牛分枝杆菌MPB51基因的克隆及其在大肠杆菌中的表达

以牛分枝杆菌Vallee111染色体DNA为模板，以MPB51成熟蛋白基因特异性引物进行PCR扩增，获得约800bp的DNA片段。通过TA克隆技术，将PCR产物克隆至pGEMT Vector中，成功地构建出克隆质粒pGEMT51。以BamHⅠ和EcoRⅠ双酶切pGEMT51和pET28a(+)，并将纯化的MPB51基因亚克隆至pET28a (+)中，构建出原核表达质粒pET28a51。将pET28a51转化至感受态E.coli BL21（DE3）中，经IPTG诱导和SDSPAGE分析，可见约30kD外源蛋白带。Western blot分析发现，该蛋白具有牛分枝杆菌抗原性，从而为进一步研究MPB51的亚单位疫苗及DNA疫苗奠定基础。

Cloning and expression of Mycobacterium bovis secreted protein MPB51 in Escherichia coli

The gene encoding MPB51 was amplified from M. bovis Valleel11 chromosomal DNA using PCR technique, and the PCR product was approximately 800 bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and cloning plasmid pGEM-T-51 was thus constructed successfully. pGEM-T-51 and pET28a( + ) were digested by BamH I and EcoR I double enzymes. The purified MPB51 gene was subcloned into the expression vector pET28a( + ), and the prokaryotic expression vector pET28a-51 was thus constructed. Plasmid containing pET28a-51 was transformed into competence E. coli BL21 (DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE, approximately 30 kD exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western blot. The results indicate that the protein is antigenic activity of MB. The results are expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB51 gene in their prevention of bovine tuberculosis.