rop18 ups参与调控弓形虫毒力因子的分子机制研究

Toxoplasma gondii, an obligate intracellular protozoan parasite, can infect most animals and humans which causes serious public health problems worldwide. T.gondii rhoptry protein ROP18 is a key virulence factor which plays an important role in the invasion and proliferation. Based on the verification that T.gondii rop18 ups inhibits ROP18 protein expression in type Ⅲ strain, this project is trying to make clear of the core area of rop18 ups by the method of deletion mutations and compare the differences between the core area and the regulatory sequences known. Intending to find the protein factor which interacts with rop18 ups by EMSA and analyze its structure and function. To verify the interaction between rop18 ups and the protein factor by the method of gene knockout, real-time RT-PCR and protein expression in vitro, analyze the molecular mechanisms in the regulation of T.gondii virulence factor. This provides a theoretical basis for further understanding the signal pathway of T.gondii virulence regulation and looking for a better method of preventing toxoplasmosis and inspecting animal-origin food safety.

弓形虫是一种世界性分布的专性胞内寄生原虫，严重危害多种哺乳动物和人类健康。弓形虫棒状体蛋白ROP18是虫体重要的毒力因子，在弓形虫入侵、增殖过程中起到关键作用，本项目在验证了弓形虫rop18 ups抑制Ⅲ型虫株ROP18蛋白表达的基础上，拟运用缺失突变等分子生物学手段弄清rop18 ups的核心区域，比较该基因片段与已知调控序列之间的基因结构特征的异同，通过凝胶迁移实验（EMSA）找到与rop18 ups作用的蛋白因子，分析该蛋白结构及其功能，推测该蛋白因子行使功能的可能方式，采用基因敲除、荧光定量RT-PCR、体外蛋白表达等技术验证rop18 ups与该蛋白因子共同作用及其方式，综合分析rop18 ups参与调控弓形虫毒力因子的分子机制，从而为进一步探明弓形虫毒力调控的信号通路、寻求更好的弓形虫病防治手段及动物源性食品安全检测措施提供理论基础。

弓形虫是一种世界性分布的专性胞内寄生原虫，严重危害多种哺乳动物和人类健康。弓形虫棒状体蛋白ROP18是虫体重要的毒力因子，在弓形虫入侵、增殖过程中起到关键作用。本项目的开展完成了以下工作：（1）三种基因型弓形虫rop18基因上游序列差异及其启动子功能异同研究，获得了Ⅲ型虫株rop18 ups序列；（2）弓形虫rop18 ups的功能验证，表明rop18 ups是需要其它转录因子共同调控Ⅲ型虫株ROP18蛋白的表达；（3）探明了弓形虫rop18 ups核心功能区域；（4）获得了与弓形虫rop18 ups相互作用的蛋白因子TgNF4，并分析了其结构功能；（5）验证了rop18 ups与该蛋白因子共同作用及其方式，探明其参与毒力调控作用的分子机制。本项目的研究为进一步探明弓形虫毒力调控的信号通路及分子机制、寻求更好的弓形虫病防治手段及动物源性食品安全检测措施提供理论基础。