro-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BATWGBRIZANGPN-ZPFDUUQYSA-N 0.000 description 1
- ZYVTXBXHIKGZMD-QSFUFRPTSA-N Ile-Val-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ZYVTXBXHIKGZMD-QSFUFRPTSA-N 0.000 description 1
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- UCOCBWDBHCUPQP-DCAQKATOSA-N Leu-Arg-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O UCOCBWDBHCUPQP-DCAQKATOSA-N 0.000 description 1
- LAGPXKYZCCTSGQ-JYJNAYRXSA-N Leu-Glu-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LAGPXKYZCCTSGQ-JYJNAYRXSA-N 0.000 description 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- ODRREERHVHMIPT-OEAJRASXSA-N Leu-Thr-Phe 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)CC1=CC=CC=C1 ODRREERHVHMIPT-OEAJRASXSA-N 0.000 description 1
- FDBTVENULFNTAL-XQQFMLRXSA-N Leu-Val-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N FDBTVENULFNTAL-XQQFMLRXSA-N 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
- LOGFVTREOLYCPF-RHYQMDGZSA-N Lys-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-RHYQMDGZSA-N 0.000 description 1
- OHXUUQDOBQKSNB-AVGNSLFASA-N Lys-Val-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OHXUUQDOBQKSNB-AVGNSLFASA-N 0.000 description 1
- 206010027201 Meningitis aseptic Diseases 0.000 description 1
- QGQGAIBGTUJRBR-NAKRPEOUSA-N Met-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCSC QGQGAIBGTUJRBR-NAKRPEOUSA-N 0.000 description 1
- LRALLISKBZNSKN-BQBZGAKWSA-N Met-Gly-Ser Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LRALLISKBZNSKN-BQBZGAKWSA-N 0.000 description 1
- 101100378640 Mus musculus Adgrg6 gene Proteins 0.000 description 1
- 206010028372 Muscular weakness Diseases 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000033952 Paralysis flaccid Diseases 0.000 description 1
- BKWJQWJPZMUWEG-LFSVMHDDSA-N Phe-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 BKWJQWJPZMUWEG-LFSVMHDDSA-N 0.000 description 1
- HTKNPQZCMLBOTQ-XVSYOHENSA-N Phe-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N)O HTKNPQZCMLBOTQ-XVSYOHENSA-N 0.000 description 1
- QPVFUAUFEBPIPT-CDMKHQONSA-N Phe-Gly-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O QPVFUAUFEBPIPT-CDMKHQONSA-N 0.000 description 1
- IWZRODDWOSIXPZ-IRXDYDNUSA-N Phe-Phe-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C1=CC=CC=C1 IWZRODDWOSIXPZ-IRXDYDNUSA-N 0.000 description 1
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 1
- VGTJSEYTVMAASM-RPTUDFQQSA-N Phe-Thr-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VGTJSEYTVMAASM-RPTUDFQQSA-N 0.000 description 1
- XALFIVXGQUEGKV-JSGCOSHPSA-N Phe-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 XALFIVXGQUEGKV-JSGCOSHPSA-N 0.000 description 1
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 1
- CDGABSWLRMECHC-IHRRRGAJSA-N Pro-Lys-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O CDGABSWLRMECHC-IHRRRGAJSA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- BKOKTRCZXRIQPX-ZLUOBGJFSA-N Ser-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N BKOKTRCZXRIQPX-ZLUOBGJFSA-N 0.000 description 1
- QGMLKFGTGXWAHF-IHRRRGAJSA-N Ser-Arg-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QGMLKFGTGXWAHF-IHRRRGAJSA-N 0.000 description 1
- BCKYYTVFBXHPOG-ACZMJKKPSA-N Ser-Asn-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N BCKYYTVFBXHPOG-ACZMJKKPSA-N 0.000 description 1
- BGOWRLSWJCVYAQ-CIUDSAMLSA-N Ser-Asp-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BGOWRLSWJCVYAQ-CIUDSAMLSA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- IOVBCLGAJJXOHK-SRVKXCTJSA-N Ser-His-His Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IOVBCLGAJJXOHK-SRVKXCTJSA-N 0.000 description 1
- LWMQRHDTXHQQOV-MXAVVETBSA-N Ser-Ile-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LWMQRHDTXHQQOV-MXAVVETBSA-N 0.000 description 1
- MOINZPRHJGTCHZ-MMWGEVLESA-N Ser-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N MOINZPRHJGTCHZ-MMWGEVLESA-N 0.000 description 1
- ZSLFCBHEINFXRS-LPEHRKFASA-N Ser-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N ZSLFCBHEINFXRS-LPEHRKFASA-N 0.000 description 1
- RXSWQCATLWVDLI-XGEHTFHBSA-N Ser-Met-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RXSWQCATLWVDLI-XGEHTFHBSA-N 0.000 description 1
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 1
- PLQWGQUNUPMNOD-KKUMJFAQSA-N Ser-Tyr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O PLQWGQUNUPMNOD-KKUMJFAQSA-N 0.000 description 1
- XSLXHSYIVPGEER-KZVJFYERSA-N Thr-Ala-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XSLXHSYIVPGEER-KZVJFYERSA-N 0.000 description 1
- LXWZOMSOUAMOIA-JIOCBJNQSA-N Thr-Asn-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O LXWZOMSOUAMOIA-JIOCBJNQSA-N 0.000 description 1
- DOBIBIXIHJKVJF-XKBZYTNZSA-N Thr-Ser-Gln Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DOBIBIXIHJKVJF-XKBZYTNZSA-N 0.000 description 1
- KAJRRNHOVMZYBL-IRIUXVKKSA-N Thr-Tyr-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAJRRNHOVMZYBL-IRIUXVKKSA-N 0.000 description 1
- XKKBFNPJFZLTMY-CWRNSKLLSA-N Trp-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O XKKBFNPJFZLTMY-CWRNSKLLSA-N 0.000 description 1
- AWYXDHQQFPZJNE-QEJZJMRPSA-N Trp-Gln-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N AWYXDHQQFPZJNE-QEJZJMRPSA-N 0.000 description 1
- OSYOKZZRVGUDMO-HSCHXYMDSA-N Trp-Lys-Ile Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OSYOKZZRVGUDMO-HSCHXYMDSA-N 0.000 description 1
- OHOVFPKXPZODHS-SJWGOKEGSA-N Tyr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N OHOVFPKXPZODHS-SJWGOKEGSA-N 0.000 description 1
- NKMFRGPKTIEXSK-ULQDDVLXSA-N Tyr-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N NKMFRGPKTIEXSK-ULQDDVLXSA-N 0.000 description 1
- YKBUNNNRNZZUID-UFYCRDLUSA-N Tyr-Val-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YKBUNNNRNZZUID-UFYCRDLUSA-N 0.000 description 1
- LTFLDDDGWOVIHY-NAKRPEOUSA-N Val-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N LTFLDDDGWOVIHY-NAKRPEOUSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- JLFKWDAZBRYCGX-ZKWXMUAHSA-N Val-Asn-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N JLFKWDAZBRYCGX-ZKWXMUAHSA-N 0.000 description 1
- DDNIHOWRDOXXPF-NGZCFLSTSA-N Val-Asp-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N DDNIHOWRDOXXPF-NGZCFLSTSA-N 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- PQSNETRGCRUOGP-KKHAAJSZSA-N Val-Thr-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(N)=O PQSNETRGCRUOGP-KKHAAJSZSA-N 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 1
- 108010029539 arginyl-prolyl-proline Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 206010061811 demyelinating polyneuropathy Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 208000028331 flaccid paralysis Diseases 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 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
- 108010077515 glycylproline Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 108010031424 isoleucyl-prolyl-proline Proteins 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 108010016686 methionyl-alanyl-serine Proteins 0.000 description 1
- 108010022588 methionyl-lysyl-proline Proteins 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 108010084525 phenylalanyl-phenylalanyl-glycine Proteins 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Images
Classifications
-
- 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- 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
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种柯萨奇病毒A组19型VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用,属于生物工程技术领域。本发明的技术方案要点为:一种柯萨奇病毒A组19型VP1基因,其碱基序列如序列表SEQ ID NO.1所示;一种柯萨奇病毒A组19型VP1基因重组表达蛋白,其氨基酸序列如序列表SEQ ID NO.2所示;一种基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体。本发明利用原核表达CV‑A19VP1基因重组蛋白制备免疫原,方法简便易行,具有生物安全性。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种柯萨奇病毒A组19型(coxsackievirus A19,CV-A19)VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用。
背景技术
CV-A19属于小核糖核酸病毒科(Picornaviridae)肠道病毒属(Enterovirus)肠道病毒C组(Enterovirus C,EVC)[1]。CV-A19的原型株为源于日本的NIH-8663(GenBank登录号为AF499641),可以引起吉兰-巴雷综合征(Guillain-Barrésyndrome,GBS)[1]。GBS,又称格林巴利综合征、急性炎症性脱髓鞘性多发性神经病,通常发生在免疫反应产生抗体的传染病之后,抗体与神经膜上的神经节苷脂发生交叉反应,是一种潜在的威胁生命的感染后疾病,是急性弛缓性麻痹(acute flaccid paralysis,AFP)的常见病因,其特征是迅速进展,四肢对称性无力,在4周内达到最严重程度[2]。目前已有研究报道表明,从呼吸道和胃肠道疾病[3-5]、无菌性脑膜炎[6]、AFP[7]患者的标本和废水[8]样本中可以检测到CV-A19。澳大利亚Isaacs等[9]对收集自遗传风险病毒(Viruses in the Genetically at Risk,VIGR)前瞻性队列研究参与者的临床粪便样品进行近全长EV基因组的扩增与测序,亦扩增到CV-A19。本发明于2019年秋冬季从新乡市手足口病患儿的粪便中检测到一种肠道病毒,经过序列测定与分析发现,其VP1核苷酸序列与原型株8663(GenBank登录号为AF499641)的相似性为86.4%。该毒株与GenBank中已有的CV-A19毒株存在差异,其VP1核苷酸序列与Coxsackievirus A19 strain BBD26 polyprotein gene,complete cds(GenBank登录号为KC785531)相似性最高,为93.1%,其全长核苷酸序列与Coxsackievirus A19 strainBBD26polyprotein gene,complete cds(GenBank登录号为KC785531)相似性最高,为89.5%,而目前尚未见CV-A19检测与诊断的方法报道。
参考文献:
[1]Pallansch M A,Oberste M S,Whitton J L.Enteroviruses:Polioviruses,Coxsackieviruses,Echoviruses,and Newer Enteroviruses[M]//Knipe D M,Howley PM.Fields Virology.6th ed.Philadelohia:LIPPINCOTT WILLIAMS&WILKINS,a WOLTERSKLUWER business.2013:510.
[2]van den Berg B,Walgaard C,Drenthen J,Fokke C,Jacobs B C,van DoornP A.Guillain-Barre syndrome:pathogenesis,diagnosis,treatment and prognosis[J].Nat Rev Neurol,2014,10(8):469-482.
[3]Lipson S M,Walderman R,Costello P,Szabo K.Sensitivity ofrhabdomyosarcoma and guinea pig embryo cell cultures to field isolates ofdifficult-to-cultivate group A coxsackieviruses[J].J Clin Microbiol,1988,26(7):1298-1303.
[4]Iritani N,Kaida A,Abe N,Kubo H,Sekiguchi J,Yamamoto S P,Goto K,Tanaka T,Noda M.Detection and genetic characterization of human entericviruses in oyster-associated gastroenteritis outbreaks between 2001and 2012inOsaka City,Japan[J].J Med Virol,2014,86(12):2019-2025.
[5]Melica G,Langlois A L,Le Goff J,Viglietti D,Glotz D,Molina J M,Peraldi M N.Acute enteritis associated with Coxsackievirus A19 in a kidneytransplant patient[J].Transpl Infect Dis,2014,16(2):344-346.
[6]Kapusinszky B,Szomor K N,Farkas A,Takacs M,Berencsi G.Detection ofnon-polio enteroviruses in Hungary 2000-2008and molecular epidemiology ofenterovirus 71,coxsackievirus A16,and echovirus 30[J].Virus Genes,2010,40(2):163-173.
[7]Donbraye E,Olasunkanmi O I,Opabode B A,Ishola T R,Faleye T O C,Adewumi O M,Adeniji J A.Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria isgeographically defined[J].J Med Microbiol,2018,67(6):854-865.
[8]Brinkman N E,Fout G S,Keely S P.Retrospective Surveillance ofWastewater To Examine Seasonal Dynamics of Enterovirus Infections[J].mSphere,2017,2(3):e00099-17.
[9]Isaacs S R,Kim K W,Cheng J X,Bull R A,Stelzer-Braid S,Luciani F,Rawlinson W D,Craig M E.Amplification and next generation sequencing of nearfull-length human enteroviruses for identification and characterisation fromclinical samples[J].Sci Rep,2018,8(1):11889.
发明内容
本发明解决的技术问题是提供了一种柯萨奇病毒A组19型(coxsackievirus A19,CV-A19)VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用。
本发明为解决上述技术问题采用如下技术方案:
一种柯萨奇病毒A组19型VP1基因,其特征在于:该柯萨奇病毒A组19型VP1基因的碱基序列如序列表SEQ ID NO.1所示,可以CV-A19基因组cDNA为模板PCR扩增获得或人工合成。
一种柯萨奇病毒A组19型VP1基因重组表达蛋白,其特征在于:该柯萨奇病毒A组19型VP1基因重组表达蛋白的氨基酸序列如序列表SEQ ID NO.2所示。
一种基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体。
本发明所述的基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体的制备方法,其特征在于具体过程为:将携带柯萨奇病毒A组19型VP1基因的重组载体表达柯萨奇病毒A组19型VP1重组蛋白,再经纯化后免疫ICR小鼠制备而得。
本发明所述的基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备柯萨奇病毒A组19型特异性检测试剂中的应用。
本发明所述的基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备小鼠后肢样品或骨骼肌细胞中柯萨奇病毒A组19型特异性检测试剂中的应用。
本发明具有以下优点:
1.本发明利用原核表达CV-A19 VP1基因重组蛋白制备免疫原,方法简便易行,具有生物安全性。
2.本发明制备的小鼠多克隆抗体anti-CV-A19 VP1能特异性地检测CV-A19感染乳鼠后肢样品中的CV-A19 VP1蛋白,说明其能够很好地用于感染组织中CV-A19的检测。
3.本发明利用制备的小鼠多克隆抗体anti-CV-A19 VP1建立了一种免疫过氧化物酶单层试验(immunoperoxidase monolayer assay,IPMA)检测新生小鼠骨骼肌细胞中CV-A19 VP1蛋白的方法,该方法特异性好,操作简便,普通光学倒置显微镜下即可观察,实用性强。
附图说明
图3是12%(m/V)SDS-PAGE分析CV-A19 VP1基因重组蛋白的表达。M:ThermoScientificTM PageRulerTM Prestained Protein Ladder;1:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌未诱导;2:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌0.5mM IPTG 37℃诱导6h。
图4是12%(m/V)SDS-PAGE分析CV-A19 VP1基因重组表达蛋白的纯化。M:ThermoScientificTM PageRulerTM Prestained Protein Ladder;1:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌未诱导;2:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌诱导;3:菌体沉淀第一次破碎后9384×g离心30min获得的上清;4:沉淀第二次破碎后3000×g离心10min获得的沉淀;5:沉淀第五次破碎后3000×g离心10min获得的沉淀;6:切胶回收、液氮研磨获得的重组CV-A19VP1蛋白。
图5是蛋白质免疫印迹(Western blot)测定CV-A19 VP1基因重组表达蛋白免疫ICR小鼠血清效价。M:Thermo ScientificTM PageRulerTM Prestained Protein Ladder;1~6:一抗分别是免疫小鼠血清1:1000、1:2000、1:4000、1:8000、1:16000、1:32000稀释;8:一抗是未免疫小鼠血清1:1000稀释。二抗用AP标记羊抗小鼠IgG(1:8000),NBT/BCIP工作液显色。
图6是Western blot检测感染乳鼠后肢样品中的CV-A19 VP1蛋白。M:ThermoScientificTM PageRulerTM Prestained Protein Ladder;1:CV-A19感染乳鼠后肢样品;2:模拟感染乳鼠后肢样品。一抗用小鼠多克隆抗体anti-CV-A19 VP1(1:1000稀释),二抗用AP标记羊抗小鼠IgG(1:8000稀释),NBT/BCIP工作液显色。
图7是IPMA检测新生小鼠骨骼肌细胞中的CV-A19 VP1蛋白。左侧:模拟感染细胞;右侧:含CV-A19粪便上清感染细胞。一抗用小鼠多克隆抗体anti-CV-A19 VP1(1:1000稀释),二抗用HRP标记羊抗小鼠IgG(1:1000稀释),AEC工作液显色。
具体实施方式
下面通过实施例对本发明的上述内容做进一步阐述,这些实施例只用于说明本发明,并非用于限制本发明的范围。以下实施例中未注明具体条件的实验方法,通常按照常规条件进行。
需特别指出的是,尽管本发明阐述的是一种CV-A19 VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用,针对感染乳鼠后肢样品和新生小鼠骨骼肌细胞中的CV-A19分别进行Western blot和IPMA检测,但应用本发明对感染任何其他细胞的CV-A19的检测也包括在本发明所要求的权利范围之内。
实施例1
CV-A19 VP1基因扩增及克隆
用QIAGENViral RNA Kit提取含有CV-A19的手足口病患儿粪便标本中的病毒RNA。用TaKaRa RNA LA PCRTM Kit(AMV)Ver.1.1将RNA逆转录成cDNA。以该cDNA为模板,用TOYOBO高成功率PCR酶KOD FX扩增CV-A19 VP1基因。所用引物由生工生物工程(上海)股份有限公司合成(如表1所示,单下划线表示酶切位点BamHI,双下划线表示酶切位点XhoI)。PCR反应体系如表2所示(3管加cDNA为模板,1管加灭菌超纯水作为对照)。PCR反应条件为:94℃,2min,(98℃,10sec;55℃,30sec;68℃,1min)35个循环,68℃终延伸10min。PCR产物用0.8%(m/V)琼脂糖凝胶电泳检测有扩增基因目的大小片段后,剩余PCR产物用OmegaCycle Pure Kit纯化。琼脂糖凝胶电泳检测纯化的PCR产物(如图1所示),发现纯化的PCR产物中有比1000bp稍小的片段,与预期的产物大小相符。
表1扩增CV-A19 VP1基因的引物
表2扩增CV-A19 VP1基因的PCR反应体系
纯化的PCR产物用NEB限制性内切酶BamHI和XhoI双酶切、OmegaCyclePure Kit纯化后,采用NEB T4 DNA连接酶与BamHI和XhoI双酶切后、切胶回收的pET-28a(+)载体片段相连,热激法转化CaCl2法制备的大肠杆菌DH5α感受态细胞,菌落PCR筛选出阳性克隆,再经BamHI和XhoI双酶切鉴定(见图2)、生工生物工程(上海)股份有限公司测序验证准确无误后用于下一步实验。
实施例2
CV-A19 VP1基因重组蛋白表达及纯化
将上述测序验证后的重组表达质粒pET-28a(+)-CV-A19 VP1通过热激法转化至CaCl2法制备的大肠杆菌BL21(DE3)感受态细胞,筛选出表达CV-A19 VP1基因重组蛋白的阳性克隆。阳性克隆划线,挑取线上单个菌落,接种于5mL含10μg/mL硫酸卡那霉素的LB液体培养基,37℃振荡过夜培养。分别取2mL菌液接种于2瓶200mL含10μg/mL硫酸卡那霉素的LB液体培养基,待菌液生长至吸光度A600为0.6~0.8时,加入终浓度为0.5mM的IPTG诱导或不加入IPTG作为对照。37℃振荡培养6h后,测定A600,收集菌液,离心收集菌体,用20mM Tris(pH7.4)洗涤3次后,加入合适体积(15×A600/mL)20mM Tris(pH7.4)重悬沉淀。用12%(m/V)SDS-PAGE电泳检测重组蛋白表达情况(如图3所示),发现与未诱导菌相比,诱导菌在35~40kDa之间有明显的蛋白表达条带,其大小与预期CV-A19 VP1基因重组表达蛋白(37kDa)相符。
上述重悬的菌体沉淀反复冻融三次,冰上超声波破碎后,差速离心分离沉淀和上清。沉淀重复超声波破碎和差速离心5次,最终将4℃3000×g离心10min获得的沉淀重悬于5mL pH7.4的磷酸盐缓冲液(phosphate buffered saline,PBS)中。取最后的重悬液加入适量5×SDS-PAGE上样缓冲液后煮沸10min,用12%(m/V)SDS-PGAE分离蛋白,5%(m/V)氯化钾(KCl)溶液染色,切胶回收目标蛋白条带,加入适量PBS后,液氮研磨三次,获得纯化的CV-A19 VP1基因重组表达蛋白。取样对纯化蛋白进行12%(m/V)SDS-PAGE分析(如图4所示),发现:第一次超声破碎后,9384×g离心30min的上清中几乎看不到目标蛋白的存在;在后续超声波破碎后,3000×g离心10min的沉淀中有较多的目标蛋白存在;最后切胶回收、液氮研磨获得的目标蛋白条带单一。结果表明,CV-A19 VP1基因重组蛋白主要以包涵体形式表达,纯化后的蛋白纯度较高,可以用于免疫ICR小鼠制备多克隆抗体。
实施例3
小鼠anti-CV-A19 VP1多克隆抗体的制备和效价测定
实施例2纯化的CV-A19 VP1基因重组表达蛋白分别以200μL/只剂量腹腔注射免疫8周龄ICR小鼠,以后每隔三周进行第二次和第三次免疫,间隔7周后进行第四次免疫,加强免疫方式均为腹腔注射,剂量约为100μL/只。第四次免疫后第10天摘眼球取血,断颈处死小鼠,分离血清,用Western blot测定抗体效价(如图5所示)。结果发现,未免疫小鼠血清1:1000稀释与纯化的CV-A19 VP1基因重组表达蛋白杂交未出现条带,免疫小鼠血清1:1000~1:32000稀释后与纯化的CV-A19 VP1基因重组表达蛋白杂交均出现位于35~40kDa之间的条带,其大小与目标蛋白(37kDa)相符,说明免疫小鼠血清中含有anti-CV-A19 VP1多克隆抗体,其效价可以达到1:32000。
实施例4
小鼠anti-CV-A19 VP1多克隆抗体用于Western blot检测感染乳鼠后肢样品中的CV-A19
取出冻存于-80℃的CV-A19感染乳鼠后肢样品和模拟感染(注射0.9wt%NaCl溶液)乳鼠后肢样品,按2mL/0.1g重量加入含有1mM PMSF的RIPA裂解液(强),液氮研磨后,冰上裂解30min。4℃14000×g离心10min后,吸取上清液,加入适量5×SDS-PAGE上样缓冲液,煮沸10min。然后用12%(m/V)SDS-PAGE分离蛋白,湿法转膜将蛋白转移到PVDF膜上,用实施例3中获得的小鼠anti-CV-A19 VP1多克隆抗体作为一抗(1:1000稀释)进行Western blot分析(见图6)。结果发现,感染样品在大约35kDa处有条带,而模拟感染样品在相应位置无明显条带,说明小鼠anti-CV-A19 VP1多克隆抗体能够特异性地检测CV-A19感染乳鼠后肢样品中的CV-A19 VP1蛋白,可用于CV-A19感染乳鼠后肢样品中的CV-A19检测。
实施例5
小鼠anti-CV-A19 VP1多克隆抗体用于IPMA检测感染新生小鼠骨骼肌细胞中的CV-A19
参考Siwar Nsaibia等人(Virus Research,2007,123:30–39)的方法,略微修改,分离新生小鼠骨骼肌细胞。在无Ca2+、Mg2+D-Hanks溶液中分离新生(1-2天)小鼠后肢,去除皮肤后,将后肢肌肉等组织剪碎成肉糜状,然后吸取至50mL离心管中,轻轻摇晃2min,再静置5min,弃去含血污的上清液,沉淀加入含有100U/mL胶原酶Ⅳ、2.4U/mL中性蛋白酶和0.036mM CaCl2的D-Hanks溶液摇匀后,于37℃消化20~30min。取出静置2min后,上清液转移至无菌洁净50mL离心管中,加入1/10(V/V)胎牛血清。沉淀的肌肉组织再消化一次,同样收集上清,加入胎牛血清。两次消化获得的细胞悬液分别用70μm孔径的尼龙膜过滤,滤液300×g离心10min后,用含有25%(V/V)Medium 199、20%(V/V)FBS、1%(V/V)100×青链霉素和2μg/mL两性霉素B的MEM培养基重悬细胞。计数后,以1.6×105个细胞/孔接种于96孔板中,37℃、体积分数5%CO2培养箱中贴壁培养1h后,上清转移至新孔中继续培养过夜,第二天用含CV-A19的粪便上清液感染或用上述培养基模拟感染新孔中的细胞1h,弃感染液或培养基,加入上述培养基继续培养。待4天后,感染细胞与模拟感染细胞出现明显差异时,进行IPMA检测:首先弃上清液,加入200μL/孔PBS洗3次;然后加入100μL/孔冰冻的无水乙醇室温固定10min,再用200μL/孔PBS清洗1次;接下来用含有5%(m/V)脱脂奶粉的PBST 37℃封闭1h,弃封闭液,用PBST清洗1次;再加入用封闭液稀释的小鼠anti-CV-A19 VP1(1:1000)作为一抗,4℃孵育过夜,PBST清洗3次;然后加入封闭液稀释HRP标记羊抗小鼠IgG(1:1000)作为二抗,37℃孵育1h,PBST清洗5次;最后加入AEC工作液充分显色,弃显色液,超纯水洗2次后,加入适量超纯水,在倒置光学显微镜下观察,拍照分析(如图7所示)。结果发现,与模拟感染细胞相比,用含CV-A19的粪便上清液感染细胞部分出现聚集皱缩,染色后呈现红棕色。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
序列表
<110> 新乡医学院
<120> 一种柯萨奇病毒A组19型VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用
<130> 2021
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 888
<212> DNA
<213> 人工序列(artificial sequence)
<400> 1
ggtattgatg acattataga taatgtagtg actaatgctc tgaaagtgtc catgccacag 60
gtccaagaca cacagtctag cggaccggtg aactcaaaag aagtgcctgc gttgaccgct 120
gttgaaactg gagcaactag ccaggttgac ccgtcagact taatagagac cagacatgta 180
ataaacaacc gtcttagatc agagtgcact attgaatctt tctttgggag atccgcttgt 240
gtggctataa ttggcctttc caatcagaaa cccaccgatg acaatgcagc taaacttttt 300
gctacatgga aaataagtta ccttgatacg taccagctga gaaggaaatt agaattcttc 360
acctactcca gatttgacct tgaattgacc tttgtaatat cagagagatt cttcacctcc 420
acttcagctg cagctagaga ttatgtgtac caaatcatgt acattcctcc aggagcccca 480
attccacagg catgggatga ttacacttgg cagtcgtcaa ccaatccatc aatattttac 540
accacaggga atgcatgtcc cagagtgtcc atcccttttg ttggtatcgg tgcagcgtac 600
tcccacttct acgatgggtt ctctctagtt cctttcaata ccattgatgc cggggcatcg 660
aacaggtatg gttacaccac cattaatgac tttggaacca tggctatcag gatagtgaat 720
gaatacgacc cagtcacaat cgatgctaaa gttagggtat acatgaaacc aaaacacatc 780
aaggtttggt gtcctagacc accacgggct gtggcataca atgggccaac tgtgaatttc 840
aacgaaaacc cacatgtgat gacagcagtt gcagatatta ggacttat 888
<210> 2
<211> 331
<212> PRT
<213> 人工序列(artificial sequence)
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Gly Ile Asp Asp Ile Ile Asp Asn Val Val Thr Asn Ala
35 40 45
Leu Lys Val Ser Met Pro Gln Val Gln Asp Thr Gln Ser Ser Gly Pro
50 55 60
Val Asn Ser Lys Glu Val Pro Ala Leu Thr Ala Val Glu Thr Gly Ala
65 70 75 80
Thr Ser Gln Val Asp Pro Ser Asp Leu Ile Glu Thr Arg His Val Ile
85 90 95
Asn Asn Arg Leu Arg Ser Glu Cys Thr Ile Glu Ser Phe Phe Gly Arg
100 105 110
Ser Ala Cys Val Ala Ile Ile Gly Leu Ser Asn Gln Lys Pro Thr Asp
115 120 125
Asp Asn Ala Ala Lys Leu Phe Ala Thr Trp Lys Ile Ser Tyr Leu Asp
130 135 140
Thr Tyr Gln Leu Arg Arg Lys Leu Glu Phe Phe Thr Tyr Ser Arg Phe
145 150 155 160
Asp Leu Glu Leu Thr Phe Val Ile Ser Glu Arg Phe Phe Thr Ser Thr
165 170 175
Ser Ala Ala Ala Arg Asp Tyr Val Tyr Gln Ile Met Tyr Ile Pro Pro
180 185 190
Gly Ala Pro Ile Pro Gln Ala Trp Asp Asp Tyr Thr Trp Gln Ser Ser
195 200 205
Thr Asn Pro Ser Ile Phe Tyr Thr Thr Gly Asn Ala Cys Pro Arg Val
210 215 220
Ser Ile Pro Phe Val Gly Ile Gly Ala Ala Tyr Ser His Phe Tyr Asp
225 230 235 240
Gly Phe Ser Leu Val Pro Phe Asn Thr Ile Asp Ala Gly Ala Ser Asn
245 250 255
Arg Tyr Gly Tyr Thr Thr Ile Asn Asp Phe Gly Thr Met Ala Ile Arg
260 265 270
Ile Val Asn Glu Tyr Asp Pro Val Thr Ile Asp Ala Lys Val Arg Val
275 280 285
Tyr Met Lys Pro Lys His Ile Lys Val Trp Cys Pro Arg Pro Pro Arg
290 295 300
Ala Val Ala Tyr Asn Gly Pro Thr Val Asn Phe Asn Glu Asn Pro His
305 310 315 320
Val Met Thr Ala Val Ala Asp Ile Arg Thr Tyr
325 330
Claims (6)
1.一种柯萨奇病毒A组19型VP1基因,其特征在于:该柯萨奇病毒A组19型VP1基因的碱基序列如SEQ ID NO.1所示。
2.一种柯萨奇病毒A组19型VP1基因重组表达蛋白,其特征在于:该柯萨奇病毒A组19型VP1基因重组表达蛋白的氨基酸序列如SEQ ID NO.2所示。
3.一种基于权利要求2所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体。
4.一种权利要求3所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体的制备方法,其特征在于具体过程为:将携带权利要求1所述的柯萨奇病毒A组19型VP1基因的重组载体表达柯萨奇病毒A组19型VP1重组蛋白,再经纯化后免疫ICR小鼠制备而得。
5.权利要求3所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备柯萨奇病毒A组19型特异性检测试剂中的应用。
6.权利要求3所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备小鼠后肢样品或骨骼肌细胞中柯萨奇病毒A组19型特异性检测试剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110338067.6A CN113122551B (zh) | 2021-03-30 | 2021-03-30 | 一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110338067.6A CN113122551B (zh) | 2021-03-30 | 2021-03-30 | 一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113122551A CN113122551A (zh) | 2021-07-16 |
CN113122551B true CN113122551B (zh) | 2022-07-12 |
Family
ID=76775374
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110338067.6A Active CN113122551B (zh) | 2021-03-30 | 2021-03-30 | 一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113122551B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015179979A1 (en) * | 2014-05-28 | 2015-12-03 | National Health Research Institutes | Viral particles as immunogens against enterovirus infection and production thereof |
CN108283631A (zh) * | 2018-01-08 | 2018-07-17 | 南京中医药大学 | 一种肠道病毒的抑制剂及其应用 |
CN109234241A (zh) * | 2018-08-22 | 2019-01-18 | 武汉生物制品研究所有限责任公司 | 一种柯萨奇病毒cva16型强毒株cva16-b6-714及其应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11339387B2 (en) * | 2018-05-22 | 2022-05-24 | The Government Of The United States Of America As Represented By The Secretary Of Homeland Security | Modified foot-and-mouth disease virus 3C proteases and methods for processing FMDV P1 precursor polypeptide |
-
2021
- 2021-03-30 CN CN202110338067.6A patent/CN113122551B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015179979A1 (en) * | 2014-05-28 | 2015-12-03 | National Health Research Institutes | Viral particles as immunogens against enterovirus infection and production thereof |
CN108283631A (zh) * | 2018-01-08 | 2018-07-17 | 南京中医药大学 | 一种肠道病毒的抑制剂及其应用 |
CN109234241A (zh) * | 2018-08-22 | 2019-01-18 | 武汉生物制品研究所有限责任公司 | 一种柯萨奇病毒cva16型强毒株cva16-b6-714及其应用 |
Non-Patent Citations (4)
Title |
---|
"Coxsackievirus A19 strain 2019103106/XX/CHN/2019, complete genome,ACCESSION: MT175706.1";NCBI;《GenBank》;20210618;第1-3页 * |
"Detection of non-polio enteroviruses in Hungary 2000-2008 and molecular epidemiology of enterovirus 71, coxsackievirus A16, and echovirus 30";Beatrix Kapusinszky 等;《Virus Genes》;20100101;第40卷;第163-173页 * |
"Genomic analysis of a recombinant coxsackievirus A19 identified in Xinxiang, China, in 2019";Liang Yi 等;《Archives of Virology》;20220410;第167卷;第1405-1420页 * |
"柯萨奇病毒A组19型基因组特征和细胞感染能力研究";易亮;《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》;20220115(第01期);第E069-142页 * |
Also Published As
Publication number | Publication date |
---|---|
CN113122551A (zh) | 2021-07-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112409496B (zh) | 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用 | |
CN110759973B (zh) | 一种表达非洲猪瘟病毒CD2v蛋白的细胞株及其应用 | |
CN113564131B (zh) | 柯萨奇病毒a6型毒株及其应用 | |
CN111471089B (zh) | 一种重组的非洲猪瘟病毒cd2v亚单位蛋白及其制备方法和应用 | |
CN110684116B (zh) | 一种结核分枝杆菌eec融合蛋白、制备方法及其应用 | |
CN105296521B (zh) | 表达可溶性人***瘤病毒16亚型l1蛋白的重组质粒及其表达方法 | |
CN109970851B (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 | |
CN113564130B (zh) | 柯萨奇病毒a10型毒株及其应用 | |
CN104211784B (zh) | 用于制备戊型肝炎病毒样颗粒的蛋白质和方法 | |
CN110551212A (zh) | 抗gii.4型诺如病毒衣壳蛋白vp1和病毒样颗粒vlp单克隆抗体的制备方法和应用 | |
CN112920278A (zh) | 一种新型冠状病毒特异性融合蛋白抗原及其制备方法和应用 | |
WO2021206638A1 (en) | Vaccine and/or antibody for viral infection | |
CN113748203A (zh) | 重组经典猪瘟病毒 | |
WO2022179535A1 (zh) | 抗SARS-CoV-2核衣壳蛋白的单克隆抗体及其制备方法和用途 | |
CN113564133A (zh) | 柯萨奇病毒a16型毒株及其免疫原性组合物和应用 | |
TW201938578A (zh) | 腸病毒疫苗 | |
Chang et al. | Development and characterization of monoclonal antibody against the critical loop structure of african swine fever virus P72 protein | |
CN106226520B (zh) | 结核分枝杆菌抗原蛋白Rv0865及其B细胞表位肽的应用 | |
CN113122551B (zh) | 一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 | |
US11767356B1 (en) | Canine parvovirus nanobody CPV-VHH-E3 and application thereof | |
CN107602672A (zh) | 重组表达的腺病毒纤毛蛋白肽、腺病毒亚单位疫苗及其制备方法 | |
CN105085672B (zh) | 3d蛋白特异性单克隆免疫球蛋白a抗体及其组合物 | |
JP7085531B2 (ja) | 強毒性エンテロウイルス71の安定的生産およびその利用 | |
CN107365365A (zh) | 重组表达的腺病毒纤毛蛋白肽、腺病毒亚单位疫苗及其制备方法 | |
CN109553680B (zh) | 一种单克隆抗体ZKns4B8及其应用 |
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 |