中国兽医科学2024，Vol.54Issue(2)：226-232,7.DOI:10.16656/j.issn.1673-4696.2024.0030
猪圆环病毒2型ORF9蛋白的原核表达及其多克隆抗体制备
Prokaryotic expression of porcine circovirus virus type 2 ORF9 protein and preparation of its polyclonalantibodies
摘要
Abstract
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.关键词
猪圆环病毒2型/ORF9蛋白/原核表达/多克隆抗体Key words
porcine circovirus type 2/ORF9 protein/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
边孟婷,田红利,梁海英,曾智勇,汤德元,王彬,叶泥,柳佳佳,黄书,潘向英..猪圆环病毒2型ORF9蛋白的原核表达及其多克隆抗体制备[J].中国兽医科学,2024,54(2):226-232,7.基金项目
贵州省科技支撑计划项目(黔科合支撑[2021]一般162项目) （黔科合支撑[2021]一般162项目）
