生殖支原体MgPa受体的鉴定及其结构与功能研究

生殖支原体（Mg）是一种最小的原核细胞型微生物，能引起泌尿生殖道和呼吸道感染。Mg必须黏附于泌尿生殖道或呼吸道黏膜上皮细胞才能感染宿主或侵入细胞内，Mg黏附蛋白（MgPa）在Mg黏附或侵入宿主细胞中起关键作用。迄今为止，尚未明确Mg是依赖MgPa和宿主细胞膜上何种受体分子结合从而黏附或侵入细胞的。本课题拟采用噬菌体展示cDNA文库、亲和蛋白质组学和基于Split-Ubiquitin的膜蛋白酵母双杂交技术鉴定人尿道上皮细胞（HUEC）和人支气管上皮细胞（BEAS-2B）膜上MgPa的受体；X射线晶体衍射法解析受体的三维结构；胰蛋白酶敏感试验等证实受体的膜定位；RNA干扰等方法验证受体的功能；用基因突变技术和免疫共沉淀试验确定受体的功能域及其关键氨基酸。旨在了解Mg与宿主细胞相互作用的分子机制，阐明Mg可能的致病机制，揭示MgPa受体的结构与功能，为开发新型靶向药物与基因工程疫苗提供新的思路。

Mycoplasma genitalium (M.genitalium), the smallest prokaryotic microorganism, can cause urinary tract and respiratory tract infection. Previous studies have demonstrated that M.genitalium must adhere to the epithelial cells of genitourinary tract or respiratory of human to infect hosts or penetrate into the cells. M.genitalium adhesion protein (MgPa) is the major membrane protein that plays a critical role in the adhesion and invasion of M.genitalium into host cells. So far, it is still unknown which receptor molecules that exist on the membrane of host cells can be combined by MgPa and thus lead to the adhesion and penetration of M.genitalium into host cells. In this study, the phage display cDNA library technique, affinity proteomics and the split-ubiquitin membrane based yeast two-hybrid technique will be used to screen and identificate the MgPa receptors that exist on the membrane of human uro-epithetial cells (HUEC) and human bronchial epithelial cell line (BEAS-2B). The three-dimensional structure of MgPa receptors will be analyzed by crystal X-ray diffraction. The membrane localization of MgPa receptors will be confirmed by Co-Immunoprecipitation, trypsin sensitivity test and indirect immunofluorescence. The function of MgPa receptors will also be validated by cell adherence test and RNA interfere technique. And the domains and key amino acids of MgPa receptors will also be determined by using the genetic mutation technique and Co-Immunoprecipitation technique. The aims of this study are to understand the molecular mechanisms of the interaction between M.genitalium and host cells, elucidate the possible pathogenic mechanism of M.genitalium, reveal the structure and function of MgPa receptors, and provide new ideas for the development of novel targeted drugs and gene engineering vaccines.