摘要
目的构建结核分枝杆菌EIS基因表达质粒,探讨其对人肺腺癌A549细胞自噬的影响。方法采用PCR技术扩增EIS基因片段并插入空载质粒pcDNA3.1-3flag中,双酶切、测序鉴定克隆质粒的正确性;将重组质粒与对照空载质粒分别与自噬体荧光表达质粒GFP-LC3共转染细胞并以自噬诱导剂雷帕霉素处理,荧光显微镜下观察自噬荧光并计数,Western blot检测EIS蛋白及自噬蛋白LC3Ⅱ/Ⅰ的表达水平。结果成功构建EIS基因重组表达质粒PcDNA3.1-EIS-3flag,该质粒共转染细胞组自噬荧光小点较对照组明显减少(P<0.05),重组质粒表达42 ku看到蛋白并可抑制自噬蛋白表达水平。结论结核分枝杆菌EIS基因可抑制A549细胞自噬的形成,其抑制作用与结核分枝杆菌持留性相关。
Objective To identify the role of EIS gene of Mycobacterium tuberculosis in the process of autophage in human pulmonary adenocarcinoma cell line A549. Methods Recombinat plasmid pcDNA3.1-EIS-3flag was con- structed and cotransfected with GFP-LC3 which is a flurescent autophagosome expression plasmid in human pulmo nary adenocarcinoma cells A549. Twenty - four hrs after cotransfection and treatmont with rapamycin which is aclassical autophage inducer,the autophagy fluorescent dots were examined quantitatively. We further detected the EIS protein and autophagic protein by Western blot. Results The recombinant plasmid Pcdna3.1-EIS-3flag was successfully constructed, and it decreased the fluorescene dots of autophage. Moreover,the EIS protein could inhibit autophagical protein by Western blot result. Conclusions EIS gene of Mycobacterium tuberculosis can inhibit the initiation of autopha^e, which plays a key role in the persistency of tuberculosis.
出处
《基础医学与临床》
CSCD
北大核心
2013年第2期129-132,共4页
Basic and Clinical Medicine
基金
国家自然科学基金(30972582
30500428)
重庆市自然科学基金(CSTC 2009BB5276)
关键词
结核分枝杆菌
EIS基因
自噬
Mycobacterium tuberculosis
EIS gene
autophage
