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目的构建弗氏志贺菌Htr A蛋白的可控表达菌株。方法构建自杀质粒同源重组载体,利用重组方法在基因组htr A前插入阿拉伯糖启动子,同时构建并导入外源表达Ara C蛋白的表达载体,实现对Htr A蛋白的可控表达;在此基础上,通过蛋白印迹实验,检测诱导前后全菌以及周质中Htr A蛋白的表达情况。结果测序结果表明,自杀质粒同源重组载体以及外源表达载体构建成功;蛋白印迹实验结果显示,对比阿拉伯糖诱导菌株,未诱导菌株中基本未检测到Htr A蛋白的表达。结论自杀质粒同源重组方式可在无阿拉伯糖时有效阻遏Htr A蛋白的表达;同时在诱导后,还可恢复Htr A蛋白的正常表达,有利于后续功能研究。
Objective To construct a controllable expression strain of Shigella flexneri Htr A protein. Methods A suicide plasmid homologous recombination vector was constructed. The promoter of arabinose was inserted before the genome htr A by recombinant method. At the same time, the expression vector of exogenous Ara C protein was constructed and introduced to realize the controllable expression of Htr A protein. Based on this, The expression of Htr A protein in whole bacteria and periplasm before and after induction was detected by Western blotting. Results The sequencing results showed that the suicide plasmid homologous recombination vector and the exogenous expression vector were successfully constructed. Western blotting results showed that the expression of Htr A protein was not detected in uninduced strains compared with those induced by arabinose. Conclusion The suicide plasmid homologous recombination can effectively suppress the expression of Htr A protein in the absence of arabinose. At the same time, the normal expression of Htr A protein can be restored after induction, which is in favor of subsequent functional studies.