Cloning And Expression Of P32 Gene Of Sheep Poxvirus And Its Preparation Of Monoclonal Antibody

The genus Capripoxvirus includes Sheep poxvirus(SPPV),Goat poxvirus(GTPV)and Lumpy Skin Disease virus(LSDV).LSDV generally affects cattle and occurs in African most countries and Middle East.In contrast,SPPV and GTPV affect both sheep and goat,and mainly occur in Africa,the Middle East and Asia.The morbidity and mortality caused by the infection of SPPV can reach to 100%.Although several methods for detection of SPPV have been developed,the commercial and specific diagnosis techniques for the SPPV is not available.As a major structural protein,the envelope protein P32 of SPPV not only plays viral roles in the pathogenicity of SPPV,but also induces efficient humoral and cellular immunity against SPPV.Notably,the P32 protein derived from the genus Capripoxvirus generally carries species-specific B cell epitopes.Therefore,P32 protein can be as an efficient target for developing specific diagnosis for detection of SPPV.In this study,the p32 gene of the SPPV was cloned and expressed using the eukaryotic and prokaryotic vector pGEX-6p-1 and pcDNA3.1 respectively.The polyclonal antibodies against the P32 was prepared by immunizing the BALB/c mice with the purified GST-P32 and the monoclonal antibodies against P32 were generated by screening using the peptide-based ELISA.The preparation of the P32 fusion protein,peptide-based ELISA and the monoclonal antibodies against P32 pave the way for further developing the specific diagnosis for the detection of SPPV.1.Cloning and expression p32 gene of sheep poxvirusTo construct the prokaryotic and eukaryotic vectors for p32,the synthesized p32 gene and its truncations were cloned into the vector pGEX-6p-1 and pcDNA3.1,and the recombinant plasmids were designated as pGEX-6p-1-P32 and pcDNA3.1-P32,respectively.The recombinant pGEX-6p-I-P32 transformed into the Rosetta competent cells and induced by 1M IPTG.SDS-PAGE and Western-blot analysis revealed that the GST-P32 fusion protein could be efficiently expressed in the form of inclusion bodies and soluble forms.The GST-P32 proteins expressed in the soluble form were then purified by GST agarose gel column,and immunized into BALB/c mice for preparing the polyclonal antibody against P32.Indirect immunofluorescence assay and Western blot showed that the polyclonal antibody against P32 generated could efficiently react not only with the GST-P32 fusion protein,but also with the cell transfected with the eukaryotic recombinant plasmid pcDNA3.1-P32,highlighting the good antigenicity of the GST-P32 fusion protein expressed in E.coli.All these expressing vectors for p32 constructed and the polyclonal antibody against P32 protein generated provide materials for further generating monoclonal antibodies against P32.2.Development of monoclonal antibodies against P32 protein of sheep poxvirusTo screen the monoclonal antibody against P32 of SPPV,a peptide-based ELISA for detection of antibodies against P32 was first developed.Total six peptides(P32-P1,P32-P2,P32-P3,P32-P4,P32-P5 and P32-P6)derived from P32 protein of SPPV were selected based on the analysis by Protean Software(DNAStar),and synthesized.ELISA detection using mice polyclonal antibody against P32 revealed that peptide P32-P1 and P32-P4 showed strong reaction with the mice sera against P32 with high OD450 values in the ELISA.Therefore,peptides P32-PI and P32-P4 were selected as coating antigen in ELISA for screening monoclonal antibody against P32.After the fusion of the myeloma cells SP2/0 with the spleen cells of the mice immunized with the GST-P32 fusion protein,the hybridoma cells secreting antibody against P32 were screened by the established peptide-based ELISA.Through screening and subcloning,two positive hybridoma cells secreting antibody specific to P32 protein were generated,designated as mAb 1D5 and mAb 3A6 respectively.mAb1D5 and mAb 3A6 not only showed ELISA reaction,but also recognized the P32 protein expressed in DF-1 through IFA.The establishment of the peptide-based ELISA for detection of antibody against P32 and the development of the monoclonal antibody against P32 pave the way for further generating efficient and specific diagnosis for the detection of SPPV.