猪圆环病毒2型去核定位信号Cap蛋白的原核表达及多克隆抗体制备OA

To generate polyclonal antibodies targeting the Cap protein of Porcine Circovirus type 2(PCV2),a Cap gene fragment without the nuclear localization signal(NLS)was amplified using the pMD18-T-ORF2 plasmid as a template.This fragment was subsequently cloned into the prokaryotic expression vector pET-28a(+)to construct the recombinant plasmid pET-28a-N-Cap.The recombinant plasmid was then transformed into Escherichia coli Rosetta(DE3)competent cells.After induction with IPTG,SDS-PAGE analysis revealed that the expressed recombinant protein was predominantly present in an insoluble form.The target protein was purified via nickel-affinity chromatography and used as an antigen to immunize rabbits for the production of rabbit-derived polyclonal antibodies.The resulting antibody titer reached up to 1∶204,800,as determined by serological testing.Furthermore,indirect immunofluorescence assay(IFA)and Western blot analysis confirmed that the obtained polyclonal antibodies specifically recognized PCV2.These findings indicate that the NLS-deleted Cap protein expressed in a prokaryotic system retains strong immunogenicity,and the acquired polyclonal antibodies can be utilized in assays related to the Cap protein.