生殖支原体MgPa受体的鉴定及其结构与功能研究

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.

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。

研究背景：生殖支原体（Mycoplasma genitalium, Mg）是一种最小的原核细胞型微生物，能引起泌尿生殖道感染。Mg必须黏附于泌尿生殖道黏膜上皮细胞才能感染宿主或侵入宿主细胞内，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是通过和宿主细胞上何种受体分子结合从而黏附或侵入细胞内的。.主要研究内容：利用噬菌体展示肽库技术、基于改进的病毒铺膜印记技术（VOPBA）、液相色谱质谱（LC-MS）分析、基于Split-Ubiquitin的膜蛋白酵母双杂交等技术筛选和鉴定能与MgPa相互作用的受体肽或位于人尿道上皮细胞膜上的MgPa受体。.重要结果与关键数据：1.利用表达并纯化的重组生殖支原体黏附蛋白（rMgPa）为靶分子，从噬菌体展示随机肽库中筛选到能与rMgPa特异结合的3条多肽：V-H-W-D-F-R-Q-W-W-Q-P-S、D-W-S-S-W-V-Y/H-R-D-P-Q-T/S和H-Y-I-D-F-R-W，经固相法合成的3条多肽均能与rMgPa特异结合，且3条多肽均能明显抑制rMgPa和Mg对SV-HUC-1细胞的黏附，说明这3条多肽是MgPa潜在的受体肽；2.以rMgPa为靶分子，从构建的人尿道上皮细胞（SV-HUC-1）T7噬菌体展示cDNA文库中筛选到能与rMgPa相互作用的蛋白——RPL35，说明Mg经与宿主细胞膜上的受体结合而侵入细胞内后，能与胞内RPL35结合从而影响细胞的功能；3.利用基于改进的病毒铺膜印记技术（VOPBA）和液相色谱质谱（LC-MS）分析等技术筛选到能与rMgPa相互作用的受体蛋白：亲环素A（CyPA）和组蛋白H2B1，两者均可定位于SV-HUC-1细胞膜表面，且均能部分阻止rMgPa和Mg对SV-HUC-1细胞的黏附，说明Cyclophilin A 和 Histone H2B可能是MgPa的受体。4.从构建的人尿道上皮细胞基于Split-ubiquitin的膜蛋白酵母双杂交cDNA文库中筛选到可能与rMgPa相互作用的23种不同的蛋白。.科学意义：本研究结果对从分子水平上阐明Mg感染与免疫的分子机制，揭示Mg与宿主细胞相互作用的关系，了解Mg的致病性和致病机制，设计受体模拟分子或拮抗分子用于研制新型靶向药物、基因工程疫苗与诊断试剂，有效预防和控制Mg感染等具有重要的理论与临床实际意义。