结核分枝杆菌H37Rv菌株Hsp16．3基因缺失突变株的构建与鉴定

Construction and identification on a strain of Mycobacterium tuberculosis H37Rv with Hsp16.3 gene knock-out

目的构建及鉴定结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因缺失突变株。方法体外培养结核分枝杆菌国际标准强毒株H37Rv菌株,并提取基因组DNA,扩增Hsp16.3基因两侧序列,分别插入到pKO质粒载体预定位点中,构建Hsp16.3基因置换型打靶载体,电转入结核分枝杆菌H37Rv菌株内,并与其基因组中的Hsp16.3基因同源交换,筛选出Hsp16.3基因缺失突变株。结果通过卡那霉素筛选和蔗糖反筛选及PCR鉴定,并在含有潮霉素培养基上不能生长的菌株为Hsp16.3基因缺失突变株。结论成功构建出结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因缺失突变株。

A strain of Mycobacterium tuberculosis H37Rv with Hspl6.3 gene knock-out were constructed in the study. Mycobacterium tuberculosis strain H37Rv was cultured in vitro and the genome DNA was extracted. In order to obtain a gene targeting vector pKO-Hspl6.3, two fragments （5＇-Hspl6.3 and 3CHspl6.3） flanking the Hsp16.3 region were amplified by PCR and inserted into pKO plasmid, and the recombinant pKO-Hspl6. 3 plasmid was obtained. Gene exchange took place within the genome of Mycobacterium tuberculosis H37Rv after pKO-Hsp16.3 plasmid was transformed into it, and the strain of Mycobacterium tuberculosis H37Rv with Hspl6.3 gene knock-out was selected. Results indicated that the target strain was the positive one identified by two step polymerase chain reaction （PCR） and one step sucrose counter selection simultaneously and could not grow in hygomycin culture media. Finally, the strain of Mycobacterium tuberculosis H37Rv with Hspl6. 3 gene knock-out is constructed successfully in our laboratory.