摘要
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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2009年第1期38-41,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金(30471285)
关键词
副结核分枝杆菌
SOD基因
原核表达
Mycobacterium paratuberculosis
SOD gene
Prokaryotic expression