鹦鹉热嗜衣原体两个TMH家族蛋白结构与功能研究

Chlamydophila psittaci, an obligate intracellular organism, is a human zoonotic pathogen. Studies have showed that inclusion membrane proteins, secreted by Chlamydophila psittaci, play important roles in maintaining intracellular growth and reproduction and the pathogenesis. Based on our previously study, we have found that CPSIT_0844 and CPSIT_0846 are newly inclusion membrane proteins, belonged to the transmembrane head proteins (TMH) family proteins. However their structure and functions are still unclear. Therefore, we expect to study the proteins structure, then we aim to employ heterologous secretion system to study the secretion pathway. Then both yeast two hybrid and targeted proteomic were used to screen host cell interaction partner. Interaction protein will be identified by GST-pull down and Co-Immunoprecipitation technique, Confocal laser scanning technique,Cross-neutralization test, and silencing target molecules were adopted to explore their co-localization and the roles on Chlamydophila psittaci infection of the interaction proteins. The results will help us to study the infection mechanism of Chlamydophila psittaci, and may provide new ideas for targeting the chlamydia drugs and engineering vaccine development.

鹦鹉热衣原体（Cps）是一种严格活细胞内寄生的人畜共患病病原体。研究表明，Cps分泌的包涵体膜（Inc）蛋白在维持衣原体胞内生长发育以及致病过程中发挥重要作用。CPSIT_0844和CPSIT_0846是申请人首次发现的新型Inc蛋白，属于TMH家族蛋白，然而其结构与功能目前尚不明确。本研究拟从其结构解析入手，应用异源分泌系统研究其分泌途径，随后通过酵母双杂交和靶向蛋白质组学技术等筛选宿主细胞相互作用蛋白，并采用GST-pull down、免疫共沉淀技术进行验证，然后通过激光共聚焦技术、交叉中和实验以及RNA干扰等方法观察其定位情况以及在Cps感染中的作用。本研究的完成有利于进一步阐明Cps感染的致病机制，并可能为研制新型靶向药物与基因工程疫苗提供新的思路。

鹦鹉热嗜衣原体（Chlamydophila psittaci, Cps）是一种严格细胞内寄生、多宿主性的人兽共患病病原体。目前对其确切的致病机制不清楚，也没有有效的疫苗。CPSIT_0844和CPSIT_0846是我们前期研究Cps分泌性蛋白中发现的新型Inc蛋白，属于TMH家族蛋白。本项目先构建了CPSIT_0844和CPSIT_0846的原核表达载体，表达、纯化相应重组蛋白，免疫小鼠制备多克隆抗体。将CPSIT_0846重组蛋白经腹腔注射途径免疫小鼠，检测其诱导产生的体液免疫与细胞免疫应答水平，然后经呼吸道感染Cps 6BC，检测小鼠肺脏病变情况，检测CPSIT_0846的免疫保护效果。结果发现，CPSIT_0846重组蛋白免疫小鼠血清中产生较高水平的抗体，脾细胞上清中IFN-γ产生水平明显高于对照组，并能保护Cps 感染后引起的肺组织损伤，提示CPSIT_0846具有较好的免疫保护效果。CPSIT_0846处理HeLa细胞后无明显的细胞毒性作用，并可抵抗STS诱导的细胞凋亡；抑制ERK1/2或SAPK/JNK信号通路可使CPSIT_0846处理的HeLa细胞中Bax/Bcl-2比值以及p53的表达水平显著上调，同时Cleaved-caspase-3/9和Cleaved PARP水平增加，MMP下降并伴随Cyt. c释放到细胞质中，提示CPSIT_0846可通过激活ERK/JNK信号通路调控线粒体途径介导的HeLa细胞凋亡。