绵羊支原体HSP70的原核表达及抗体间接ELISA检测方法的建立

Prokaryotic Expression of Mycoplasma ovis Hsp70 Protein and Development of Indirect ELISA for Antibody Detection

为建立绵羊支原体(Mycoplasma ovis)的血清学检测方法,根据GenBank收录的绵羊支原体HSP70蛋白C端的基因序列(登录号:CP006935.1),构建了重组原核表达质粒。通过原核表达获取重组蛋白,并将纯化的重组蛋白作为包被抗原,建立了绵羊支原体间接ELISA抗体检测方法。结果显示,成功构建了绵羊支原体HSP70蛋白的重组原核表达质粒;重组蛋白在大肠埃希氏菌BL21(DE3)感受态细胞中,同时以可溶性蛋白和包涵体蛋白的形式表达;以重组蛋白作为包被抗原建立的绵羊支原体间接ELISA抗体检测法,抗原包被最佳浓度为4μg/mL,血清最佳稀释度为1∶100,酶标二抗最佳稀释度为1∶2000。建立的ELISA方法具有良好的特异性、灵敏性和重复性,为绵羊支原体的检测及试剂盒的开发提供了技术支持。

To establish a serological method for detection of Mycoplasma ovis,a recombinant prokaryotic expression plasmid of HSP70 was constructed based on the C-terminal gene sequence of HSP70 protein of Mycoplasma ovis(Accession number:CP006935.1)in GenBank.The purified recombinant protein obtained by prokaryotic expression was used as the coating antigen to establish an indirect ELISA antibody detection method for Mycoplasma ovis.The study suggested that the recombinant prokaryotic expression plasmid was successfully constructed.The recombinant protein was expressed in Escherichia coli BL21(DE3)competent cells as both soluble protein and inclusion body protein,used as coating antigen to establish an indirect ELISA antibody detection method for Mycoplasma ovis.It revealed that the optimal concentration of antigen coating is 4μg/mL,the optimal dilution of serum is 1∶100,and the optimal dilution of enzyme-labeled secondary antibody is 1∶2000.This method has great specificity,sensitivity and repeatability,providing certain reference for detection of Mycoplasma ovis and the development of reagent kits.