Development Of Monoclonal Antibodies Specific To Cap Protein Of Porcine Circorvirus Type 2d And Identification Of The Epitope Recognized By MAbs

Porcine circovirus type 2(PCV2)is etiology of porcine circovirus associated diseases(PCVAD)in recent years,resulting in immunosuppression in swine herds,causing PCVAD outbreaks all over the world.At present,the PCV2b and PCV2d are the main epidemic genotypes in pigs,while there is a growing trend of PCV2d in pigs,implying that PCV2d tends to be a prevalent genotype surpassing current PCV2b in the infected pig herds.Now,antibodies against PCV2 subjected to PCV2 detection or PCVAD diagnosis are available usually specific to either PCV2b or PCV2a,so it is necessary to develop robust antibodies suitable to identify and detect PCV2d strains.The development of monoclonal antibody(MAb)specific to PCV2d is of great significance for the diagnosis and serological epidemiology surveinance of PCVAD.In this study,a pair of primers were designed based on the capsid protein encoding gene(orf2)of porcine circovirus genotype 2d(PCV2d/Changzhou/JS/20150728CZ),and the orf2 gene without nuclear localization signal peptide encoding sequence was amplified by PCR technology,and the amplicon was inserted into the prokaryotic expression plasmid pET28a to construct the recombinant expression plasmid pET28a-orf2.Recombinant Cap protein was expressed in E.coli strain BL21(DE3)transformed with plasmid pET28a-orf2 induced by isopropy-?-D-thiogalactoside(IPTG).SDS-Polyacrylamide gel electrophoresis(SDS-PAGE)and Western-blot detection confirmed that the recombinant Cap protein was properly expressed,and the soluble expression form made up the majority of the total expressed proteins under the optimized culture and induction conditions.The recombinant Cap protein was designated as His-rCapThe His-rCap recombinant protein was purified by Ni-NTA affinity chromatography and administered to 6-week-old female Balb/c mice.After three inoculations,the spleen cells of the immunized mice were fused with myeloma cells SP2/0,and titers of antibody specific to Cap protein in the supernatant of hybridoma were detected by an indirect enzyme-linked immunosorbent assay(iELISA)and indirect immunofluorescence assay(IFA).After three serial subclonings,four hybridomas stably secreting antibody specific to Cap protein were obtained,namely 1D9,2C3,3F9 and 4D11 respectively.The specificity of the four monoclal antibodies(MAbs)was identified by Western-blot.The results of IFA showed that the two MAbs 1D9,3F9 and 4D11 reacted with both PCV2d and PCV2b strains,1D9,3F9 seemed to react with PCV2d strain stronger than PCV2b strain,and 4D11 seemed to react with PCV2b strain stronger than PCV2d strain,while MAb 2C3 reacted with neither PCV2d nor PCV2b strain in IFA.The developed MAbs might be robust antibodies for identification of currently prevalent PCV2d and PCV2b genotype strains,and also serological epidemiology surveinance of PCVAD.In this study,to stepwisely truncate the orf2 gene of PCV2d strain,thirteen pairs of specific primers were designed to amplify orf2 fragments with different sizes,and construct a serial of recombinant plasmids with truncated orf2 fragments.A serial of truncated Cap proteins were expressed in Escherichia coli BL21(DE3)induced by IPTG at 37?.MAbs 1D9,2C3,3F9and 4D11 were employed to recongnize the antigenic epitopes in truncated Cap proteins by Western blot.The results suggested that MAbs 1D9 and 3F9 recognized 75NIND78 epitopeof Cap protein,Mab 2C3 recognized 90TVPFEY95 epitope of Cap protein,Mab 4D11 recognized 225NLKDPPLNP233 epitope of Cap protein.The virus neutralization assay showed that none of the four MAbs exhibited neutralizing activity.