牛分枝杆菌溶血磷脂酶基因的克隆及在大肠杆菌中的表达

Cloning and expression of Mycobacterium bovis secreted protein the Lysophospholipase gene in Escherichia coli

为研究牛分枝杆菌(M.bovis)溶血磷脂酶基因在致病机理中的作用,本研究构建了表达M.bovis的溶血酶(LIP)基因的重组质粒pET30a-LIP,经IPTG诱导在大肠杆菌BL21(DE3)中高效表达,SDS-PAGE分析表明,重组蛋白表达量占菌体总蛋白的20%;该蛋白经电洗脱纯化后,纯度达95%以上;免疫印迹实验表明,原核表达的蛋白可与兔抗牛分枝杆菌多克隆抗体结合,具特异的免疫反应性。

The Lysophospholipase gene of Mycobacterium bovis plays an important role in catalysis of lysophosphatide inactivation,and is considered a key player in regulating the biological activity on lipoids level. In order to study the role of Lysophospholipase gene in the M. bovis pathogenesis,the Lysophospholipase gene was cloned into prokaryotic expression vector pET30a and expressed in BL2l（DE3） cells by inducing with IPTG. The expressed product was analyzed by SDS-PAGE and western blot. The results showed that the expressed recombinant protein could reach 20 percent of the whole bacterial protein. Western blot analysis showed the protein had the antigenic activity of M. bovis.