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
边孟婷 1梁海英 1曾智勇 1汤德元 1王彬 1叶泥 1柳佳佳 1黄书 1潘向英 1田红利1
作者信息
- 1. 贵州大学动物科学学院,贵州贵阳 550025
- 折叠
摘要
为了鉴定并深入研究ORF9蛋白在猪圆环病毒2型感染中的作用,本研究制备了小鼠抗ORF9蛋白多克隆抗体并进行了初步应用.首先通过扩增克隆ORF9基因,构建原核表达质粒pET-32a(+)-PCV2-ORF9并转化至BL21(DE3)感受态细胞中,再利用SDS-PAGE对可能影响ORF9蛋白表达效果的因素(温度、IPTG浓度、时间)进行优化研究,随后将重组蛋白经纯化复性后免疫BALB/c小鼠制备多克隆抗体,用ELISA测定小鼠多抗效价,并用IFA和Western-blot方法对抗ORF9多克隆抗体特异性进行研究.结果显示,重组质粒pET-32a(+)-PCV2-ORF9构建成功;ORF9重组蛋白在IPTG浓度为0.1mmol/L、37 ℃条件下诱导4h表达量最高,且主要以包涵体形式表达,分子量大小约24.2 ku;经ELISA检测制备的多抗效价为1:6400;IFA和Western-blot试验结果均证明ORF9蛋白能被PCV2阳性血清和小鼠多克隆抗体特异性识别.本研究成功表达并纯化了具备抗原性的PCV2 ORF9重组蛋白,制备的特异性抗PCV2 ORF9多克隆抗体为探究ORF9蛋白在PCV2感染中的作用奠定了基础材料.
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引用本文复制引用
基金项目
贵州省科技支撑计划项目(黔科合支撑[2021]一般162)
出版年
2024
国家科技期刊平台
NSTL
万方数据