CN114014916B - Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection - Google Patents
Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection Download PDFInfo
- Publication number
- CN114014916B CN114014916B CN202111422353.7A CN202111422353A CN114014916B CN 114014916 B CN114014916 B CN 114014916B CN 202111422353 A CN202111422353 A CN 202111422353A CN 114014916 B CN114014916 B CN 114014916B
- Authority
- CN
- China
- Prior art keywords
- recombinant protein
- 3cap
- gene
- porcine circovirus
- protein
- 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 111
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 76
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 76
- 238000002965 ELISA Methods 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 38
- 241001432873 Porcine circovirus 3 Species 0.000 title claims description 12
- 241000202347 Porcine circovirus Species 0.000 claims abstract description 28
- 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 claims abstract description 8
- 238000000576 coating method Methods 0.000 claims description 30
- 239000011248 coating agent Substances 0.000 claims description 27
- 239000013612 plasmid Substances 0.000 claims description 26
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 13
- 230000006698 induction Effects 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 46
- 238000000034 method Methods 0.000 abstract description 37
- 239000012634 fragment Substances 0.000 abstract description 20
- 241000282887 Suidae Species 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000001042 affinity chromatography Methods 0.000 abstract description 6
- 238000000746 purification Methods 0.000 abstract description 6
- 210000004777 protein coat Anatomy 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 62
- 241000282898 Sus scrofa Species 0.000 description 29
- 239000000427 antigen Substances 0.000 description 24
- 102000036639 antigens Human genes 0.000 description 24
- 108091007433 antigens Proteins 0.000 description 24
- 238000010790 dilution Methods 0.000 description 24
- 239000012895 dilution Substances 0.000 description 24
- 238000012360 testing method Methods 0.000 description 16
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 15
- 238000007789 sealing Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 238000005457 optimization Methods 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 239000000758 substrate Substances 0.000 description 10
- 241001673669 Porcine circovirus 2 Species 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 235000020183 skimmed milk Nutrition 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 230000000903 blocking effect Effects 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 229960005486 vaccine Drugs 0.000 description 6
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 5
- 101150009852 ORF2 gene Proteins 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 101150044789 Cap gene Proteins 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 3
- 241000710188 Encephalomyocarditis virus Species 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 3
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 3
- 102100024407 Jouberin Human genes 0.000 description 3
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 3
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 3
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 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 3
- 238000012258 culturing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 239000012474 protein marker Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- PUBLUECXJRHTBK-ACZMJKKPSA-N Ala-Glu-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O PUBLUECXJRHTBK-ACZMJKKPSA-N 0.000 description 2
- LFWOQHSQNCKXRU-UFYCRDLUSA-N Arg-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 LFWOQHSQNCKXRU-UFYCRDLUSA-N 0.000 description 2
- GGRSYTUJHAZTFN-IHRRRGAJSA-N Asp-Pro-Tyr Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O GGRSYTUJHAZTFN-IHRRRGAJSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- RGNMNWULPAYDAH-JSGCOSHPSA-N Gln-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N RGNMNWULPAYDAH-JSGCOSHPSA-N 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- FOBUGKUBUJOWAD-IHPCNDPISA-N Leu-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FOBUGKUBUJOWAD-IHPCNDPISA-N 0.000 description 2
- 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 2
- PGLGNCVOWIORQE-SRVKXCTJSA-N Lys-His-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O PGLGNCVOWIORQE-SRVKXCTJSA-N 0.000 description 2
- JHNOXVASMSXSNB-WEDXCCLWSA-N Lys-Thr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JHNOXVASMSXSNB-WEDXCCLWSA-N 0.000 description 2
- VWJFOUBDZIUXGA-AVGNSLFASA-N Lys-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCCCN)N VWJFOUBDZIUXGA-AVGNSLFASA-N 0.000 description 2
- KYXDADPHSNFWQX-VEVYYDQMSA-N Met-Thr-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O KYXDADPHSNFWQX-VEVYYDQMSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010066427 N-valyltryptophan Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- GPLWGAYGROGDEN-BZSNNMDCSA-N Phe-Phe-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O GPLWGAYGROGDEN-BZSNNMDCSA-N 0.000 description 2
- JMVQDLDPDBXAAX-YUMQZZPRSA-N Pro-Gly-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 JMVQDLDPDBXAAX-YUMQZZPRSA-N 0.000 description 2
- VZKBJNBZMZHKRC-XUXIUFHCSA-N Pro-Ile-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O VZKBJNBZMZHKRC-XUXIUFHCSA-N 0.000 description 2
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 2
- DWUIECHTAMYEFL-XVYDVKMFSA-N Ser-Ala-His Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 DWUIECHTAMYEFL-XVYDVKMFSA-N 0.000 description 2
- MQQBBLVOUUJKLH-HJPIBITLSA-N Ser-Ile-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MQQBBLVOUUJKLH-HJPIBITLSA-N 0.000 description 2
- VZQRNAYURWAEFE-KKUMJFAQSA-N Ser-Leu-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VZQRNAYURWAEFE-KKUMJFAQSA-N 0.000 description 2
- RXUOAOOZIWABBW-XGEHTFHBSA-N Ser-Thr-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RXUOAOOZIWABBW-XGEHTFHBSA-N 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- GFRIEEKFXOVPIR-RHYQMDGZSA-N Thr-Pro-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O GFRIEEKFXOVPIR-RHYQMDGZSA-N 0.000 description 2
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp 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](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 2
- GQHAIUPYZPTADF-FDARSICLSA-N Trp-Ile-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 GQHAIUPYZPTADF-FDARSICLSA-N 0.000 description 2
- XGFGVFMXDXALEV-XIRDDKMYSA-N Trp-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N XGFGVFMXDXALEV-XIRDDKMYSA-N 0.000 description 2
- PGEFRHBWGOJPJT-KKUMJFAQSA-N Tyr-Lys-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O PGEFRHBWGOJPJT-KKUMJFAQSA-N 0.000 description 2
- RYQUMYBMOJYYDK-NHCYSSNCSA-N Val-Pro-Glu Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N RYQUMYBMOJYYDK-NHCYSSNCSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 108010062796 arginyllysine Proteins 0.000 description 2
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000011841 epidemiological investigation Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000001932 seasonal effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 101150038172 1.2 gene Proteins 0.000 description 1
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 1
- PEFFAAKJGBZBKL-NAKRPEOUSA-N Arg-Ala-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PEFFAAKJGBZBKL-NAKRPEOUSA-N 0.000 description 1
- OVVUNXXROOFSIM-SDDRHHMPSA-N Arg-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O OVVUNXXROOFSIM-SDDRHHMPSA-N 0.000 description 1
- WKPXXXUSUHAXDE-SRVKXCTJSA-N Arg-Pro-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O WKPXXXUSUHAXDE-SRVKXCTJSA-N 0.000 description 1
- AWMAZIIEFPFHCP-RCWTZXSCSA-N Arg-Pro-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O AWMAZIIEFPFHCP-RCWTZXSCSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 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
- 241000522195 Dalbergia Species 0.000 description 1
- SOBBAYVQSNXYPQ-ACZMJKKPSA-N Gln-Asn-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SOBBAYVQSNXYPQ-ACZMJKKPSA-N 0.000 description 1
- MYXNLWDWWOTERK-BHNWBGBOSA-N Gly-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN)O MYXNLWDWWOTERK-BHNWBGBOSA-N 0.000 description 1
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 1
- FOKISINOENBSDM-WLTAIBSBSA-N Gly-Thr-Tyr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FOKISINOENBSDM-WLTAIBSBSA-N 0.000 description 1
- HXIDVIFHRYRXLZ-NAKRPEOUSA-N Ile-Ser-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)O)N HXIDVIFHRYRXLZ-NAKRPEOUSA-N 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- ZYLJULGXQDNXDK-GUBZILKMSA-N Leu-Gln-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ZYLJULGXQDNXDK-GUBZILKMSA-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
- SQRLLZAQNOQCEG-KKUMJFAQSA-N Lys-Tyr-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 SQRLLZAQNOQCEG-KKUMJFAQSA-N 0.000 description 1
- VIZLHGTVGKBBKO-AVGNSLFASA-N Met-Arg-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N VIZLHGTVGKBBKO-AVGNSLFASA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- GCFNFKNPCMBHNT-IRXDYDNUSA-N Phe-Tyr-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)NCC(=O)O)N GCFNFKNPCMBHNT-IRXDYDNUSA-N 0.000 description 1
- 101100382437 Porcine circovirus 2 Cap gene Proteins 0.000 description 1
- KIDXAAQVMNLJFQ-KZVJFYERSA-N Pro-Thr-Ala Chemical compound C[C@@H](O)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](C)C(O)=O KIDXAAQVMNLJFQ-KZVJFYERSA-N 0.000 description 1
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- GJFYFGOEWLDQGW-GUBZILKMSA-N Ser-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GJFYFGOEWLDQGW-GUBZILKMSA-N 0.000 description 1
- CRJZZXMAADSBBQ-SRVKXCTJSA-N Ser-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO CRJZZXMAADSBBQ-SRVKXCTJSA-N 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- PCMDGXKXVMBIFP-VEVYYDQMSA-N Thr-Met-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMDGXKXVMBIFP-VEVYYDQMSA-N 0.000 description 1
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 1
- QAYSODICXVZUIA-WLTAIBSBSA-N Tyr-Gly-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O QAYSODICXVZUIA-WLTAIBSBSA-N 0.000 description 1
- CLEGSEJVGBYZBJ-MEYUZBJRSA-N Tyr-Thr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CLEGSEJVGBYZBJ-MEYUZBJRSA-N 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000009385 viral infection Effects 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
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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)
- Molecular Biology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a porcine circovirus type 3Cap recombinant protein, a coding gene thereof and application thereof in ELISA antibody detection, wherein a PCR method is adopted to delete a 60 th-120 aa gene fragment expressed in a PCV 3Cap gene to obtain a coding gene of the PCV 3Cap recombinant protein, the coding gene is cloned to pET32a and then transformed to Rosseta competent cells, and the PCV 3Cap recombinant protein can be obtained through IPTG induced expression. The protein is soluble and expressed, can be combined with His tag antibody, and is convenient for purification by adopting an affinity chromatography method. The recombinant protein is adopted to immunize pigs, so that polyclonal antibodies can be obtained, and the polyclonal antibodies have high titers and antigenicity similar to PCV3. The recombinant protein coats the ELISA plate, and the PCV3 is subjected to indirect ELISA antibody detection, so that the ELISA plate has the advantages of good sensitivity, specificity, repeatability and the like.
Description
The application is a divisional application of the invention with the application number of 201910046124.6, the application date of 2019, 1 month and 18 days, and the invention name of porcine circovirus 3Cap recombinant protein, and the coding gene and application thereof.
Technical Field
The invention relates to a porcine circovirus type 3Cap recombinant protein and a gene encoding the same, and also relates to application of the obtained porcine circovirus type 3Cap recombinant protein in PCV3 indirect ELISA antibody detection.
Background
Porcine circovirus disease is caused by porcine circovirus, which is classified into three serotypes, PCV1, PCV2, and PCV3. PCV1 is generally considered to be non-pathogenic to animals. At present, the most clear of the virus infection characteristics and pathogenicity studies is PCV2, and the damage caused by PCV2 to the pig industry is serious, and can cause the circular ring virus related disease (PCVAD) to cause immunosuppression. PCV2 has mainly two reading frames: ORF1 and ORF2, ORF1 expresses Rep protein and cutter Rep', ORF2 encodes Cap protein of virus, cap protein constitutes main component of virus nucleocapsid, is main structural protein of virus, determines antigenicity of virus, and can self-assemble to form virus-like particle. The ORF2 gene is a main target protein for vaccine development and pathogen detection, and is also a gene with main differences between subtypes. The American scholars Rachel Palinski et al, 2016 reported a novel virus PCV3, which subsequently emerged in many countries and regions such as Asia, america, europe. PCV3 genome length 2000bp, ORF1 encodes Rep protein, length 297aa (amino acid), ORF2 encodes Cap protein, length 214aa (amino acid). PCV3 can cause PDNS and exhibits symptoms like reproductive disorders, multisystemic inflammation, etc. similar to PCV 2. The Zhan ocean analyzed the PCV 3Cap protein structure and found that there was no common linear epitope with the PCV2 Cap protein structure. Also, it was found that the ORF2 gene of PCV3 and the ORF2 gene of PCV2 show a large difference, homology of only about 37%, and thus it was predicted that PCV2 vaccine might not have a cross-protective effect on PCV3.
For the above reasons, neither the current PCV2 vaccine nor the detection method of PCV2 is directly applicable to PCV3, and a specific study on the ORF2 gene of PCV3 is required to be suitable for its detection method and vaccine. Serological monitoring is often used clinically to understand the distribution of antibodies or pathogens in animals. The indirect immunofluorescence experiment and the immunoperoxidase monolayer cell experiment have complex operation, high cost and high equipment requirement, and are not suitable for clinical large-scale detection. The ELISA method can be used for simply monitoring the infection condition of the pig herd and evaluating the immune effect of the vaccine, and in the ELISA method, the concentration and the purity of the coating antigen are key to influence the sensitivity and the specificity of the detection result, so that the research on the soluble expression and the purification of PCV 3Cap protein is of great significance to obtain the ELISA method suitable for PCV3 detection.
Disclosure of Invention
One of the purposes of the invention is to provide a coding gene of porcine circovirus type 3Cap recombinant protein, which is obtained by deleting the gene fragment expressing amino acids 1-120 or 60-120 of PCV3ORF2 (open reading frame) gene, and the gene successfully realizes the soluble expression of porcine circovirus type 3Cap recombinant protein, namely PCV 3Cap recombinant protein.
The PCV ORF2 gene codes Cap protein, which is an important structural protein of the virus, determines the antigenicity of the virus and is a main antigen for vaccine immune targeting. The colibacillus expression system has the features of high expression efficiency of target protein, simple operation, low cost, etc. and has the demerits of easy influence on the expression of the vector, especially the transmembrane region and signal peptide of the target gene, so that the target protein may not be expressed. According to the invention, through research, the high-efficiency expression of the target protein is realized by truncating the gene fragments of the 1 st to 120 th amino acids or 60 th to 120 th amino acids of the expression of the PCV3ORF2 gene with stronger hydrophobicity and weaker antigenicity, and the target protein is expressed in a soluble way. The His tag is fused and expressed at the tail end of the target protein gene, which is favorable for purifying the target protein by adopting nickel column affinity chromatography.
Furthermore, the encoding gene of the porcine circovirus type 3Cap recombinant protein is obtained by deleting gene fragments expressing amino acid fragments from 1 st to 120 th or 60 th to 120 th amino acid fragments in the PCV3ORF2 gene. The encoding gene of the porcine circovirus 3 type Cap recombinant protein has a gene sequence shown as SEQ ID NO.1 or SEQ ID NO.2, wherein SEQ ID NO.1 is a sequence for expressing truncated aa (amino acid) fragments at positions 1-120, and SEQ ID NO.2 is a sequence for expressing truncated aa at positions 60-120.
Furthermore, the invention also provides a recombinant plasmid which contains the coding gene of the porcine circovirus 3Cap recombinant protein. Preferably, the recombinant plasmid is obtained by recombining the encoding gene of the porcine circovirus 3 type Cap recombinant protein and a pET32a vector.
The invention also aims to provide a porcine circovirus type 3Cap recombinant protein which is expressed in a soluble way, can be combined with a His tag antibody and is convenient to purify and prepare. Compared with the porcine circovirus 3 type Cap protein, the recombinant protein lacks aa at the 1 st to 120 th positions or lacks aa at the 60 th to 120 th positions.
Furthermore, the porcine circovirus 3-type Cap recombinant protein is obtained by expressing the recombinant plasmid. Specifically, the recombinant plasmid is obtained by transforming the recombinant plasmid into Rosseta competent cells, and then IPTG (Isopropyl Thiogalactoside) inducing expression.
Furthermore, the amino acid sequence of the porcine circovirus 3 type Cap recombinant protein is shown as SEQ ID NO. 3or SEQ ID NO. 4. Wherein the amino acid sequence shown in SEQ ID NO.3 is the amino acid sequence of deletion of aa at positions 1-120, and the amino acid sequence shown in SEQ ID NO.4 is the amino acid sequence of deletion of aa at positions 60-120.
Further, the preparation method of the purified porcine circovirus type 3Cap recombinant protein comprises the following steps:
(1) Transforming the recombinant plasmid into Rosseta competent cells, picking single colony, inoculating into 5-10 mL LB liquid medium, and culturing at 37 ℃ overnight;
(2) Inoculating the LB liquid medium obtained in the step (1) into a new LB medium in a volume ratio of 1:100, and taking the OD as the OD 600 When the value reaches 0.6-0.8, adding IPTG with the final concentration of 1.0mmol/L, and culturing for 4 hours at 37 ℃ to obtain a culture solution;
(3) Centrifuging the obtained culture solution, collecting thalli, re-suspending the thalli by using PBS, performing ultrasonic wave pyrolysis, and centrifuging to collect supernatant fluid to obtain porcine circovirus 3 type Cap recombinant protein;
(4) Purifying the obtained porcine circovirus type 3Cap recombinant protein by using an affinity chromatography method to obtain the purified porcine circovirus type 3Cap recombinant protein.
Another object of the present invention is to provide a PCV3 porcine polyclonal antibody, which PCV3 porcine polyclonal antibody is prepared according to the following method:
(1) Taking purified porcine circovirus 3 type Cap recombinant protein as an antigen, immunizing a pig by using the antigen, and enhancing the immunity once every 3 weeks;
(2) And taking pig blood when the titer of the antibody in the pig serum is greater than 1:10000, separating the serum, centrifuging the serum, and taking the supernatant to obtain the PCV3 pig polyclonal antibody.
Further, when preparing PCV3 porcine polyclonal antibody, porcine circovirus 3Cap recombinant protein (PCV 3Cap recombinant protein for short, hereinafter referred to as PCV 3Cap recombinant protein) is immunized after the following treatments: the purified porcine circovirus 3Cap recombinant protein is diluted to 2mg/mL by PBS, then mixed with an equal volume of ISA-206 adjuvant, fully emulsified, and then immunized to pigs.
Furthermore, when PCV3 porcine polyclonal antibody is prepared, the immune dose of each pig is 1mg of porcine circovirus 3Cap recombinant protein.
Another object of the present invention is to provide a PCV3 indirect ELISA antibody detection kit comprising an ELISA plate coated with the above PCV 3Cap recombinant protein.
It is another object of the present invention to provide an indirect ELISA antibody detection method for PCV3, which comprises the step of coating an ELISA plate with purified PCV 3Cap recombinant protein.
Further, the coating concentration of PCV 3Cap recombinant protein is preferably 4 μg/mL; the coating mode is preferably as follows: coating at 37 ℃ for 2 hours and then coating at 4 ℃ overnight.
Furthermore, except for adopting the PCV 3Cap recombinant protein coated ELISA plate special for the invention, the steps of the indirect ELISA antibody detection method are all conventional in the prior art, and the conditions used in each step are optimized by the invention. For example, the blocking solution used in the method of the present invention is preferably 5wt% skim milk, the dilution factor of the serum used is preferably 1:100, the dilution of the enzyme-labeled antibody is preferably 1:10000, etc.
Description and explanation of related terms in the present invention
In the present invention, unless otherwise indicated, scientific and technical terms used have the meanings commonly understood by one of ordinary skill in the art. Also, the techniques used herein, such as cell culture, cell transformation, plasmid recombination, fusion PCR, immunological experiments, polyclonal antibody preparation, indirect ELISA antibody detection methods, etc., are all reported in the art, and the steps thereof can be performed using conventional procedures disclosed in the prior art. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
According to the invention, the coding gene of the porcine circovirus type 3Cap recombinant protein refers to a gene for coding the porcine circovirus type 3Cap recombinant protein, and can be simply called a coding gene, a truncated 3Cap gene and a coding gene of PCV 3Cap recombinant protein.
According to the invention, the porcine circovirus type 3Cap recombinant protein refers to a protein obtained by soluble expression of a coding gene of the porcine circovirus type 3Cap recombinant protein, and can be abbreviated as recombinant protein, 3Cap recombinant protein, cap recombinant protein and PCV 3Cap recombinant protein.
According to the invention, the PCV3 porcine polyclonal antibody refers to an antibody obtained by immunizing a pig with purified porcine circovirus type 3Cap recombinant protein, and is called polyclonal antibody for short.
The invention adopts a PCR method to delete gene fragments expressing aa at 1-120 th or aa at 60-120 th in PCV 3Cap genes to obtain encoding genes of PCV 3Cap recombinant proteins, and clones the encoding genes to E.coli expression plasmid pET32a to construct and obtain recombinant plasmids. The recombinant plasmid is transformed into Rosseta competent cells, and the recombinant plasmid is subjected to IPTG induction expression to obtain PCV 3Cap recombinant protein. The protein is soluble and expressed, can be combined with His tag antibody, and is convenient for purification by adopting an affinity chromatography method. The recombinant protein is adopted to immunize pigs, so that polyclonal antibodies can be obtained, and the polyclonal antibodies have high titers and antigenicity similar to PCV3. The PCV 3Cap recombinant protein is coated on the ELISA plate, so that a PCV3 indirect ELISA antibody detection method is established, the sensitivity, the specificity and the repeatability of the detection method are good, the distribution situation of the PCV3 antibodies in pig groups in 2013-2017 is retrospectively investigated by adopting the method, and the result shows that the positive rate of the PCV3 antibodies in 2013-2017 in China is in an ascending trend, the positive rate in 2017 reaches 75.9%, and an important basis is provided for prevention and control of the disease.
Drawings
FIG. 1 is a diagram of SDS-PAGE and Western Blot analysis of recombinant proteins; wherein, FIG. 1A is SDS-PAGE and Western Blot analysis of 3 Cap/delta 1-120 recombinant proteins, M is a protein Marker,1 is whole recombinant escherichia coli, 2 is supernatant of recombinant bacteria lysate, 3 is precipitation of recombinant bacteria lysate, and 4 is escherichia coli control; FIG. 1B is SDS-PAGE and Western Blot analysis of 3 Cap/delta 60-120 recombinant proteins, M is a protein Marker,1 is whole recombinant E.coli, 2 is supernatant of recombinant bacterial lysate, 3 is precipitation of recombinant bacterial lysate, and 4 is E.coli control.
FIG. 2 is a purification diagram of 3Cap/Δ1-120 recombinant proteins; wherein M is a protein Marker,1 is an unpurified protein, 2 is a flow-through protein, 3-5 is a washed-out protein, and 6 is an eluted protein.
Detailed Description
The invention is further illustrated and described below by means of the figures and the specific embodiments and the immunological properties of the soluble proteins obtained according to the invention are verified. The following description is merely exemplary in nature and is in no way intended to limit the scope of the present disclosure.
Example 1
1 materials and methods
1.1 major reagents and materials
E.coli strain E.coli DH 5. Alpha. And Rosseta competent cells were all purchased and stored in this laboratory. Various restriction enzymes and T4 DNA ligases were purchased from Novain; plasmid extraction kits were purchased from biomega company; DNA gel recovery kit was purchased from BioFlux company; SPA-HRP, goat anti-mouse IgG-HRP, was purchased from Bodhisattva bioengineering; mouse anti-His-Tag monoclonal antibodies were purchased from Abmart; IPTG was purchased from dalbergia bioengineering limited; the resin Ni Sepharose 6Fast Flow (17-5318-01) was produced by GE Healthcare. The rest chemical reagents are all of domestic analytical purity. Other plasmids, cells or reagents mentioned below but not described herein are commercially available.
Reference serum: PRRSV, PRV, PCV2, CSFV, HPS, EMCV and FMDV positive serum, stored in this laboratory. PCV3 negative serum and positive serum, stored in this laboratory.
Pig serum sample: 3868 parts of 2013-2017 pig clinical serum from Shandong, jiangsu, anhui, zhejiang, henan, shanxi, fujian, sichuan, hebei and Shanghai, which are 10 provinces and cities, are stored in a refrigerator at-20 ℃.
1.2 Gene Synthesis and primer design
1.2.1 Cap gene truncation
According to 642bp of the gene sequence of PCV3-US/MO2015 (GenBank: KX 778720.1) Cap, 214 amino acids are encoded, and a recombinant plasmid pMD19T-3Cap is synthesized by Nanjing Jinsri biotechnology Co., ltd as a template for gene amplification. The amino acid sequence of PCV 3Cap protein is analyzed for antigenicity and hydrophilicity by using protein software, and the result shows that the antigenicity and hydrophilicity of aa at 1-120 th and aa at 60-120 th are lower, and the target gene determines to delete the gene fragments expressing amino acid at 1-120 th and amino acid at 60-120 th in the Cap gene respectively, so as to obtain two target gene fragments. The PCV 3Cap gene after shortening the 1-120aa fragment is defined as 3 Cap/delta 1-120 gene, the PCV 3Cap gene after shortening the 60-120aa fragment is defined as 3 Cap/delta 60-120 gene, and the amino acid sequences obtained by expressing the two gene fragments are respectively the amino acid sequence of the PCV 3Cap protein shortened by 1-120aa and the amino acid sequence of the PCV 3Cap protein shortened by 60-120 aa.
1.2.2 primer design
The 3 Cap/delta 1-120 gene and the 3 Cap/delta 60-120 gene primers are designed according to the synthesized Cap gene template, two enzyme cutting sites of BamHI and XhoI are respectively added into the upstream and downstream primers, the 3 Cap/delta 1-120 gene is obtained by only one PCR amplification, and the 3 Cap/delta 60-120 gene is obtained by adding Linker (GGATCT) sequences into two gene fragments after 60-120aa is cut off and then connecting by using a fusion PCR method. The primer sequences are shown in Table 1.
TABLE 1 PCR primer sequences
Note that: delta 1-120-F represents the upstream primer of the 3 Cap/Delta 1-120 gene, delta 1-120-R represents the downstream primer of the 3 Cap/Delta 1-120 gene, delta 60-120F1 represents the upstream primer of the previous gene obtained by truncating 60-120aa fragment, delta 60-120R1 represents the downstream primer of the previous gene obtained by truncating 60-120aa fragment, delta 60-120F2 represents the upstream primer of the next gene obtained by truncating 60-120aa fragment, and Delta 60-120R2 represents the downstream primer of the next gene obtained by truncating 60-120aa fragment.
1.2 construction of recombinant expression plasmids
The truncated 3Cap genes, namely the 3 Cap/Delta1-120 gene and the 3 Cap/Delta60-120 gene, were amplified using pMD19T-3Cap as a template and the primers of Table 1 as primers. PCR amplification system: 2 XTaq Mix 12.5. Mu.L, pMD19T-3Cap plasmid 1. Mu.L, upstream and downstream primers 1. Mu.L each, ddH 2 O was made up to 25. Mu.L. PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; 95. denaturation at 30℃for 30s, annealing at 56℃for 30s, extension at 72℃for 50s,35 cycles; and at 72℃for 10min. The PCR products were identified by 1% agarose gel electrophoresis. The 3 Cap/delta 1-120 gene can be amplified in one step. The 3 Cap/delta 60-120 gene is obtained by amplifying the front and rear fragments 1 and 2 respectively, cutting gel to recover amplified products, and performing fusion PCR with the two fragments with equal molar ratio as templates and the upstream primer of the fragment 1 and the downstream primer of the fragment 2 to obtain the 3 Cap/delta 60-120 gene.
The PCR product is recovered and purified by agarose gel electrophoresis, connected with pMD19-T vector and expanded in E.coli DH5 alpha competent cells, bamHI and XhoI double enzyme cut positive T vector, and the target fragment is selected to be connected with pET-32 a. And (3) carrying out double enzyme digestion identification on the extracted recombinant plasmid, and carrying out agarose gel electrophoresis identification on enzyme digestion products. After identification of the correct plasmid was sent to biosystems for sequencing. The constructed recombinant plasmids are named pET-Cap/delta 1-120 and pET-Cap/delta 60-120 respectively.
1.3 Induction of expression of recombinant proteins
The recombinant plasmid is transformed into Rosseta competent cells, single colony is selected and inoculated into 5-10 mL LB liquid culture medium, and the culture is carried out at 37 ℃ overnight to obtain bacterial liquid. Inoculating the bacterial liquid into a new LB culture solution in a volume ratio of 1:100 for culture, and taking the bacterial liquid as OD 600 When the value reaches 0.6-0.8, adding IPTG with the final concentration of 1.0mmol/L, and culturing for 4 hours at 37 ℃ to obtain recombinant Rosseta, and simultaneously setting a recombinant Rosseta non-induction group and an empty vector Rosseta induction group as a control. After incubation, 12000g was centrifuged for 1min, the cells were collected, resuspended in 400. Mu.L PBS, the bacteria were sonicated, the supernatant was collected by centrifugation, and 100. Mu.L of 1 XSDS-PAGE and Western-blot were added for detection, wherein one antibody was a mouse anti-His-Tag-labeled monoclonal antibody (Abmart), incubated at room temperature for 2h, and the target protein expression was detected with ECL chemiluminescent solution.
1.4 purification of the protein of interest
Collecting a sample containing target protein, purifying the protein by using a nickel column affinity chromatography method, purifying and eluting by using imidazole solution diluted by a lysis buffer according to a gradient, carrying out SDS-PAGE identification on the product, analyzing the protein purification result by using quality one software, and calculating the purity of the protein.
1.5 PCV3 pig positive serum antibody preparation
1.5.1 immunization of pigs
Pigs negative for PCV3 antigen detection and antibody detection in serum were selected and immunized with purified 3Cap/Δ1-120 protein. The immunization method comprises the following steps: diluting the purified 3 Cap/delta 1-120 protein into 2mg/mL by PBS, mixing with an equal volume of ISA-206 adjuvant, fully emulsifying, and immunizing the pig with an immune dose of 1mL (1 mg)/head in a neck intramuscular injection mode. The immunization was repeated every 3 weeks, and the vena cava blood was collected every week to measure the antibody titer. Pig killing and blood taking are carried out when the titer is greater than 1:10000, the blood is placed at 37 ℃ for 30min and then is kept stand at 4 ℃ for 1h, separated serum is centrifuged at 4000rpm and 4 ℃ for 5min, and the supernatant is the prepared PCV3 polyclonal antibody (namely PCV3 pig positive serum antibody). And (5) sub-packaging and storing at-20 ℃.
1.5.2 PCV3 pig polyclonal antibody titer determination
The ELISA plate was coated with purified 3Cap/Δ1-120 protein (1. Mu.g/mL) as a coating antigen, incubated at 37℃for 2h, and PBST was washed 3 times (3 min. Times -1 The same applies hereinafter) and then 200. Mu.L/well of 5% skim milk was used, blocking was performed at 37℃for 2 hours, PBST was washed 3 times, the PCV3 polyclonal antibody prepared as described above was added, 100. Mu.L/well was allowed to act at 37℃for 1 hour, PBST was allowed to actWashing for 3 times, adding SPA-HRP (1:10000), 100 mu L/hole, acting at 37 ℃ for 45min, adding TMB chromogenic substrate after PBST washing for 3 times, adding stop solution after developing for 10min, and detecting OD450 value by an enzyme-labeled instrument. And (3) judging positive by S/N being more than or equal to 2.1, wherein the corresponding highest dilution is the serum titer.
1.6 establishment of an Indirect ELISA method
1.6.1 ELISA procedure
Diluting the purified Cap/delta 1-120 to 8 mug/mL by using CBS, then sequentially diluting to 4 mug/mL by double ratio, and coating the ELISA plate at 4 ℃ overnight after incubation for 2h at 37 ℃; the plate was washed 3 times with 0.05M PBS (PBST for short, pH 7.2) containing 0.05wt% Tween, 5min each time; after drying, adding PBST containing 5wt% of skim milk for sealing, 200 μl/hole, and at 37deg.C for 2 hr; PBST washing plates; adding positive serum and negative serum which are diluted by 1:100, 100 μl/well, and 37 ℃ for 1h; PBST washing plates; adding HRP-SPA diluted by 1:10000, 100 μl/well, and incubating at 37deg.C for 1 hr; PBST washing plates; adding TMB substrate reaction liquid, 100 μl/hole, and reacting at 37deg.C for 10min; add 2M H 2 SO 4 OD450 values were read after termination of the substrate reaction at 50. Mu.l/well. And (3) judging positive by the fact that the P/N is more than or equal to 2.1, wherein the corresponding highest dilution is the serum titer. Or calculating the S/P value according to the following formula, wherein the S/P= (sample value-negative value)/(positive value-negative value) is determined as follows: when S/P is more than or equal to 0.46, the S/P is less than 0.33, the S/P is negative, and the S/P are suspicious.
1.6.2 Selection of PCV3 antibody negative and positive serum
3 pig blood serum positive to PCV3 antigen PCR detection result stored in a laboratory is selected as a positive control, and one pig blood serum negative to PCV3 antigen PCR detection result stored in a laboratory is selected as a negative control.
1.6.3 optimization of reaction conditions for an Indirect ELISA method
Selection of optimal coating concentration of 1.6.3.1 antigen
The antigen was coated with 0.05mol/L carbonate buffer (CBS, pH 9.6) at antigen concentrations of 0.25. Mu.g/mL, 0.5. Mu.g/mL, 1.0. Mu.g/mL, 2.0. Mu.g/mL, 4.0. Mu.g/mL, 8.0. Mu.g/mL, respectively, in the same manner as 1.6.1, with the highest P/N antigen concentration being the optimal antigen coating concentration.
Selection of 1.6.3.2 optimal antigen coating time
The ELISA plate is coated with the determined optimal antigen coating concentration, the coating method is respectively carried out at 37 ℃ for 2h, at 4 ℃ overnight, at 37 ℃ for 2h and at 4 ℃ overnight, and the other steps are the same as 1.6.1, and the coating time with the maximum P/N value is the optimal antigen coating time.
Selection of 1.6.3.3 optimal sealing liquid and sealing time
The ELISA plate is coated by an optimized coating method, 1% BSA,5% skim milk and 2% gelatin are used for sealing respectively, the rest conditions are the same as 1.6.1, and the sealing liquid with the maximum P/N value is taken as the optimal sealing liquid. And (3) sealing for 1h,2h and 3h at 37 ℃ respectively by using the optimal sealing liquid, and determining the optimal sealing time.
Selection of best serum dilution and time of action of 1.6.3.4
The negative and positive serum is diluted by 1:50, 1:100, 1:200 and 1:400 respectively, an indirect ELISA experiment is carried out, the rest conditions are the same as 1.6.1, the OD450nm value and the P/N value of each group of serum are compared, and the maximum dilution of P/N is the optimal dilution of serum. After diluting the serum with the optimal dilution, the serum is respectively acted for 0.5h, 1h, 1.5h and 2h at 37 ℃, and the maximum time of P/N is the optimal serum reaction time.
Selection of best dilution and time of action of 1.6.3.5 enzyme-labeled antibody
The enzyme labels SPA-HRP are respectively diluted 1:10000,1:15000,1:20000 and 1:30000, the rest conditions are the same as 1.6.1, the OD value and the P/N value of each group of serum are compared, and the maximum dilution of the P/N is the optimal dilution of the enzyme label. After the enzyme label is diluted according to the optimal dilution, the enzyme label acts for 0.5h, 1h and 1.5h at 37 ℃ respectively, and the maximum acting time of P/N is the acting time of the optimal enzyme label.
Selection of 1.6.3.6 optimal substrate reaction time
ELISA experiments were performed under the optimized conditions described above, with substrate reactions at 37℃for 5min,10min,15min,20min, 2mol/L H, respectively 2 SO 4 The substrate reaction was stopped, the OD values and P/N values of each group were compared, and the time of maximum P/N was selected as the optimal substrate reaction time.
Determination of 1.6.4 threshold
Detection 30Sub-standard negative and positive serum to obtain negative highest OD450N max =0.23, mean value
Positive lowest P min =0.93, mean>
Therefore, the condition for the establishment of the experiment is C P ≥0.93，C N ≤0.23。
120 clinical test S/N <2.1 serum was randomly picked as negative serum. The S/P values thereof are calculated respectively,
that is, S/p= (sample value-0.15)/(1.01-0.15), the average value x=0.080 of S/P was obtained, the standard deviation sd=0.127, and the positive threshold ∈according to the statistical law>
1.6.5 specificity test
One serum of PRRSV, PRV, PCV, CSFV, HPS, EMCV, FMDV positive for PCV3 antibody and one serum of negative for PCV3 antibody were selected and detected by ELISA method after optimization of the above conditions.
1.6.6 sensitivity experiments
Yin-yang serum in 1.6.2 was used, 1:100 dilution is followed by double dilution to 1:12600, ELSIA detection is performed, and the highest dilution factor with positive detection result is sensitivity of ELISA method.
1.6.7 repeatability test
1.6.7.1 in-batch repeat test
4 parts of PCV3 antibody positive clinical serum and 2 parts of PCV3 antibody negative serum are selected, ELISA experiments are carried out by using the same batch of purified 3 Cap/delta 1-120 protein coating strips, 3 replicates are carried out on each serum, and the average value, the standard deviation and the variation coefficient are calculated.
1.6.7.2 repeated test between batches
4 clinical sera positive for PCV3 antibodies and 2 negative sera were selected and ELISA assays were performed using three different batches of purified 3 Cap/Delta1-120 protein coated strips, 3 replicates per serum, and the mean, standard deviation, and coefficient of variation were calculated.
1.6.8 Epidemiological investigation of PCV3 antibodies
The detection of PCV3 antibodies was performed on 3868 sera of 2013-2017 stored in the present laboratory using an optimized indirect ELISA method. These serum samples were from 10 provinces in Shandong, jiangsu, anhui, zhejiang, henan, shanxi, fujian, sichuan, hebei and Shanghai.
The optimized indirect ELISA method comprises the following steps: diluting the purified Cap/delta 1-120 to 8 mug/mL by using CBS, then sequentially diluting to 4 mug/mL by double ratio, and coating the ELISA plate at 4 ℃ overnight after incubation for 2h at 37 ℃; the plate was washed 3 times with 0.05M PBS (PBST for short, pH 7.2) containing 0.05wt% Tween, 5min each time; after drying, adding PBST containing 5wt% of skim milk for sealing, 200 μl/hole, and 3h at 37deg.C; PBST washing plates; adding 1:100 diluted serum to be tested, 100 μl/well, 37 ℃ for 1.5h; PBST washing plates; adding HRP-SPA diluted by 1:10000, 100 μl/well, and incubating at 37deg.C for 1.5h; PBST washing plates; adding TMB substrate reaction liquid, 100 μl/hole, and reacting at 37deg.C for 10min; add 2M H 2 SO 4 OD450 values were read after termination of the substrate reaction at 50. Mu.l/well. Meanwhile, a positive control and a negative control were set.
2 results
2.1 Preparation and identification of 3Cap recombinant protein
2.1.1 construction of recombinant plasmid and expression of Gene of interest
Extracting recombinant plasmid, and carrying out gene sequencing identification after double enzyme digestion identification to prove that the recombinant plasmid contains target genes. The recombinant plasmids were designated pET-Cap/Delta1-120 and pET-Cap/Delta60-120. The recombinant plasmid was transformed into Rosseta competent cells for induction expression, and SDS-PAGE results showed (FIG. 1): the size of the target protein expressed by pET-Cap/delta 1-120 recombinant Rosseta is about 38kDa (defined as 3 Cap/delta 1-120 recombinant protein, which is abbreviated as 3 Cap/delta 1-120 protein), the size of the target protein expressed by pET-Cap/delta 60-120 recombinant Rosseta is about 48kDa (defined as 3 Cap/delta 60-120 recombinant protein, which is abbreviated as 3 Cap/delta 60-120 protein), and the expression forms are all soluble expression according to the expectations. Western Blot results show that the two recombinant proteins can react with PCV3 serum antibodies (Nanjing Boquan) and have clear specific bands, which indicates that the two recombinant proteins are successfully expressed, wherein pET-Cap/delta 1-120 recombinant Rosseta has higher expression efficiency and is used for subsequent further research.
2.1.2 purification of recombinant proteins
Selecting the 3 Cap/delta 1-120 recombinant protein with high expression of pET-Cap/delta 1-120, purifying by adopting an affinity chromatography method, and optimizing the imidazole concentration of a binding buffer solution, a washing solution and an eluent, wherein the result is as follows: the 300mM imidazole wash can remove the impurity protein better, and the 500mM imidazole eluate has the best effect on eluting the target protein (FIG. 2). And analyzing the purified protein electrophoresis pattern by using Quantity one software, and calculating to obtain the purified protein with the purity of 93%.
2.1.3 recombinant protein antigenicity
PCV3 pig polyclonal serum antibodies were prepared from 3 Cap/delta 1-120 recombinant protein immunized pigs and the results of the indirect ELISA assays are shown in Table 2. When S/N is more than or equal to 2.1, the antibody titer is more than 1:12800.
TABLE 2 determination of PCV3 pig polyclonal antibody titers
2.2 establishment of an Indirect ELISA method
2.2.1 optimization of Indirect ELISA reaction conditions
2.2.1.1 antigen coating concentration and coating time
The purified Cap/delta 1-120 recombinant protein is diluted to 8 mug/mL by CBS, and then diluted to 0.25 mug/mL by double ratio in turn, and 4 ℃ after 2h at 37 DEG CCoating overnight, washing with PBST washing solution for 3 times each for 5min, adding 5% skim milk sealing solution, sealing at 37deg.C for 2 hr, and removing sealing solution; adding pig serum diluted by 1:100, 100 mu L/hole, setting PCV3 positive serum and negative serum control, 100 mu L/hole, and acting at 37 ℃ for 1h; washing 3 times according to the same method; adding 1:10000 diluted SPA-HRP enzyme label, 100 mu L/hole, and reacting at 37 ℃ for 1h; washing 3 times according to the same method; adding TMB substrate reaction solution (equal volume of solution A and solution B, mixing well), and allowing reaction at 37deg.C for 10min at 100 μl/hole; adding stop solution 2M H 2 SO 4 50. Mu.L/well; the OD450nm value was determined. The results are shown in Table 3. The maximum P/N value at an antigen concentration of 4. Mu.g/mL was determined as the optimal antigen coating concentration.
TABLE 3 optimization of antigen coating concentration
ELISA was performed by coating the ELISA plate with a protein concentration of 4. Mu.g/mL for 2h at 37℃overnight at 4℃and overnight at 4℃after 2h at 37℃with the remainder of the procedure. The results are shown in Table 4. The antigen coating time with the maximum P/N value is selected as the optimal antigen coating time after 2 hours at 37 ℃ and overnight at 4 ℃.
TABLE 4 optimization of antigen coating time
2.2.1.2 selection of blocking liquid and blocking time
The ELISA plate was coated by the optimized coating method, blocked with 1% BSA,5% skim milk and 2% gelatin, respectively, and the remaining conditions were the same as 1.6.1, and the results are shown in Table 5. It can be seen that 5% skim milk is the best confining liquid.
TABLE 5 blocking fluid selection
According to the optimized antigen coating concentration and coating time, 5% skim milk is selected to be respectively sealed for 1h,2h and 3h at 37 ℃ to determine the optimal sealing time. The results are shown in Table 6, and the P/N was maximum when the cells were blocked at 37℃for 3 hours, and the optimal blocking time was determined.
TABLE 6 seal time optimization
2.2.1.3 serum dilutions and time of action
The ELISA plate was coated using an optimized procedure and the negative and positive sera were diluted 1:50, 1:100, 1:200 and 1:400, respectively, for ELSIA assay. The results are shown in Table 7, and a 1:100 ratio is determined as the optimal dilution ratio.
TABLE 7 serum dilution optimization
Serum duration was optimized and the rest was the same. The results are shown in Table 8. It can be seen that an effect of 1.5h at 37℃is the optimal serum duration.
TABLE 8 serum action time optimization
2.2.1.4 dilution of enzyme-labeled antibody and time of action
The enzyme-labeled secondary antibodies were diluted 1:10000,1:15000,1:20000 and 1:30000, respectively, and the serum OD values and P/N values of each group were compared, and the results are shown in Table 9. And the P/N is the largest when the enzyme-labeled antibody is diluted 1:10000, and the optimal dilution multiple of the enzyme-labeled antibody is determined.
TABLE 9 dilution optimization of enzyme-labeled antibodies
The enzyme-labeled antibodies were set to have the action time of 0.5h, 1h, and 1.5h, respectively, and ELSIA test was performed, and the results are shown in Table 10, and the enzyme-labeled antibodies were set to have the action time of 1.5h P/N maximum, and were determined to be the optimal action time.
Table 10 time optimization of enzyme-labeled antibodies
2.2.1.5 substrate reaction time
ELISA experiments were performed according to the above-described optimization method, and four substrate reaction times of 5min,10min,15min and 20min were set. The results are shown in Table 11, and the optimal substrate reaction time was determined to be 10min.
TABLE 11 optimization of substrate action time
2.2.2 determination of the negative-positive threshold
Detecting 30 times standard negative and positive serum to obtain negative highest OD450N max =0.23, mean value
Positive lowest P min =0.93, mean>
Therefore, the condition for the establishment of the experiment is C P ≥0.93，C N ≤0.23。
120 clinical test S/N <2.1 serum was randomly picked as negative serum. The S/P values thereof are calculated respectively,
namely S/P= (sample value-0.15)/(1.01-0.15), and the average value of S/P was obtained>
Standard deviation sd=0.127, positive critical ++according to statistical law>
Thus, the criteria for the ELISA method were: when S/P is more than or equal to 0.46, the S/P is less than 0.33, the S/P is negative, and the S/P are suspicious.
2.2.3 specificity assays
The results of ELISA assay for PRRSV, PRV, PCV, CSFV, HPS, EMCV, FMDV positive serum and PCV3 negative and positive serum are shown in the table. The ELISA method established only reacts with PCV3 antibodies, has no cross reactivity with antibodies of other pathogens, and has good specificity.
TABLE 12 specificity test
2.2.4 sensitivity test
The results of the test after double dilution of standard negative and positive serum are shown in Table 13, with S/N >2.1 at 1:3200 dilution and S/N <2.1 at 1:6400 dilution, and the sensitivity of ELISA method is 1:3200.
TABLE 13 sensitivity test
| 1:100 | 1:200 | 1:400 | 1:800 | 1:1600 | 1:3200 | 1:6400 | 1:12800 | |
| S | 1.5 | 1.33 | 1.18 | 0.82 | 0.55 | 0.33 | 0.14 | 0.11 |
| N | 0.14 | 0.08 | 0.08 | 0.08 | 0.07 | 0.08 | 0.07 | 0.07 |
| S/N | 10.71 | 16.63 | 14.75 | 10.25 | 7.86 | 4.13 | 2 | 1.57 |
2.2.5 repeatability test
2.2.5.1 in-batch repeat test
4 positive and 2 negative sera were selected and three replicates were made for each serum. The Average Value (AV), standard Deviation (SD) and Coefficient of Variation (CV) of OD450 were calculated, and the results are shown in table 14. The mean value of the variation coefficient is 3.7%, which shows that the ELISA method has better in-batch repeatability.
TABLE 14 in-batch repeat test
2.2.5.2 repeated test between batches
ELISA experiments were performed using three batches of purified 3 Cap/Delta1-120 recombinant protein coated ELISA plates, with 4 positive and 2 negative sera, and OD450 AV, SD and CV were calculated, and the results are shown in Table 15. The mean value of the variation coefficient was found to be 4.2%, which indicates that the ELISA method has good batch-to-batch reproducibility.
TABLE 15 in-batch repeat test
2.3 serum epidemiological investigation
By adopting the ELISA method with optimized conditions, 3868 pig clinical serum PCV3 antibodies are detected, the positive rate of the antibodies in the past year is counted, and the distribution of the antibodies in different seasons, regions and pig groups is counted.
2.3.1 results of different year PCV3 antibody detection
The statistical results are shown in Table 16. As can be seen from the table, the PCV3 antibody positive rate increased from 7.5% in 2013 to 75.9% in 2017, with the overall increase in antibody positive rate. The average positive rate over the years is 34.4%.
TABLE 16 results of PCV3 antibody detection at different years
| Time | Number of samples | Positive number | Positive rate |
| 2013 | 745 | 56 | 7.5％ |
| 2014 | 704 | 61 | 8.7％ |
| 2015 | 811 | 319 | 39.3％ |
| 2016 | 614 | 215 | 35.0％ |
| 2017 | 812 | 616 | 75.9％ |
| Totals to | 3686 | 1267 | 34.4％ |
2.3.2 detection results of different Swine antibodies
The results of statistics on the positive rate of the antibodies in sows, replacement pigs, boars, fattening pigs, nursery pigs and suckling pigs are shown in Table 17, the positive rate of the antibodies in sows, boars and fattening pigs is higher and can reach 43%, and the positive rate of the antibody in the suckling pigs is the lowest and is 10.2%. Indicating that the antibody positive rate has a certain relation with the age of the day.
TABLE 17 detection results of PCV3 antibodies from different pig groups
| Pig farm | Number of samples | Positive number | Positive rate |
| Sow | 1272 | 489 | 38.4％ |
| Backup pig | 429 | 127 | 29.6％ |
| Boar (boar) | 279 | 120 | 43.0％ |
| Fattening pig | 750 | 289 | 38.5％ |
| Nursery pig | 759 | 222 | 29.2％ |
| Suckling piglet | 197 | 20 | 10.2％ |
| Totals to | 3686 | 1267 | 34.4％ |
2.3.3 detection results of antibodies in different regions
Serum samples of different areas are respectively counted, the result is shown in table 18, the highest positive rate of the south China area reaches 81.8%, the second positive rate of the south China area is 46.0%, and the lowest positive rate of the north China area is 6.0%.
TABLE 18 detection results of PCV3 antibodies in different regions
| Region of | Number of samples | Positive number | Positive rate |
| North China | 133 | 8 | 6.0％ |
| East China | 3139 | 1037 | 33.0％ |
| Huazhong (Chinese style) | 326 | 150 | 46.0 |
| South China | |||
| 88 | 72 | 81.8％ | |
| Totals to | 3686 | 1267 | 34.4％ |
2.3.4 results of antibody detection in different seasons
The results of the seasonal distribution statistics of the annual samples are shown in Table 19, and the higher positive rates in summer and autumn, which indicate that the PCV3 distribution may have a certain seasonal property.
TABLE 19 detection results of PCV3 antibodies in different regions
| Season | Number of samples | Positive number | Positive rate |
| Spring of spring | 1125 | 357 | 31.7％ |
| (Summer) | 171 | 67 | 39.2％ |
| Autumn when it is | 1736 | 778 | 44.8％ |
| Winter season | 654 | 65 | 9.9％ |
| Totals to | 3686 | 1267 | 34.4％ |
Sequence listing
<110> Nanjing agricultural university
<120> porcine circovirus 3-type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 297
<212> DNA
<213> Artificial Sequence
<400> 1
ggatccctcc aagacgaccc ttatgcggaa agttccactc gtaaagttat gacttctaaa 60
aaaaaacaca gccgttactt caccccgaaa ccaattctgg cgggaactac cagcgctcac 120
ccaggacaaa gcctcttctt tttctccaga cccaccccat ggctcaacac atatgacccc 180
accgttcaat ggggagcact gctttggagc atttatgtcc cggaaaaaac tggaatgaca 240
gacttctacg gcacgaaaga agtttggatt cgttacaagt ccgttctcta actcgag 297
<210> 2
<211> 480
<212> DNA
<213> Artificial Sequence
<400> 2
ggatccatga gacacagagc tatattcaga agaagacccc gcccaaggag acgacgacgc 60
cacagaaggc gctatgccag aagaagacta ttcattagga ggcccacagc tggcacatac 120
tacacaaaga aatactccac catgaacgtc atttccgttg gaactcctca gaataataag 180
cccggatctc tccaagacga cccttatgcg gaaagttcca ctcgtaaagt tatgacttct 240
aaaaaaaaac acagccgtta cttcaccccg aaaccaattc tggcgggaac taccagcgct 300
cacccaggac aaagcctctt ctttttctcc agacccaccc catggctcaa cacatatgac 360
cccaccgttc aatggggagc actgctttgg agcatttatg tcccggaaaa aactggaatg 420
acagacttct acggcacgaa agaagtttgg attcgttaca agtccgttct ctaactcgag 480
<210> 3
<211> 94
<212> PRT
<213> Artificial Sequence
<400> 3
Leu Gln Asp Asp Pro Tyr Ala Glu Ser Ser Thr Arg Lys Val Met Thr
1 5 10 15
Ser Lys Lys Lys His Ser Arg Tyr Phe Thr Pro Lys Pro Ile Leu Ala
20 25 30
Gly Thr Thr Ser Ala His Pro Gly Gln Ser Leu Phe Phe Phe Ser Arg
35 40 45
Pro Thr Pro Trp Leu Asn Thr Tyr Asp Pro Thr Val Gln Trp Gly Ala
50 55 60
Leu Leu Trp Ser Ile Tyr Val Pro Glu Lys Thr Gly Met Thr Asp Phe
65 70 75 80
Tyr Gly Thr Lys Glu Val Trp Ile Arg Tyr Lys Ser Val Leu
85 90
<210> 4
<211> 155
<212> PRT
<213> Artificial Sequence
<400> 4
Met Arg His Arg Ala Ile Phe Arg Arg Arg Pro Arg Pro Arg Arg Arg
1 5 10 15
Arg Arg His Arg Arg Arg Tyr Ala Arg Arg Arg Leu Phe Ile Arg Arg
20 25 30
Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val
35 40 45
Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Gly Ser Leu Gln Asp
50 55 60
Asp Pro Tyr Ala Glu Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys
65 70 75 80
Lys His Ser Arg Tyr Phe Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr
85 90 95
Ser Ala His Pro Gly Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro
100 105 110
Trp Leu Asn Thr Tyr Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp
115 120 125
Ser Ile Tyr Val Pro Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr
130 135 140
Lys Glu Val Trp Ile Arg Tyr Lys Ser Val Leu
145 150 155
Claims (7)
1. A coding gene of a porcine circovirus 3 type Cap recombinant protein is characterized in that: the sequence is shown as SEQ ID NO. 2.
2. A recombinant plasmid, characterized in that: is obtained by recombining the encoding gene of the porcine circovirus 3 type Cap recombinant protein and a pET32a vector.
3. A porcine circovirus 3 type Cap recombinant protein is characterized in that: expressed from the recombinant plasmid of claim 2.
4. The porcine circovirus type 3Cap recombinant protein according to claim 3, wherein the recombinant protein is characterized by: the porcine circovirus type 3Cap recombinant protein is obtained by transforming the recombinant plasmid of claim 2 into Rosseta competent cells and then performing IPTG induction expression.
5. The porcine circovirus type 3Cap recombinant protein according to claim 3, wherein the recombinant protein is characterized by: the amino acid sequence is shown as SEQ ID NO. 4.
6. A PCV3 indirect ELISA antibody detection kit is characterized in that: an enzyme-labeled plate comprising a Cap recombinant protein coated with the porcine circovirus 3 of claim 3, 4 or 5.
7. An indirect ELISA antibody detection method of PCV3 is characterized in that: comprising the step of coating an ELISA plate with purified porcine circovirus type 3Cap recombinant protein according to claim 3, 4 or 5, wherein the detection method is aimed at diagnosis of a non-disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111422353.7A CN114014916B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111422353.7A CN114014916B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection |
| CN201910046124.6A CN109762052B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus type 3 Cap recombinant protein and coding gene and application thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910046124.6A Division CN109762052B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus type 3 Cap recombinant protein and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114014916A CN114014916A (en) | 2022-02-08 |
| CN114014916B true CN114014916B (en) | 2023-04-25 |
Family
ID=66454056
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910046124.6A Active CN109762052B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus type 3 Cap recombinant protein and coding gene and application thereof |
| CN202111422353.7A Active CN114014916B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910046124.6A Active CN109762052B (en) | 2019-01-18 | 2019-01-18 | Porcine circovirus type 3 Cap recombinant protein and coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109762052B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114814211B (en) * | 2022-04-28 | 2024-07-23 | 山东信得科技股份有限公司 | ELISA kit for detecting PCV3 |
| CN115819567B (en) * | 2022-11-19 | 2023-09-01 | 深圳盛源生物技术有限公司 | Monoclonal antibody and application thereof in detection kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
| CN108431024A (en) * | 2015-10-16 | 2018-08-21 | 堪萨斯州立大学研究基金会 | 3 type circovirus immunogenic composition of pig and its preparation and application |
| CN108627648A (en) * | 2017-03-24 | 2018-10-09 | 华南农业大学 | A kind of ELISA antibody assay kits of 3 type of novel pig circular ring virus and its application |
| CN109207441A (en) * | 2018-08-12 | 2019-01-15 | 扬州大学 | 3 type Cap protein of recombinant baculovirus expression pig circular ring virus and its construction method and primer |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108619503A (en) * | 2017-03-24 | 2018-10-09 | 华南农业大学 | A kind of pig circular ring virus genetic engineering subunit vaccine and the preparation method and application thereof |
| CN109125719B (en) * | 2017-06-19 | 2022-06-14 | 普莱柯生物工程股份有限公司 | Immunogenic composition containing porcine circovirus type 3 and porcine circovirus type 2 antigens and application thereof |
| CN108823231B (en) * | 2018-07-09 | 2022-05-31 | 荣俊 | Porcine circovirus type 3 genetic engineering subunit vaccine and preparation method thereof |
| CN109207522B (en) * | 2018-08-12 | 2022-05-27 | 扬州大学 | Recombinant baculovirus expressing truncated Cap protein of porcine circovirus type 3 and construction method and primer thereof |
-
2019
- 2019-01-18 CN CN201910046124.6A patent/CN109762052B/en active Active
- 2019-01-18 CN CN202111422353.7A patent/CN114014916B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108431024A (en) * | 2015-10-16 | 2018-08-21 | 堪萨斯州立大学研究基金会 | 3 type circovirus immunogenic composition of pig and its preparation and application |
| CN108627648A (en) * | 2017-03-24 | 2018-10-09 | 华南农业大学 | A kind of ELISA antibody assay kits of 3 type of novel pig circular ring virus and its application |
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
| CN109207441A (en) * | 2018-08-12 | 2019-01-15 | 扬州大学 | 3 type Cap protein of recombinant baculovirus expression pig circular ring virus and its construction method and primer |
Non-Patent Citations (2)
| Title |
|---|
| GenBank: ARD71120.1.capsid protein, partial [Porcine circovirus 3].GenBank.2017,全文. * |
| 王俊伟等.基于 ＰＣＶ３－Ｃａｐ蛋白间接 ＥＬＩＳＡ 检测方法的建立及临床应用.畜牧兽医学报 .2019,第50卷(第2期),454-460. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109762052A (en) | 2019-05-17 |
| CN114014916A (en) | 2022-02-08 |
| CN109762052B (en) | 2022-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110105436B (en) | ELISA detection kit for porcine circovirus type 3 antibody and preparation method and application thereof | |
| CN114230660B (en) | Monoclonal antibody for resisting micropterus salmoides iridovirus LMBV and application thereof | |
| CN112980802B (en) | Hybridoma cell secreting novel duck reovirus sigma B protein monoclonal antibody, monoclonal antibody and application | |
| CN112877348B (en) | African swine fever virus CD2v extracellular domain recombinant protein and application thereof | |
| CN114014916B (en) | Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection | |
| CN113684189A (en) | Novel chicken circovirus type 3 strain and detection system based on same | |
| CN108559754B (en) | Porcine reproductive and respiratory syndrome virus GP5 gene modified recombinant protein and application thereof | |
| CN107011417B (en) | Recombinant protein, encoding gene and application thereof, and detection kit and detection method for porcine epidemic diarrhea virus antibody | |
| CN110887963B (en) | PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof | |
| CN116333060B (en) | Porcine reproductive and respiratory syndrome virus GP5 protein, preparation method and application thereof, gene, kit and detection method | |
| US12097255B2 (en) | Recombinant porcine parvovirus antigenic protein and use thereof | |
| CN110376388B (en) | Haemophilus parasuis antibody detection method and kit thereof | |
| CN109535229B (en) | Recombinant porcine circovirus type 2Rep protein, ELISA detection kit, preparation method and application thereof | |
| CN116478302A (en) | Variant pseudorabies virus gBgCgD epitope concatemer fusion protein and application thereof | |
| CN115960250A (en) | Recombinant porcine circovirus type 3 trimer protein and preparation method and application thereof | |
| CN110698542B (en) | Artificially-modified porcine circovirus type 2 Rep' protein, ELISA (enzyme-linked immuno sorbent assay) detection kit and application thereof | |
| CN107446041B (en) | Antiserum for resisting apple necrosis mosaic virus and preparation method thereof | |
| CN114002426A (en) | Hog cholera virus E0 protein antibody ELISA detection kit | |
| Longyant et al. | Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV) | |
| CN115028688B (en) | PCV3 Cap protein antigen peptide, antibody and PCV3 detection immunohistochemical kit | |
| CN115894718B (en) | Antigen epitope peptide of African swine fever virus and application thereof | |
| CN117683127B (en) | ELISA detection kit for capturing porcine epidemic diarrhea virus antibody | |
| CN112321707B (en) | Specificity recognition antibody and detection kit of machupo virus | |
| Zheng et al. | Expression and antigenic characterization of the epitope-G 1 of the Bovine ephemeral fever virus glycoprotein in Pichia pastoris | |
| CN118813550A (en) | African swine fever virus P22 protein antibody and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |