结核分枝杆菌标准株H37Rv mazEF3基因过表达菌株的构建及初步鉴定

The Construction and preliminary identification of mazEF3 gene overexpress Mycobacterium Tuberculosis H37Rv strain

为构建H37Rv mazEF3基因的过表达菌株,检测过表达菌株mazEF3 mRNA表达水平。使用穿梭质粒pMV361,把mazEF3基因连入其中,构建重组质粒pMV361-mazEF3;利用电穿孔技术,将穿梭表达质粒导入H37Rv中,并将该菌涂在卡那霉素培养基上初步筛选,提取阳性菌株质粒进行PCR、双酶切后送公司测序进一步验证;检测不同电转电压下mazEF3 mRNA表达水平,寻找最佳电转电压。结果显示,成功构建H37Rv mazEF3基因的过表达菌株,PCR、双酶切后目的基因,测序结果与已知mazEF3基因比对,同源性100%。实时荧光定量检测,电转电压为2300V时,构建的过表达菌株mazEF3 mRNA表达量最高。由此可知,成功构建及初步鉴定了mazEF3 mRNA过表达菌株,电转电压为2300V时,构建的过表达菌株mazEF3 mRNA表达量最高。

To construct Mycobacterium tuberculosis standard strain（H37Rv） overexpressing mazEF3 gene and to detect the mazEF3 gene mRNA expression level. The shuttle- plasmid pMV361 was used, and the mazEF3 with the pMV361 was connected to construct a recombination plasmid pMV361- mazEF3. The shuttle- plasmid pMV361- mazEF3 was transfected into H37 Rv by electroporation technology, and then the bacteria was inoculated and screened initially on kanamycin culture medium.The positive strain＇s plasmid was extracted and further verified after PCR and double enzyme digestion technology. The mRNA expression levels of mazEF3 under different voltages with electrotransformation was tested by the real- time fluorescent quantitative PCR, to find the optimal voltage. As the results showed that H37 Rv overexpress mazEF3 gene was successfully constructed. Sequencing results of target genes by PCR and double digestion were with known mazEF3 gene sequences, the homology was consistent, reached 100%. At the 2300 V with electrotransformation voltage, the mRNA expression level of mazEF3 was the highest. H37 Rv overexpress mazEF3 gene was successfully constructed. At the 2300 V with electrotransformation voltage,the expression level of mazEF3 is the highest.