利用酵母双杂交技术筛选与生殖支原体黏附蛋白(MgPa)相互作用的宿主蛋白

Screening of Host Proteins Interacting with Mycoplasma genitalium Adhesion Protein of Using Yeast Double-Hybrid System

从利用基于分离的泛素介导膜蛋白酵母双杂交系统构建的人尿道上皮细胞cDNA文库中筛选生殖支原体(Mg)黏附蛋白(MgPa)的互作蛋白。以pET30a-MgPa为模板,经PCR扩增MgPa基因,将其分别连接到pBT3STE和pBT3SUC载体以构建诱饵质粒pBT3STE-MgPa和pBT3SUC-MgPa;将诱饵质粒分别转化到酵母菌株NMY32内,检测其毒性和自激活活性;将人尿道上皮细胞cDNA文库质粒pPR3-N-207-Zeng转入到含pBT3SUC-MgPa诱饵质粒的酵母菌株中,筛选阳性克隆。检测阳性克隆LacZ报告基因的活性。提取阳性克隆质粒进行DNA测序与BLAST分析。结果显示,成功构建的诱饵质粒pBT3STE-MgPa和pBT3SUC-MgPa两者都没有自激活功能,且pBT3SUC-MgPa的活性强于pBT3STE-MgPa。用pBT3SUC-MgPa转化NMY32酵母作为受体酵母细胞。经DNA测序与BLAST分析结果表明筛选到的28个阳性克隆归属于23个不同的蛋白。从利用分离的泛素介导膜蛋白酵母双杂交系统构建的人尿道上皮细胞cDNA文库中筛选到23个与MgPa相互作用的蛋白,为阐明MgPa的功能及Mg可能的致病机制提供参考。

Proteins interaction with Mycoplasma genitalium adhesion protein (MgPa) was screened using the construction of human uroepithelial cell cDNA library based on the isolated ubiquitin-mediated membrane protien yeast double-hybrid system. The MgPa gene was amplified with PCR using pET30a-MgPa as template and respectively ligated with pBT3STE and pBT3SUC vectors to construct the bait plasmids pBT3STE-MgPa and pBT3SUC-MgPa;then transformed into yeast strain NMY32 respectively, their toxicity and self-activation activity were detected. The human uroepithelial cell cDNA library plasmid pPR3-N-207-Zeng was transformed into the yeast strains containing pBT3SUC-MgPa bait plasmid and the positive clones were screened. The activity of the LacZ reporter gene was detected for the positive clones. The plasmids of positive clones were extracted and sequenced for DNA and analyzed by BLAST. The results showed that bait plasmids pBT3STE-MgPa and pBT3SUC-MgPa were successfully constructed, both had no self-activation activities, and pBT3SUC-MgPa had stronger activity than pBT3STE-MgPa. The pBT3SUC-MgPa was transformed into NMY32 yeast and used as receptor of yeast cell. A total of 28 positive clones were screened and the results of DNA sequencing and BLAST analysis showed that these clones belonged to 23 different proteins. It was concluded that 23 kinds of proteins interacting with MgPa were screened using the construction of human uroepithelial cell cDNA library based on the isolated ubiquitin-mediated membrane protien yeast double-hybrid system, and thus laid a certain experimental foundation to enunciate the function of MgPa as well as the possible pathogenic mechanism of M. genitalium.