副结核分枝杆菌map0862基因的克隆及其在大肠杆菌中的表达

Cloning and Expression of Mycobacterium avium subsp. Paratuberculosis Gene map0862 in E.coli

为探索MAP0862蛋白在分枝杆菌感染鉴别诊断中的作用,研究构建了MAP0862的原核表达map0862-pET32a(+)质粒,并对其进行了诱导表达。以副结核分枝杆菌P10基因组DNA为模板,经PCR方法扩增副结核分枝杆菌map0862基因片段,将其克隆到原核表达载体pET32a(+)中。将重组子转化到大肠杆菌BL21(DE3),在E.coli中诱导表达带组氨酸标签的map0862-pET32a(+)的融合蛋白。结果显示,经IPTG诱导表达出约55 kD的融合蛋白,用副结核阳性血清进行Western blot检测,证明MAP0862重组蛋白具有良好的免疫原性。

In order to determine the function of the MAP0862 in the prevention and diagnosis of Johne＇s disease in cattle,the vector map0862-pET32a（＋） was constructed.The gene map0862 was amplified from M.avium subsp.paratuberculosis strain P10 by using PCR technique.PCR product was cloned into pET32a（＋） plasmid,then the recombinant plasmid was transformed into E.coli BL21（DE3） and expressed.Results showed that the recombinant protein about 55 kD was expressed and could be recognized by serum of M.avium subsp.paratuberculosis infected cattle in western blot.The western blot analysis showed the expressed protein had antigenic activity.