绵羊肺炎支原体内蒙古分离株DnaJ基因克隆表达及其免疫原性研究

Cloning,Expression of DnaJ Gene and Immunogenicity Analysis of DnaJ Protein in Mycoplasma ovipneumoniae Isolated from Inner Mongolia

[目的]克隆表达绵羊肺炎支原体DnaJ基因,研究其表达产物的免疫原性。[方法]以绵羊肺炎支原体内蒙古分离株NM03-MO株基因组DNA为模板,克隆其DnaJ基因序列,对蛋白质序列进行分析比对及三维结构预测;将该基因片段连接至pColdⅠ表达载体,构建pColdⅠ-DnaJ原核表达载体表达重组DnaJ蛋白并纯化,通过Western blot技术检测其与支原体抗体阳性血清的结合活性。[结果]NM03-MO株DnaJ蛋白序列与绵羊肺炎支原体序列同源性最高,但三维结构存在一定差异,重组DnaJ蛋白与绵羊肺炎支原体阳性血清有较好的结合活性,初步证明该蛋白具有免疫原性。[结论]绵羊肺炎支原体DnaJ蛋白被首次成功克隆表达,且具有较好的免疫原性,为后续分子致病机制研究及新型疫苗研制奠定基础。

[Objective]To clone and express the DnaJ gene in Mycoplasma ovipneumoniae,and to assess the immunogenicity of its expression product.[Method]Using genomic DNA extracted from Mycoplasma ovipneumoniae NM03-MO strain isolated from Inner Mongolia as template,DnaJ gene was cloned.The DnaJ protein sequence was analyzed and its three-dimensional structure was predicted.A pColdⅠ-DnaJ prokaryotic expression vector was constructed with the cloned DnaJ gene fragment being connected to pColdⅠvector to express the recombinant DnaJ protein.The obtained DnaJ protein was purified,and its binding activity to Mycoplasma antibody positive serum was evaluated by Western blot.[Result]The NM03-MO strain′s DnaJ protein had the highest homology to Mycoplasma ovipneumoniae,while there were minor differences in the three-dimensional structure.The recombinant DnaJ protein displayed high binding activity to Mycoplasma ovipneumoniae positive serum,indicating that it was a immunogenic protein.[Conclusion]For the first time,the DnaJ protein of Mycoplasma ovipneumoniae was successfully cloned and expressed and had good immunogenicity.This research lays the groundwork for the further molecular pathogenesis research and the development of novel vaccines.