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为了研究单核细胞增生李斯特菌毒力基因启动子的结构特点与转录调控因子PrfA蛋白之间的关系,应用PCR定点突变和重组PCR技术缺失了该菌毒力基因inlC启动子上可能与PrfA蛋白结合以及诱发转录起始相关的碱基序列,构建了一系列突变启动子与lacZ报告基因融合表达质粒,使lacZ基因的表达置于inlC突变启动子下,并分别电转化单核细胞增生李斯特菌野生株P14、PrfA蛋白高表达突变株P14a和prfA基因等位缺失突变株A42中,检测相应的β-半乳糖苷酶活性。结果表明:位于inlC启动子转录起始点下游22bp处的一段17bp的类似PrfA蛋白结合序列TTAACAGCGTTTGTTAA并没有增强和抑制PrfA转录调控活性的功能;甚至将其改造成“完美的”PrfA蛋白结合序列TTAACATTTGTTAA后,也不影响inlC依赖于PrfA的转录活性地表达;但是,如果缺失inlC启动子上原始的PrfA蛋白结合序列,则使inlC依赖于PrfA的转录活性完全丧失;另外,单核细胞增生李斯特菌毒力基因inlC和plcA依赖于PrfA的转录活性的表达也与启动子上PrfA蛋白结合区(PrfA-box)距离-10区的碱基个数有关:最适为22或23bp,长于23bp或短于22bp的突变启动子的依赖PrfA的转录活性大大降低,甚至没有活性。说明除PrfA蛋白结合序列外,受PrfA调控的毒力基因启动子上还可能存在其它尚未阐明的结构和序列影响PrfA蛋白的结合以及启动转录表达。
In order to study the relationship between the structural characteristics of the promoter of Listeria monocytogenes virulence promoter and PrfA protein, PCR-based site-directed mutagenesis and recombinant PCR were used to delete the promoter of virulence gene inlC Protein binding and the initiation of transcription related to the base sequence, a series of mutant promoters and lacZ reporter gene fusion expression plasmid was constructed so that the expression of lacZ gene inlC mutant promoter, and were electrically transformed monocyte proliferation Li Si The corresponding β-galactosidase activity was detected in the wild strain P14 of Escherichia coli, the mutant P14a of high PrfA protein and the mutant A42 of prfA gene. The results showed that a 17bp similar PrfA protein binding sequence TTAACAGCGTTTGTTAA located at 22bp downstream of the inlC promoter did not function to enhance and inhibit PrfA transcriptional regulatory activity; even transformed into a “perfect” PrfA protein binding sequence TTAACATTTGTTAA , But also did not affect inlC expression dependent on PrfA's transcriptional activity; however, deletion of the native PrfA protein binding sequence on the inlC promoter resulted in complete loss of inlC PrfA-dependent transcriptional activity; in addition, Listeria monocytogenes The expression of the virulence genes inlC and plcA in PrfA dependent transcriptional activity is also related to the number of bases in the -10 region of the PrfA-binding region (PrfA-box) on the promoter: optimally 22 or 23 bp longer than 23 bp or shorter PrfA-dependent transcriptional activity at 22 bp of the mutant promoter is greatly reduced, even without activity. In addition to the PrfA protein binding sequence, there may be other unclear structures and sequences on PrfA-regulated virulence promoters that affect PrfA protein binding and initiation of transcriptional expression.