多房棘球绦虫钙网蛋白对补体经典途径启动因子C1q的作用及其在虫体免疫逃避中的作用机制研究

Interfering the complement activation plays a key role for pathogens to escape from host immune attack. Recent studies have shown that calreticulin in several parasites is involved in immune regulation of host immune system by binding to complement component C1q. But now, how Echinococcus multilocularis evade the host complement system attack and its mechanisms involved, has not been understood. In this study, the interaction between E. multilocularis calreticulin (EmCRT) will be investigated by using ELISA, MST and co-immunoprecipitation. In order to explore the influence of EmCRT over classical complement activation, the activated intermediates and hemolysis will be quantitated. Moreover, to reveal the effect of EmCRT on the functions of immune effector cells, chemotaxis and production of active substance by neutrophil and mast cell will be detected. Finally, to evaluate the effect of EmCRT on E. multilocularis metacestodes killing, in vitro, the adherence of neutrophils to E. multilocularis metacestodes and passive transfer assay will be analyzed. This research aims to demonstrate that E. multilocularis-expressed CRT binds to C1q, and EmCRT plays critical roles in host complement immune attack, thus, the mechanisms of E. multilocularis evading from immune killing could be clarified from a new perspective.

干扰补体系统的活化在病原体免疫逃避中发挥着关键作用。近年来研究发现某些寄生虫钙网蛋白可通过作用于补体组分而参与宿主的免疫调节作用，但目前关于多房棘球绦虫如何逃避宿主补体系统的杀伤及其作用机制尚不清楚。本项目以多房棘球绦虫钙网蛋白EmCRT与补体经典途径启动因子C1q的相互作用为切入点，拟应用ELISA、MST和免疫共沉淀等方法，明确EmCRT可特异性结合C1q；通过检测补体中间产物的生成，分析EmCRT与C1q的结合对经典途径下游反应的影响；通过中性粒细胞、肥大细胞迁移及活性物质释放实验，研究EmCRT对C1q介导免疫效应细胞功能的影响；通过体外中性粒细胞粘附杀伤虫体及虫体被动转移实验，评价EmCRT在补体介导虫体杀伤中的作用。本研究旨在明确EmCRT与C1q的互作对C1q介导补体免疫杀伤效应的影响，从新的视角揭示多房棘球绦虫免疫逃避的分子机制。

干扰补体系统的活化在病原体免疫逃避中发挥着关键作用。近年来研究发现某些寄生虫钙网蛋白可通过作用于补体组分而参与宿主的免疫调节作用，但目前关于多房棘球绦虫如何逃避宿主补体系统的杀伤及其作用机制尚不清楚。本课题在表达重组多房棘球绦虫钙网蛋白EmCRT的基础上，应用ELISA、Far Western blot和免疫共沉淀等方法明确EmCRT可特异性结合C1q；通过检测补体经典激活途径中间生成产物C4b和C3b的生成及补体介导细胞溶解反应证实，EmCRT与C1q的结合可抑制补体经典途径的活化；通过竞争性结合抑制实验，发现EmCRT与C1q的结合可明显抑制C1q与IgM的结合，初步探讨了EmCRT抑制经典途径活化的机制；通过肥大细胞迁移实验证实，EmCRT可抑制C1q介导的肥大细胞趋化迁移作用。本研究证实EmCRT与补体C1q存在相互作用，二者的互作抑制了C1q依赖的经典途径活化及C1q介导的肥大细胞功能，从新的视角揭示多房棘球绦虫逃避宿主免疫反应的相关分子机制，为包虫病防治新策略拓展思路，同时也为抗包虫疫苗和新药的研制提供重要的科学依据。

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