鸟分枝杆菌PhoR基因的克隆及诱导表达

目的:克隆鸟分枝杆菌的PhoR基因,构建高表达重组质粒pGEX-PhoR,确定目的蛋白表达形式,为后续对鸟分枝杆菌PhoR的功能研究打下基础。方法:以一株鸟分枝杆菌临床分离株作为DNA模板,PCR扩增PhoR基因,pGEX-4T-3为载体构建表达质粒,转化到E.coliBL21(DE3)中诱导表达。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)分析蛋白表达形式及表达量。结果:扩增出鸟分枝杆菌PhoR基因,构建了重组表达质粒pGEX-PhoR,在BL21(DE3)中以可溶性方式高效表达,经诱导产生与预期值相符的一个约81KDa的融合蛋白。结论:成功构建了pGEX-PhoR重组表达质粒,在大肠杆菌中诱导表达,为后续深入研究鸟分枝杆菌PhoR的生物活性和免疫活性奠定了基础。

To clone the gene for Mycobacterium avim PhoR, construct recombinant plasmid p GEX-PhoR, make sure the expression form, and to establish a basis for further research.The gene cloning for PhoR was ampified by polymerase chain reaction(PCR), and then inserted to vector p GEX-4T-3 to construct the recombinant plasmid p EGX-PhoR. The recombinant plasmid was transformed into E.coli BL21(DE3), the recombinant protein was analyzed by SDS-PAGE.A 81 KDa protein, as expected, was obtained evidently. The resaults indicated that the recombinant plasmid with gene of PhoR was constructed successfully. In conclusion the target gene was efficiently expressed in E.coli BL21(DE3), and these results would establish the basis for research in biology and immunology of PhoR.