卡介苗菌MDP1基因敲除技术的研究

Study on gene knock-out in Mycobacterium BCG

目的 在卡介苗中建立基因敲除技术。方法 通过分别设计两对引物 ,将靶基因[DNA结合蛋白 1(MDP1)基因 ]通过聚合酶链反应 (PCR)的方法扩增得到 2个片段 ,分别插入pKO质粒中 ,构建得到基因敲除质粒pKO MDP1,电转入至卡介苗菌株细胞内并与BCG基因组中的MDP1基因同源交换 ,筛选出敲除菌株 ,并测其生长曲线。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MDP1基因敲除菌株 ,对敲除菌株与原代菌株每 12h测定其菌液的 6 0 0nm处吸光度 (A60 0 )值 ,连续 16d。两者的生长速度曲线呈现“S”形 ,两者之间的生长速度未见明显差别。结论 pKO质粒可作为基因敲除有用的质粒载体 ,MDP1基因可能是影响BCG生长的因素之一 。

Objective To establish the methodology of plasmid for gene knock out in Mycobacterium BCG Methods We designed two pairs of primers for amplification of MDP1 gene and inserted two fragments into pKO plasmid, and then the recombinant plasmid for MDP1 gene knock out was obtained, and named pKO MDP1 Gene exchange took place within the genome of BCG after pKO MDP1 plasmid was transformed into Mycobacterium BCG The strain of Mycobacterium BCG with MDP1 gene knock out was selected, and the curve of growth rate was studied Results 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 value of A 600 per 12 hours was detected for sixteen days A “S” shaped growth curve was detected However, there was no significant difference in the growth rates between the wild type Mycobacterium BCG strain and the gene knock out Mycobacterium BCG strain Conclusions The plasmid of pKO was a useful tool for gene knock out in Mycobacteria MDP1 maybe one of the factors influencing the growth rate, but it was not the only one