Screening Of Host Proteins Interacting With MgPa Of Mycoplasma Genitalium By Yeast Two-hybrid System

[Objective] Mycoplasma genitalium(M.genitalium)is a bacterial pathogen which has the smallest genome and can self-duplicate in vitro.The epidemiology results indicated the M.genitalium is closely related to the AIDS,vaginosis cervicitis,pelvic inflammatory disease and infertility.Mycoplasma genitalium adhesion protein(Mg Pa)is the major adhesion of M.genitalium outer membrane protein which play an important role in adhesion to host cells and pathogenicity.In this study,the DUAL membrane starter kit was used to screen the ligand protein of Mg Pa in human urothelium cell,which provide experimental evidence for explaining the functions and pathogenic mechanism of Mg Pa.[Methods]The Mg Pa DNA sequence was amplified by PCR from the plasmid p ET-30a(+)-Mg Paandinserted into thep BT3 STE and p BT3 SUCvector to generate bait plasmidp BT3STE-Mg Pa and p BT3SUC-Mg Pa,respectively.The plasmids were transformed into the NMY32 yeast competent cells.The self-activations of p BT3STE-Mg Pa and p BT3SUC-Mg Pa were then tested.The c DNA library of Human urothelium cell was transformed into the yeast NMY32 with plasmid p BT3SUC-Mg Pa.The yeast cells were spread on the SD-Trp-Leu-His+5 m M 3AT plates to screen the positive clones.The plasmids were extracted from positive yeasts and had been sequenced and analyzed by BLAST.And the location of these proteins were analyzed by bioinformatics tool.[Results]The 870 bp DNA fragment of Mg Pa sequence was ligated into the p BT3 STE and p BT3 SUCto construct the bait plasmid p BT3STE-Mg Pa and p BT3SUC-Mg Pa,which then were identified by PCR and DNA sequencing.Both plasmids were nontoxic to yeast cells and no self-activation.The p BT3SUC-Mg Pa has much more activity than the p BT3STE-Mg Pa.The yeast cell NMY32 gained the plasmid p BT3SUC-Mg Pa was established.28 positive clones were harvested and been sequenced.23 different proteins were screened out,which including Tumor necrosis factor receptor superfamily member 6B,cytochrome c oxidase,metallothionein 1X,serum-inducible kinase and so on.The H3 histone,metallothionein and tumor necrosis factor receptor superfamily members 6B are located outside the cell membrane.The serum-induced kinase and FK506 binding protein are located in the cytoplasm and the cytochrome c oxidase is located in mitochondria.[Conclusion]In this study,23 proteins fromc DNA library of human urothelium cell were screened by membrane yeast two-hybrid system,which can interact with Mg Pa from Mycoplasma genitalium.