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【目的】对鸟分枝杆菌PhoP的功能进行分析及构建PhoP基因突变株,为深入研究PhoP的调控机制打下基础。【方法】利用PCR扩增出鸟分枝杆菌PhoP DNA结合区(PhoPC)编码序列,与表达载体p GEX-4T-3连接后,转化入大肠杆菌BL21(DE3)中表达GST-PhoPC融合蛋白。用凝血酶去除GST标签,制备PhoPC蛋白;利用PCR扩增出鸟分枝杆菌PhoP基因及其下游基因MAV0127、PhoU和Amt的启动子片段,采用凝胶迁移率移动试验(EMSA)分别检测PhoPC与PhoP、MAV0127、PhoU和Amt的启动子结合的情况。通过PCR扩增PhoP基因上、下游片段,构建PhoP基因缺失性同源核苷酸片段,与自杀质粒p GMB151连接后,通过电转化导入鸟分枝杆菌进行同源交换,利用PCR筛选出PhoP基因缺失突变株。【结果】EMSA结果显示,鸟分枝杆菌PhoP能与PhoP、MAV0127及Amt基因启动子结合,不能与PhoU结合。通过PCR和序列分析证实基因突变株的PhoP基因缺失了309个碱基。【结论】PhoP不仅可调控其下游基因MAV0127和Amt的转录水平,还可调控其自身基因的转录,但不参与调节PhoU二元调控系统。构建了PhoP基因缺失突变株,为进一步研究其在鸟分枝杆菌的调控功能奠定了基础。
【Objective】 The purpose of this study is to analyze the function of PhoP and to construct the PhoP gene mutant, which lays the foundation for further study on the regulatory mechanism of PhoP. 【Method】 The PhoPC DNA binding region (PhoPC) coding region of Mycobacterium phlei was amplified by PCR and ligated with the expression vector pGEX-4T-3. The fusion protein was transformed into E. coli BL21 (DE3) to express GST-PhoPC fusion protein. Phosphatidylinositol PhoP gene and its downstream genes MAV0127, PhoU and Amt promoter fragment were amplified by PCR using GST tag with thrombin to remove the GST tag, and the PhoPC and PhyP were detected by EMSA PhoP, MAV0127, PhoU and Amt promoters. The upstream and downstream fragments of PhoP gene were amplified by PCR, and the homologous nucleotide fragment of PhoP gene was constructed. After being linked with suicide plasmid pGMB151, the Mycobacterium avium was electroporated into homologous exchanging and the PhoP gene was screened by PCR Deletion mutant. 【Result】 EMSA results showed that Mycobacterium phlei PhoP could bind with PhoP, MAV0127 and Amt promoter and could not bind with PhoU. PCR and sequence analysis confirmed that the mutant gene PhoP gene deletion of 309 bases. 【Conclusion】 PhoP not only regulates the transcription of its downstream genes MAV0127 and Amt, but also regulates the transcription of its own genes, but it is not involved in the regulation of PhoU binary regulatory system. The deletion mutant of PhoP gene was constructed, which lays the foundation for the further study of its regulatory function in Mycobacterium avium.