猪肺炎支原体解螺旋酶RuvA的原核表达、多克隆抗体制备及活性鉴定

Prokaryotic Expression,Polyclonal Antibody Preparation and Activity Identification of Helicase RuvA from Mycoplasma hyopneumoniae

猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA,经诱导表达和纯化获得RuvA M hp重组蛋白;免疫家兔制备抗RuvA M hp多克隆抗体,再利用间接ELISA和Western blot试验进行效价测定和特异性验证;利用凝胶迁移试验结合表面等离子共振技术分析RuvA M hp的DNA结合活性;利用凝胶迁移试验结合Western blot试验鉴定RuvA M hp的寡聚特性。结果显示:原核表达的RuvA M hp重组蛋白约为26 ku;制备的抗RuvA M hp多克隆抗体效价为1∶256000,且具有良好的特异性;RuvA M hp具有较强的DNA结合活性,对霍利迪连接体的亲和力高达624.4 pmol·L^(-1)(K D),并且主要以八聚体形式与其形成稳定复合物。通过RuvA M hp的原核表达、多克隆抗体制备及活性鉴定,为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。

Homologous recombination mediated antigenic variation plays an important role in the frequent persistent infection of Mycoplasma hyopneumoniae of pigs.Allosteric regulation of Holliday junctions by helicase RuvA is a key step involved in homologous recombination.Given that,this study aims at expressing RuvA Mhp recombinant protein,identifying its DNA binding activity and preparing polyclonal antibody against RuvA Mhp.Prokaryotic expression plasmid pET21a-RuvA was constructed by molecular cloning.Following expression induction and purification,the RuvA Mhp recombinant protein was obtained;The polyclonal antibody against RuvA Mhp was prepared by immunizing rabbit,and the titer and specificity were determined by indirect ELISA and Western blot;The DNA binding activity of RuvA Mhp was analyzed by electrophoretic mobility shift assay and surface plasmon resonance technique;The oligomerization of RuvA Mhp was determined by electrophoretic mobility shift assay combined Western blot.RuvA Mhp recombinant protein expressed by prokaryotic expression was about 26 ku;The prepared polyclonal antibody against RuvA Mhp had good specificity and high titer of 1:256000;RuvA Mhp has strong DNA binding activity with an affinity of 624.4 pmol·L^(-1)(K D)for Holliday junctions,and forms stable complexes with it mainly in octamers.In this study,the prokaryotic expression,polyclonal antibody preparation and activity identification of RuvA Mhp were achieved,which laid a foundation for further exploration of the molecular mechanism of helicase RuvA mediating antigenic variation of Mycoplasma hyopneumoniae by homologous recombination regulation.