结核分枝杆菌EF4敲除菌株的构建

EF4是一个由lepA基因编码的与蛋白质翻译密切相关的延伸因子，在细菌中高度保守，但其确切功能和分子机制尚不清楚，在结核分枝杆菌中的功能至今未见报道。为探索EF4在结核分枝杆菌中的功能，需构建一株结核分枝杆菌lepA基因敲除株。本研究以结核分枝杆菌H37Ra全基因组DNA为模板，设计并通过聚合酶链反应(polymerase chain reaction，PCR)扩增lepA基因左、右臂，连接到p0004S质粒，构建同源重组质粒p0004S-ΔlepA。然后，通过噬菌体体外包装，将p0004S-ΔlepA质粒连接到phAE159质粒，构建phAE159-ΔlepA噬菌体包装质粒。在耻垢分枝杆菌mc2155中大量扩增噬菌体并受结核分枝杆菌侵染进行同源重组，筛选阳性克隆，从基因组和蛋白质表达水平检测该突变株中lepA基因及EF4蛋白表达。PCR结果显示，敲除株基因组中lepA基因已被潮霉素抗性基因成功替换，蛋白免疫印迹结果显示该敲除株中无EF4表达，表明其为成功构建的RaΔlepA。生长曲线分析显示，正常培养条件下，结核分枝杆菌野生株与敲除株生长趋势一致。敲除株与野生株在菌落形态上有一定差异，相比于野生株，RaΔlepA菌落颜色发黄，凸起偏厚，生长过程中生物膜皱褶较少。耐胁迫能力分析显示，与野生株相比，RaΔlepA耐热、抗去垢剂、抗氧化能力无显著差异，但耐酸性环境能力明显增强。本研究利用噬菌体介导的重组法成功构建了结核分枝杆菌lepA基因敲除株，为后续研究结核分枝杆菌EF4的功能提供了重要基础。

Construction of EF4 deletion mutant in Mycobacterium tuberculosis

EF4 encoded by lepA gene is an elongation factor related to protein translation. To study the function of EF4 in Mycobacterium tuberculosis (M. tuberculosis), a lepA gene knockout strain was constructed in this study. The whole genome DNA of M. tuberculosis H37Ra was used as a template to amplify the left and right arms of lepA gene respectively by polymerase chain reaction (PCR). The fragments were cloned into p0004S plasmid to construct homologous recombinant p0004S-ΔlepA plasmid. p0004S-ΔlepA plasmid was cloned into phAE159 plasmid by phage packaging in vitro to construct a phAE159-ΔlepA phage packaging plasmid. The phage was amplified in M. smegmatis mc2155 and infected M. tuberculosis for homologous recombination. The positive colonies were selected and the EF4 protein were detected. The PCR results showed that lepA gene in the knockout strain was successfully replaced by the hygromycin resistance gene and the results of Western blotting showed that there was no EF4 expression in the knockout strain, indicating that RaΔlepA strain was successfully constructed. Growth curve analysis showed that lepA knockout in M. tuberculosis had no phenotype change under the normal growth conditions. Comparing to the wild-type strain, colonies of RaΔlepA had yellowish color and thicker protrusions. Stress resistance analysis showed that there was no difference in heat resistance, detergent resistance and oxidant resistance between the wild-type strain and RaΔlepA strain, but RaΔlepA strain had enhanced ability to withstand acidic environment.