绿僵菌素A结合蛋白的分离鉴定与功能研究

Destrxin, a kind of depsicyclopeptidic toxins, is an important pathogenic factor of the entomopathogenic fungus, Metarhizium anisopliae. It is found that destruxin damages the insect host's hemolymph immunity system. However, the secret of the toxicological mechanism of destruxin is not unclosed yet. Originally, we discovered destruxin-A influences silkworm's hemocytes and hemolymph proteome, on the basis, the researches will be focused on the aspects in this proposal: 1) isolating destruxin-A binding proteins by means of affinity chromatography or isotopic tracing, analyzing their amino sequences and homology with other proteins using mass spectrum and bioinformatics techniques, defining their mitifs and domains, then, expressing its gene in Escherichia coli and purifing the protein again. 2) positioning the destruxin-A binding protein in silkworm's tissues and hemocytes with the help of GFP fusion expression and immunofluorescence techniques. 3) making clear the interaction of destruxin-A binding protein and other hemolymph proteins by aid of twohybridsystem and co-immunoprecipitation. In this proposal, we use interdiscipline techniques of chemistry and biology to isolate and identify the destruxin-A binding proteins, to find the key protein and its biological function in host hemolymph immunity regulation network. Through this reseach, the applicants hope to clarify the molecular toxicological mechanism of destruxin acts on insect host's hemolymph immunity system. Meanwhile, with the experiments results, the applicants expect that give some creative thinkings about new insecticides class-insect immunity inhibitants to collegues.

绿僵菌素是绿僵菌产生的环缩羧肽类毒素，能抑制寄主昆虫的血淋巴免疫功能，是绿僵菌的重要致病因子，然而，绿僵菌素作用的分子机理尚不明确。本申请项目在前期明确绿僵菌素A对家蚕血细胞及血淋巴蛋白质组影响的基础上，拟采用亲和层析或同位素示踪技术从家蚕的血淋巴中分离绿僵菌素A结合蛋白，运用质谱与生物信息学技术分析鉴定其氨基酸序列及其与其他蛋白的同源关系，确定其motifs与domains，进而在大肠杆菌中异源表达其基因并分离纯化出该蛋白；通过GFP融合表达及免疫荧光技术，定位该蛋白在家蚕组织与血细胞中的存在部位；应用酵母双杂交与免疫共沉淀技术研究该蛋白与其他血淋巴蛋白的互作关系。本项目运用化学与生物学交叉技术，分离鉴定绿僵菌素A结合蛋白，明确其中的关键蛋白，阐明该蛋白在昆虫血淋巴免疫调控网络中的作用与功能。研究结果可揭示绿僵菌素作用的分子机理，为新型杀虫剂-昆虫免疫抑制剂研发提供新思路。

绿僵菌素A（destruxin A, DA)是一种环缩羧肽类化合物，是金龟子绿僵菌Metarhizium anisopliae产生的主要毒素，在其致病寄主昆虫的过程中发挥重要作用，同时DA也具有较强的杀虫活性，然而，DA作用的分子机理仍不明确。本项目以家蚕Bm12细胞为材料，以DA处理该细胞，然后提取细胞蛋白，再用蛋白酶处理这些蛋白，通过电泳发现了特异性条带，从特异性条带中鉴定了113种蛋白质，然后采用MOE2014软件对这些蛋白进行同源建模与DA分子对接分析，预测其中34种蛋白与DA分子的结合自由能<-10 kCal/mol，展示出较强的结合作用。进而，异源表达了其中结合力预测较强的14种蛋白质，然后采用生物膜光干涉技术Bio-Layer Interferometry (BLI)测定了DA与这些蛋白的结合力，亲和力系数KD值介于10^-4--10^-14M之间，其中，结合力最强的是一种moesin-like蛋白，其KD值达到10^-14M数量级。研究了moesin-like蛋白在家蚕中的时空表达模式，证明该蛋白在家蚕各虫态与各器官组织均有表达，其功能有待进一步深入研究。本研究分离鉴定了DA结合蛋白，发现结合力最强的是一种moesin-like蛋白，该蛋白可能是DA的靶标分子。本研究结果从分子水平提高了对绿僵菌素作用机理的认识。