卡介苗ERP基因缺失突变株的构建

目的构建卡介苗ERP基因缺失突变株。方法分别设计两对引物,通过聚合酶链反应（PCR）的方法扩增ERP基因两侧的2个目的片段,分别插入pKO质粒中,构建基因敲除质粒pKO-ERP,电转入至卡介苗菌株细胞内并与BCG基因组中的ERP基因同源交换,筛选出ERP基因敲除菌株。结果通过2次PCR筛选和1次蔗糖反筛选后得到的、并在含潮霉素培养基上不能生长的菌株为ERP基因敲除菌株。结论 pKO质粒可作为基因敲除有用的质粒载体,成功构建了卡介苗ERP基因缺失突变株。

Construction of the strain of Mycobacterium BCG with ERP gene knock-out

To establish the strain of Mycobacterium BCG with ERP gene knock-out.two pairs of primers were designed for amplification of the targeted gene with PCR and inserted two fragments into pKO plasmid,and then the recombinant plasmid for ERP gene knock-out was obtained,and named pKO-ERP gene exchange took place within the genome of BCG after pKO-ERP plasmid was transformed into Mycobacterium BCG.The strain of Mycobacterium BCG with ERP gene knock-out was selected.The target strain was that of the positive strain by two step PCR and one step sucrose counter selection,without growth in culture media with hygomycin.The strain of Mycobacterium BCG with ERP gene knock-out was constructed successfully.The plasmid of pKO was a useful tool for gene knock-out in Mycobacteria.