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【目的】检测铜绿假单胞菌基因pfm对三型分泌系统效应蛋白的影响。【方法】构建pfm基因互补菌株pfmC。提取野生株PAO1、敲除株Δpfm和互补株pfmC的RNA,利用Real-time PCR从转录水平检测效应蛋白ExoS、ExoT和ExoY转录水平的变化。以ExoS为代表,检测细胞内和分泌到细胞外效应蛋白的含量。收集铜绿假单胞菌PAO1、Δpfm和pfmC菌体内和分泌到细胞外的总蛋白,利用ExoS多克隆抗体进行Western杂交,特异检测ExoS的蛋白水平。【结果】与野生型相比,Δpfm中exoS、exoT和exoY转录水平明显降低,而pfmC中这3个蛋白的转录水平得到回补。Δpfm菌体内和分泌到细胞外的ExoS量均明显低于野生株PAO1,pfmC细胞内和细胞外分泌的ExoS蛋白量均得到恢复。【结论】铜绿假单胞菌基因pfm会影响三型分泌系统效应蛋白的水平。
【Objective】 To detect the effect of pfm gene of Pseudomonas aeruginosa on the type 3 secretion system effector protein. 【Method】 The pfmC-complementary strain pfmC was constructed. RNA of wild-type strain PAO1, knockout strain Δpfm and the complementary strain pfmC was extracted, and transcription of ExoS, ExoT and ExoY were detected by Real-time PCR. ExoS as the representative, detection of intracellular and secretory extracellular effector protein content. The total proteins in and out of the cells of Pseudomonas aeruginosa PAO1, Δpfm and pfmC were collected and subjected to Western blot with ExoS polyclonal antibody to detect the protein level of ExoS. 【Result】 Compared with the wild type, the transcript levels of exoS, exoT and exoY in Δpfm decreased significantly, while the transcriptional levels of these three proteins in pfmC were replenished. The amount of ExoS within and outside of the Δpfm was significantly lower than that of the wild-type PAO1, and both the intracellular and extracellular secretion of ExoS in pfmC were recovered. 【Conclusion】 Pseudomonas aeruginosa gene pfm can affect the level of secreted system effector protein.