摘要
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.
作者
谢青云
邢蕙萱
于岩飞
袁厅
熊祺琰
熊富强
冯志新
XIE Qingyun;XING Huixuan;YU Yanfei;YUAN Ting;XIONG Qiyan;XIONG Fuqiang;FENG Zhixin(Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture/Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Animal Science,Tibet Agricultural and Animal Husbandry University,Linzhi 850400,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第1期271-281,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然基金(32202812)
江苏省农业科技自主创新项目(CX(22)3195)。
