Prokaryotic expression of porcine circovirus virus type 2 ORF9 protein and preparation of its polyclonalantibodies
In order to identify and further investigate the role of the ORF9 protein in porcine circovirus type 2 infection,a murine polyclonal antibody against ORF9 protein was prepared and carried out preliminary application.First,the ORF9 gene was cloned and the ORF9 protein prokaryotic expression plasmid pET-32a(+)-PCV2-ORF9 was constructed and transformed into Transetta(DE3)expression strains.The optimum concentration of IPTG induction temperature and induction time of the ORF9 expression were investigated by SDS-PAGE method.BALB/c mice were immunized with the target protein after purification and renatu-ration.The titer of polyclonal antibody against ORF9 was determined by ELISA,and the specificity of polyclonal antibody against ORF9 was studied by IFA and Western-blot.The results showed that the recom-binant plasmid pET-32a(+)-PCV2-ORF9 is constructed successfully.The optimum IPTG concentration,temperature and induction time of ORF9 protein expression were 0.1 mmol/L,37 ℃ and 4 h,respectively,and it was mainly expressed in the form of inclusion bodies with molecular weight of about 24.2 ku.The antibody titer detected by ELISA could reach 1∶6 400.Both IFA and Western-blot results showed that the protein could be specifically recognized by PCV2 positive serum and murine polyclonal antibody.The recombinant ORF9 protein of PCV2 with antigenicity was successfully expressed and purified,the murine polyclonal antibodies laid the basis for an in-depth study of the role of ORF9 in PCV2 infection.
porcine circovirus type 2ORF9 proteinprokaryotic expressionpolyclonal antibody
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