家蚕C型凝集素介导细胞免疫反应的机理研究

Cellular immunity and humoral immunity constitute two major branches of the insect innate immune system. Previous studies have shown that some C-type lectins (CTLs) can function as opsonins to enhance various cellular immunity responses, such as phagocytosis, nodulation and encapsulation, but the mechanism by which CTLs enhance cellular immunity is unclear. Our group has reported the classification and features of all CTLs encoded by the Bombyx mori genome. On this basis, this project plans to choose four secreted CTLs (CTL-S2/S3, IML-1/3) as objects of the study. We plan to construct deletion mutants and site-directed mutants to analyze their binding to hemocytes and bacteria, respectively. We will also analyze their effects on phagocytosis and encapsulation by fluorescence localization, flow cytometry and western blotting, to identify the functional domains and key residues that mediate cellular immunity. The interaction between CTLs and integrin β subunit family members will be analyzed by co-immunoprecipitation, far-Western and RNAi. The unknown membrane receptors will be identified by pull-down assay, mass spectrometry and glycosylation assays. This project will help us to uncover the mechanism by which CTLs mediate cellular immunity. And it will provide theoretical and practical foundations for regulating the immune system of insects for purposes of protection or control.

细胞免疫和体液免疫构成了昆虫先天免疫系统的两个主要分支。前期研究显示某些C型凝集素（CTL）能够作为调理素增强各种细胞免疫反应，如吞噬、集结和包囊，但是对于其增强细胞免疫的作用机理并不清楚。项目组前期已经报道过家蚕基因组所编码的所有C型凝集素的类型和特征，在此基础上，本项目拟以其中4个分泌型CTL（CTL-S2/S3、IML-1/3）为研究对象，构建缺失突变体和定点突变体，通过荧光定位、流式细胞和蛋白印迹分析它们分别与血细胞和细菌的结合，以及对吞噬和包囊的影响，明确序列中参与介导细胞免疫的功能区域和关键位点；通过免疫共沉淀、far-Western和RNAi分析CTL与整合素β亚基成员间的互作关系；通过pull-down联合质谱和糖基化位点分析鉴定CTL的未知膜蛋白受体。本项目有助于揭示C型凝集素参与介导细胞免疫反应的机理，为调控昆虫的免疫系统以达到保护或防治的目的提供理论依据和应用基础。

C型凝集素（C-type lectin）是动植物中普遍存在的一类免疫受体家族，具有糖识别域（Carbohydrate recognition domain），能识别特定的糖基结构，介导各种免疫反应，对抗病原物的入侵。对C型凝集素的研究过去主要集中在病原识别相关的功能，而对其介导不同免疫反应的研究较少。本项目主要研究了家蚕C型凝集素BmCTL10和BmCTL5的免疫功能，研究内容包括：（1）生物信息学分析；（2）基因的发育期、组织和诱导表达模式；（3）重组蛋白表达纯化和免疫功能分析；（4）调控血细胞基因表达；（5）与整合素β亚基的互作。主要结果如下：（1）BmCTL10和BmCTL5分别含有2个和1个CRD结构域，分别属于免疫凝集素（Immulectin）和单CRD类型的C型凝集素。（2）BmCTL10和BmCTL5基因的表达在不同时间点和组织受到细菌、真菌和蜕皮激素的显著诱导，但整体诱导水平比较低。（3）BmCTL10和BmCTL5重组蛋白都可以识别不同病原菌，并定位于血细胞上。BmCTL10能促进血细胞集结、吞噬和包囊反应，BmCTL5能增强血细胞吞噬。（4）注射BmCTL10和BmCTL5重组蛋白能诱导幼虫血细胞中许多免疫相关基因的显著上调。（5）酵母双杂实验证明BmCTL10可以与整合素β3和β4亚基互作，其中CRD1只与β4互作，而CRD2与β3和β4都有互作。BmCTL5全长和截短型蛋白（ΔN，ΔC和ΔNC）都可以和β2-β5互作。本项目揭示了家蚕BmCTL10和BmCTL5除了具有识别病原物的功能外，还可以与血细胞的整合素β亚基互作，增强细胞免疫反应，并调控血细胞中免疫基因的表达，增强体液免疫反应。因此，C型凝集素在家蚕的体液和细胞免疫反应中发挥多种功能。相关研究结果为研究昆虫与病原互作提供了借鉴，为蚕病的防治提供了新思路。