鹦鹉热嗜衣原体持续性感染的分子机制研究

鹦鹉热嗜衣原体（Cps）是一种严格细胞内寄生，多宿主性的人畜共患病病原体，常引起机体的持续性感染。目前，对于Cps持续性感染机制知之甚少。随着Cps 基因组的完整测序及注释，以及人畜共患病的频发，使得人们对人畜共患病病原体越来越关注。本项目针对这一前沿科学问题，采用间接免疫荧光、RT-PCR、Western Blot、免疫共沉淀等分子生物学和免疫学手段，建立Cps持续性感染的体外感染细胞模型，筛选引起持续性Cps感染的相关基因，从抑制宿主细胞凋亡和逃避机体的免疫机制等方面入手，研究Cps致病的关键蛋白和细胞因子以及引起Cps感染宿主细胞改变的Raf/MEK/ERK和PI3K/AKT等调控信号通路，探讨Cps持续性感染发生的分子机制。本项目的开展将进一步揭示衣原体和真核细胞内环境相互作用的分子机制，从而为研究阻断Cps持续性感染，设计新的药物和新型疫苗的提供科学依据。

Chlamydophila psittaci (C. psittaci) is an obligate intracellular bacterial pathogen and multiply in a wide range of eukaryotic hosts including animals and humans. However, molecular mechanism of persistent C. psittaci infection haven't been best-studied yet. With available results of completed C. psittac genome sequencing and annotated its whole genome and frequent occurrence of zoonosis, Zoonotic pathogens became of great interest. Herein, this project will construct in vitro cell modelings of persistent C. psittac infection, screen related genes resulting in sustained C. psittac infection through Bioinformatics methods, harvest a great amount of expressive products of these found genes by PCR amplification and isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced protein overexpression, search for new crucial proteins and cytokines involving in its continued infection, and monitor several related signal-regulated pathways which can cause infected host cell to transform in some aspects and explore potential molecular mechanism associated with persistent C. psittac infection. In this project, Series of techniques of molecular biology and immunology including indirect immunofluorescence, RT-PCR, Western Blotting, and Co-immuno precipitation are adopted. Moreover, we will focus on apoptosis suppression and immune escape of host cell and other feasible channels which can make host suffer from C. psittac infection continually. Briefly, my group will perform analysis of apoptotic pathway from the death receptor pathway, mitochondrial pathway and pathway of endoplasmic reticulum, characterize apoptosis related proteins (Bcl-2 family, IAPs and CPAF), and inhibit Raf/MEK/ERK pathway and Phosphoinositide 3-kinase (PI3K)/AKT pathway to detect the apoptotic signals. Furthermore, in vitro A549 cell, PBMC cell and HeLa229 cell are assaulted by C.psittaci to establish models of persistent C.psittaci infection. Then we detect expressive level of CD1d, amount of secreted cytokines (including IL-8, IL-10 and TNF-α) and analyze activity of IκBα, p65/RelA and other functionally related proteins in NF-κB signal pathway and relationship between Nuclear transcriptional factor (RFX5 and USF-1) and MHC (MHCⅠand MHCⅡ), respectively. Generally, the launching of this project will further reveal the molecular mechanism of chlamydial and eukaryotic intracellular environment interactions and provide a scientific basis for blocking the C.psittaci continued infection and design of drugs and new vaccines..