牛结核分枝杆菌MPB70蛋白分泌性载体的构建与蛋白表达

Construction of MPB70’s Mycobacterium bovis secretion expression vector and protein expression

为了研究牛结核病新型诊断抗原,试验根据GenBank中Mycobacterium bovis基因序列设计1对引物,将牛结核分枝杆菌MPB70基因构建到pET22b(+)原核表达载体上,将重组载体转到E.coliBL21(DE3)中,经IPTG诱导获得高效表达,并进行SDS-PAGE和Westen-blot分析。结果表明:MPB70蛋白以可溶形式在细胞周质中表达,有部分蛋白以包涵体形式在细胞质中表达,其分子质量约为30ku,蛋白表达量占菌体总蛋白的20%;重组MPB70蛋白可与牛分枝杆菌阳性血清发生特异性反应。说明重组MPB70蛋白能够作为诊断抗原。

To improve Mycobacterium bovis diagnosis,according to genetic sequences of Mycobacterium bovis from GenBank design a primer,the MPB70 of Mycobacterium tuberculosis was constructed into the gene expression vector pET22b（＋）.The plasmid containing pET22b-MPB70 was transformed into the competence E.coil BL21（DE3）.The transformed bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE.The proteins in cells with were expressed as solable forms in the periplasm,a part of the protein expression in form of inclusion body in the cytoplasm,which an about 30 ku protein was observed.As demonstrated by Western-blot,this protein could be identified only by antiserum against M.bovis.It concludes that MPB70 recombinant protein reaction can be as a diagnostic antigen for further research.