不同类型Cry毒素抗独特型单链抗体分子的杀虫结构特征分析

Bacillus thuringiensis (Bt) a uniquely specific, safe, and effective biological insecticide that has been used for more than 50 years. The increased use of Bt toxins in biological control have encouraged researchers to find novel strains with high specific activity and new functional genes. Consequently, we found some kinds of bioactive peptides depended on the principle of anti-idiotype antibodies with specific structural and functional mimic of antigen in our lab, and the mimic Bt toxin single chain fragment variable (scFv) anti-idiotypic antibody is an new way to develop novel insecticidal protein. However, the low affinity of genetically engineered antibodies was a common deficiency, then it is an important foundation to explore and search for high insecticidal activity of genetic material by systematic researching on differences between structure and bioactivity of different of types scFv anti-idiotypic antibodies. Therefore, the research project will plan to determine which receptors (cadherin, aminopeptide N or alkaline phosphatase) in the midgut brush-border membrane vesicles (BBMV) of the rice leaffolder, Cnaphalocrocis medinalis (Guenée) can bind to to scfv anti-idiotypic antibodies serve as their receptors on the basis of early obtaining different types of Cry toxin (Cry1Ac, Cry1B, Cry1C and Cry1F) scFv anti-idiotypic antibodies. Here, the Bioinformatics methods such as sequence alignment, homology model, molecular docking, molecular dynamics optimization and conformation superposition were used to get commond binding domains for scFv anti-idiotypic antibodies against 4 types of Cry toxin, and structural characterization of similar domains was identified and compared. Moreover, highly bioactive general binding domains were obtained theoretically by analysed and computer-aided design structural characterization of scFv anti-idiotypic antibodies against Bt Cry toxins; additionally the affinity and bioactivity of synthetic peptides were confirmed, thereby higher affinity Ab2β type regional features of key structures were showed. In conclusion, these results will provide the directions of regularity for molecular modification of antibodies.

新的Bt毒素抗虫资源开发是国内外研究的热点。本实验室前期研究发现基于抗独特型抗体可以获得具有模拟特定抗原结构及功能的肽活性材料，抗独特型抗体制备技术是开发新型杀虫蛋白的一个新途径。但基因工程抗体常常存在亲和力低的不足，系统研究不同毒素抗独特型抗体的活性与结构差异是探索获得高活性杀虫基因材料的重要基础。本课题拟在前期获得不同类型Cry毒素（Cry1Ac、Cry1B、Cry1C、Cry1F）抗独特型单链抗体的基础上，确定其与特异靶标（BBMV）结合类型，利用同源建模、分子对接、动力学优化、构象叠加分析，对获得的4类Cry毒素抗独特型抗体同受体蛋白共性结合区域进行结构特征鉴定与比较研究。分析Bt Cry毒素抗独特型抗体分子结构特征，然后借助计算机辅助设计，建立高活性的结合区域；并对人工合成短肽进行亲和性和生物活性验证，从而提示高亲和力Ab2β型关键结构区域特征。为抗体分子改造提供规律性指导。

基因工程抗体常常存在亲和力低的不足，系统研究不同毒素抗独特型抗体的活性与结构差异是探索获得高活性杀虫基因材料的重要基础。在前期获得不同类型Cry 毒素（Cry1Ac、Cry1B、Cry1C）抗独特型单链抗体的基础上，对其进行DNA家族改组，结合噬菌体突变库筛选，获得同昆虫中肠BBMV结合能力更高的Cry毒素Ab2β型Anti-Id scFv新基因E1和G4，氨基酸序列比对表明，E1、G4的氨基酸序列同亲本序列相比，都是在重链和轻链的CDR2、3区发生了突变，而且各自的FR区都未改变，属于人源化抗体。 Cry1B毒素抗独特型单链抗体C7与昆虫BBMV结合能力不高，利用定点饱和突变对其进行分子改造提高亲和力。对C7与受体稻纵卷叶螟APN进行同源建模、分子对接，对获得的C7同APN结合区域进行热点预测。利用C7的热点（A123、Y124、S204和S207）构建定点饱和突变库并筛选，获得突变株Y124G。BIAcore SPR分析表明突变株Y124G与稻纵卷叶螟中肠BBMV具有一定的结合能力，而且Ab2β型Anti-Id scFv突变株对稻纵卷叶螟的杀虫效果较野生型C7显著提高。在人源化抗体分子改造的基础上，扩大杀虫蛋白基因资源的来源动物并进行高效筛选也能获得具有应用价值的抗虫基因，本研究利用将人源抗体动物源化这种改型抗体制备的方式，获得了具有杀虫活性的猪源和牛源杀虫基因资源。通过生物信息学预测猪源和牛源化抗体VH/VL的8个FR，植入Cry1Ab毒素抗独特型单链抗体A12的6个CDR以确定动物源化改型抗体的序列，人工合成猪源化swine-A12-PUC57和牛源化bovine-A12-pUC57这2种改型抗体，并通过替换表达载体获得猪源化swine-A12-pIT2质粒和牛源化bovine-A12-pIT2质粒。结合实验表明动物源化改型抗体swine-A12-pIT2和bovine-A12-pIT2都与小菜蛾中肠BBMV具有一定的结合能力，而且这2种Ab2β型Anti-Id改型抗体对小菜蛾幼虫都具有杀虫活性，但是与亲本抗体A12的杀虫活性相比无显著差异。本研究通过DNA家族改组、定点饱和突变、动物源化改型抗体制备等抗体分子改造策略及高效的鉴定方法探索获得高活性杀虫基因材料的设计制备规律。为抗体分子改造提供新的思路。