發(fā)布時間：2018-03-02 11:39

  本文選題：鳥分枝桿菌　切入點：PhoP　出處：《微生物學(xué)通報》2016年09期 　論文類型：期刊論文

【摘要】：【目的】對鳥分枝桿菌PhoP的功能進行分析及構(gòu)建PhoP基因突變株,為深入研究PhoP的調(diào)控機制打下基礎(chǔ)。【方法】利用PCR擴增出鳥分枝桿菌PhoP DNA結(jié)合區(qū)(PhoPC)編碼序列,與表達載體p GEX-4T-3連接后,轉(zhuǎn)化入大腸桿菌BL21(DE3)中表達GST-PhoPC融合蛋白。用凝血酶去除GST標簽,制備PhoPC蛋白;利用PCR擴增出鳥分枝桿菌PhoP基因及其下游基因MAV0127、PhoU和Amt的啟動子片段,采用凝膠遷移率移動試驗(EMSA)分別檢測PhoPC與PhoP、MAV0127、PhoU和Amt的啟動子結(jié)合的情況。通過PCR擴增PhoP基因上、下游片段,構(gòu)建PhoP基因缺失性同源核苷酸片段,與自殺質(zhì)粒p GMB151連接后,通過電轉(zhuǎn)化導(dǎo)入鳥分枝桿菌進行同源交換,利用PCR篩選出PhoP基因缺失突變株�！窘Y(jié)果】EMSA結(jié)果顯示,鳥分枝桿菌PhoP能與PhoP、MAV0127及Amt基因啟動子結(jié)合,不能與PhoU結(jié)合。通過PCR和序列分析證實基因突變株的PhoP基因缺失了309個堿基�！窘Y(jié)論】PhoP不僅可調(diào)控其下游基因MAV0127和Amt的轉(zhuǎn)錄水平,還可調(diào)控其自身基因的轉(zhuǎn)錄,但不參與調(diào)節(jié)PhoU二元調(diào)控系統(tǒng)。構(gòu)建了PhoP基因缺失突變株,為進一步研究其在鳥分枝桿菌的調(diào)控功能奠定了基礎(chǔ)。
[Abstract]:[objective] to analyze the function of Mycobacterium avium PhoP and construct the mutant of PhoP gene so as to lay a foundation for further study on the regulatory mechanism of PhoP. [methods] the coding sequence of PhoP DNA binding region of Mycobacterium birdis was amplified by PCR. After ligation with expression vector p GEX-4T-3, GST-PhoPC fusion protein was expressed in Escherichia coli BL21 (DE3). The PhoPC protein was prepared by removing GST tag by thrombin, and the promoter fragments of PhoP gene and its downstream gene MAV0127 PhoU and Amt were amplified by PCR. Gel mobility shift assay (EMSA) was used to detect the binding of PhoPC to the promoter of PhoPMAV0127 Phou and Amt. The upstream and downstream fragments of PhoP gene were amplified by PCR, and the homologous nucleotide fragment of PhoP gene was constructed, and the homologous nucleotide fragment of PhoP gene was ligated with suicide plasmid p GMB151. The deletion mutant of PhoP gene was screened by PCR. [results] EMSA results showed that PhoP could bind to the promoter of PHOPMAV0127 and Amt gene. It is proved by PCR and sequence analysis that the PhoP gene of the mutant lacks 309 bases. [conclusion] PhoP can not only regulate the transcription level of the downstream gene MAV0127 and Amt, but also regulate the transcription of its own gene. The mutant of PhoP gene deletion was constructed, which laid a foundation for further study of its regulatory function in Mycobacterium avium.
【作者單位】： 廣西醫(yī)科大學(xué)生物化學(xué)與分子生物學(xué)教研室廣西高校生物分子醫(yī)學(xué)研究重點實驗室;

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