黄曲霉毒素通用抗体特异性机制及其与半抗原结构的关联性

Contaminants such as mycotoxins, threatening heavily the national agro-products safety, are usually a group of compounds with high toxicity. Their antibodies in the current screening assays recogernize just one or a small number of them, and the range of these antibodies' specific recognitions is narrow. To solve the problem that it is difficult to design a general specific antibody, an effect of hapten attachment site (on its antibody's specificity) was previously investigated and discovered, and the 4 kinds of aflatoxin haptens and the 47 kinds of monoclonal antibodies induced by them were prepared. Based on these, cross-reactivities to a series of aflatoxins (B, G, M, P, Q, etc) and the variable sequences of the prepared antibodies will be tested, and then, we will search for the amino acids contributed on the corresponding specificity difference through comparison. with the data including the antibodies' sequences, the cross-reactivities, and the inflexible structure of the targets, the homologous modeling and molecular docking will help us to search for the key sites and main forces of the antibody-target molecular interactions and to clarify the molecular mechanism of the specificities. Through the horizontal (among those antibodies induced by the same antigen) and the vertical (among those antibodies induced by different antigen) comparisons of the specificity mechanisms, we will try to explore the internal relationship between the specificity mechanisms and the immunizing hapten structures. Finally, some antibodies against the early warning target molecules (precursors) for aflatoxin formation will be desinged, prepared, and their specificities will be tested to verify the scientificalness of the developed theory. The results will become a guidance for a development of general specific antibodies against contaminants such as other mycotoxins.

严重威胁我国农产品安全的真菌毒素等污染物常为一类毒性大、结构类似的化合物，现有筛查方法中抗体往往仅识别其中一种或少数几种，抗体特异性识别范围窄。针对污染物通用抗体分子设计难题，前期研究揭示了半抗原连接臂位点效应，同时积累了4种黄曲霉毒素半抗原偶联物及其诱导的47种单克隆抗体，在此基础上，测定抗体与系列黄曲霉毒素（B、G、M、P、Q等）交叉反应率和抗体可变区序列，经比较，寻找引起特异性差异的氨基酸位点；利用抗体序列、交叉反应率和靶分子刚性结构，经同源建模与分子对接，探寻抗体与靶分子互作关键位点与主要作用力，解析特异性分子机制；通过对特异性机制纵向（同一抗原诱导的抗体之间）和横向（不同抗原诱导的抗体之间）比对分析，探寻抗体特异性机制与诱导抗原结构之间内在联系；以黄曲霉毒素发生预警靶分子（前体物）为对象，经抗体设计、制备、特异性测定等，验证理论科学性，为其它真菌毒素等通用抗体研制提供理论指导。

项目组按照项目任务书计划执行，全部圆满完成了项目计划任务，达到或部分超额完成预期考核目标。针对污染物通用抗体分子设计难题，在前期研究基础上，通过对黄曲霉毒素B1、G1、M1累计共60株单克隆抗体的半抑制浓度（IC50）、交叉反应率特性、功能区序列比对、同源建模、分子对接等研究工作，解析了黄曲霉毒素通用抗体特异性分子机制，探明了抗体重链变异较大的可变区序列于抗体IC50，而抗体轻链相对保守的可变区序列则贡献于抗体的簇特异性（即通用抗体特性），其中轻链L106等位点成为黄曲霉毒素通用抗体关键位点。通过深入研究抗原-抗体互作关系，探明了抗原结构与抗体灵敏度及特异性之间呈现的共性规律，归纳总结提出了诱导广谱特异性（通用）抗体的抗原分子设计一般规则（抗原设计连接臂位点选择规则）:抗原-抗体分子互作中，与半抗原连接臂位点反向的靶标分子基团总是深陷在抗体活性区内侧，因此研制簇特异性抗体（即一类小分子通用抗体），连接臂位点应选择在与一类物质共性基团反向的靶标分子末端基团上，检测抗原应该避免采用与免疫抗原完全相反的链接方向。在此基础上，指导研制出黄曲霉毒素生物合成前体杂色曲菌素通用抗体、黄曲霉毒素G族通用抗体、辣椒素类通用抗体和I型菊酯通用抗体、黄曲霉毒素纳米抗体共6种，系列抗体特性与设计预期基本一致，从而验证了上述理论的科学性。这一发现为其它真菌毒素等通用抗原设计与抗体研制提供重要理论指导，同时为这些污染物快速检测方法研发与应用提供了自主核心材料。项目部分成果实现了转化或应用（用于毒素与农残检测技术）；在Analyical Chemistry等发表SCI收录论文5篇(均已第一标注本项目编号)，出版著作2部；申请中国发明专利6件、获得授权中国发明专利2件、欧盟与美国专利4件；培养博、硕士研究生6名，其中已毕业3名；应邀在美国等地做国际学术会议报告6次，先后出任SCI期刊Current Organic Chemistry和World Mycotoxin Journal专刊主编；获得了中国专利优秀奖和美国化学会会员奖等各类奖励共4项。