副结核分枝杆菌SOD基因在大肠杆菌中的表达

Expression of the SOD gene of Mycobacterium paratuberculosis in E.coli

目的构建副结核分枝杆菌SOD基因的原核表达载体,并在大肠杆菌中获得表达。方法以BamHⅠ和EcoRⅠ双酶切pGEM-T-SOD和pET-28a(+),并将纯化的SOD基因亚克隆至pET-28a(+)中,构建原核表达质粒pET-28a-SOD,并将其转化至感受态E.coli BL21(DE3)中,经IPTG诱导后,进行SDS-PAGE和Western bloting分析。结果成功地构建出原核表达质粒pET-28a-SOD,并表达了约26.5kDa融合外源蛋白带,且具有牛副结核分枝杆菌抗原反应性。结论副结核分枝杆菌SOD基因在大肠杆菌中获得了表达,并具有抗原反应性,为进一步研究SOD作为诊断试剂或亚单位疫苗奠定了基础。

The purpose of this study is to construct the prokaryotic expression plsmid of SOD gene from Mycobacterium paratuberculosis and to express this gene in E. coli in which, pGEM-T-SOD and pET-28a（＋） were digested in which doubIe enzymes BamH I and EcoR I . The prokaryotic expression vector pET-28a-SOD was constructed by using the purified SOD gene that was subcloned into the expression vector pET-28a（＋）. Plasmid containing pET-28a-SOD was transformed into competence E. coli BL21 （DE3）. The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. An approximately 26.5kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blot- ting and it had antigenic reactivity of Mycobacterium paratuberculosis. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and diagnostic reagent as bovine paratuberculosis.