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目的构建pup/mpa/dop/pafA基因过表达结核杆菌国际标准强毒株H37Rv和无毒株H37Ra。方法提取已构建的结核杆菌-大肠埃希菌重组穿梭质粒PMV361pup,PMV361mpa,PMV361dop和PMV361pafA,利用电转化方法分别转入H37Rv和H37Ra感受态细胞,构建pup/mpa/dop/pafA基因过表达H37Rv和H37Ra,同时优化电转化脉冲电压,并应用QRT-PCR进行检测及鉴定。结果成功构建了pup/mpa/dop/pafA基因过表达H37Rv和H37Ra,重组菌均能表达目的基因,在2 100~2 300V脉冲电压范围内构建的菌株目的基因相对表达量与电压呈正相关。结论构建的pup/mpa/dop/pafA基因过表达H37Rv和H37Ra均能表达相应目的基因,以电压为2 300V电转化构建的菌株表达量较高。
Objective To construct the international standard virulent strain H37Rv and non-virulent strain H37Ra of pup / mpa / dop / pafA gene overexpression. Methods Recombinant shuttle plasmid of Mycobacterium tuberculosis-Escherichia coli PMV361pup, PMV361mpa, PMV361dop and PMV361pafA were extracted and transformed into H37Rv and H37Ra competent cells respectively by electroporation to construct H37Rv and H37Rv of pup / mpa / dop / pafA gene H37Ra, while optimizing the electrical conversion pulse voltage, and the application of QRT-PCR detection and identification. Results The overexpression of H37Rv and H37Ra gene were successfully constructed in pup / mpa / dop / pafA gene. All the recombinant plasmids could express the target gene. The relative gene expression of target gene in voltage range of 2 100-2 300 V was positively correlated with voltage. Conclusion The constructed pup / mpa / dop / pafA gene overexpression H37Rv and H37Ra can express the corresponding gene of interest, the voltage of 2 300V electric transformation of the constructed strain higher expression.