与结核分枝杆菌PE25/PPE41和PE35/PPE68相互作用的宿主蛋白的筛选

摘要

To screen the host proteins interacting with the two heterodimers of PE25/PPE41 and PE35/PPE68 of Mycobacterium tuberculosis,the two pairs of heterodimers were used as bait proteins to screen the host protein partners.First,PCR-amplified PE25/PPE41 or PE35/PPE68 genes were cloned into a modified pcDNA3.1 (+) (containing ZZ and Flag tags sequences jointed by a protease cleavage site sequence from tobacco etch virus at the 5'terminal) to construct the bait recombinant plasmids of pcDNA-PE25-PPE41 and pcDNA-PE35-PPE68,which were transfected into 293T cells,respectively.The interacting host proteins were captured by ZZ and Flag tagged protein affinity chromatography from the cell lysates.The trapped proteins were further analyzed by mass spectrometry.Of which 15 proteins were identified for PE25/PPE41 and 29 proteins for PE35/PPE68.Among them,10 were most likely to interact with the two heterodimers,respectively.These results provide basis for further identification and study of the biological function of PE25/PPE41 and PE35/PPE68 protein partners.%为筛选与结核分枝杆菌(MTB)的PE25/PPE41和PE35/PPE68两对异源二聚体相互作用的宿主蛋白,本研究以这两对异源二聚体为诱饵蛋白筛选与其相互作用的宿主蛋白.首先将PCR扩增的PE25/PPE41或PE35/PPE68基因串联克隆于改造的pcDNA3.1(+)中(在5'端引入了以编码烟草蚀纹病毒蛋白酶切位点的序列连接的ZZ和Flag标签序列),构建诱饵重组质粒pcDNA-PE25-PPE41和pcDNA-PE35-PPE68.分别将构建的诱饵重组质粒转染293T细胞后提取细胞总蛋白,经ZZ和Flag标签蛋白两步串联亲和层析钓取宿主蛋白,SDS-PAGE电泳分离后切取差异蛋白胶条进行质谱分析,结果显示,PE25/PPE41鉴定到15个差异蛋白,PE35/PPE68鉴定到29个差异蛋白,并分别挑选了与PE25/PPE41和PE35/PPE68相互作用的3个和7个差异蛋白,为进一步验证与PE25/PPE41和PE35/PPE68直接作用的宿主蛋白及研究其生物学功能奠定了基础.