对虾隐蔽态T-2毒素的多维表征及其依赖JAK/STAT通路的免疫毒性分子标记识别

Trace residues of T-2 toxin in shrimp causes immune toxicity in mice, this abnormal phenomenon reflects that there are masked T-2 toxins（mT-2s）existed in shrimp. mT-2s has become a potential food safety hazard. The multi-dimensional characterization and the toxicity molecular mark recognition of mT-2s have been the the bottlenecks of risk assessment because of the lack of cognition of mT-2s. In this project, by preparing A-family specific antibody against the modified parent nucleus structure of T-2 toxin, building immunomagnetic purification technology and ELISA quantification technology, quantifing the whole/monomer components of shrimps seperated by immune-fishing and HSCCC (high-speed counter-current chromatography) with LC/MS/MS and NMR(nuclear magnetic resonance), and analyzing the change rules of shrimps before and after heat/enzyme/acid and base treatment, mT-2s in shrimps are characterized multi-dimensionally. For the whole/abundance components, with RAW264.7 mononuclear macrophages being models, MTT method is applied to determine the ED50 of mT-2s that affects the cell proliferation, and real-time fluorescent quantitative PCR(RFQ-PCR) and Weatern blot method are adopted to detect the family genes and protein expression in the JAK/STAT(Janus kinase- signal transducer and activator of transcription) signal pathway to find out the family members that have dose-dependent effect, dertermin molecular markers of shrimp mT-2s immunotoxicity and provide scientific basis for molecular toxicological evaluation of mT-2s.

T-2毒素痕量残留的对虾引起小鼠免疫毒性反应，该异常现象说明对虾中存在具有免疫毒性的隐蔽态T-2毒素（mT-2s）。mT-2s已成为食品安全隐患。但对mT-2s的认知缺乏，其多维表征及毒性分子标记识别成为风险评估的瓶颈问题。本项目以修饰后T-2毒素母核结构为A族抗原表位，制备A族特异抗体，结合免疫磁珠，建立mT-2s免疫磁净化ELISA定量技术，以串联质谱和核磁鉴定，定量检测经免疫垂钓和高速逆流色谱分离的染毒对虾中整/单体组分，软件分析经热/酶/酸碱等处理组的前后变化规律，多维表征对虾mT-2s。针对整体/高丰度组分，以RAW264.7单核巨噬细胞为模型，用MTT法确定其抑制细胞增殖的ED50，用实时荧光定量 PCR和免疫印迹法分别检测JAK/STAT信号通路家族基因和蛋白表达，找出具有剂量依赖效应的家族成员，确定对虾mT-2s免疫毒性的分子标记，为mT-2s分子毒理学评价提供科学依据。

隐蔽态T-2毒素（mT-2s）残留及其危害识别始终是准确评估T-2毒素引发食品安全风险的难题，主要难点在于mT-2s组成复杂、理化性质各异、残留量少但危害较大。课题组在T-2毒素系统研究基础上，对mT-2s进行了深入探讨。课题组先以修饰后T-2毒素母核结构为A族抗原表位，制备A族特异抗体，结合免疫磁珠，建立mT-2s免疫磁净化ELISA定量技术，申报专利3项，获得2项；再利用ELISA试剂盒分析了对虾中mT-2s整体组分的分布规律，体外合成了两种mT-2s的单体物质，即脱甲基T-2（已获得专利）和T-2-葡萄糖苷酸（T-2-GluA），然后从酶/酸碱/高压角度，多维表征了对虾中整体组分和目标单体组分的免疫毒性，发现人工肠液能够使mT-2s解离为前体毒素，并从小鼠免疫器官指数、免疫细胞（RAW264.7）和免疫分子方面解析其毒性。mT-2s能够引起小鼠肝肾脏功能、巨噬细胞吞噬功能下降。首次发现mT-2s和T-2对RAW264.7中JAK/STAT 信号通路家族成员（细胞炎性因子、负反馈调节因子、信号通路关键分子）的影响不同。与T-2 和 T-2-GluA相比，肝胰腺和肌肉mT-2s对STAT2的 mRNA表达增强，肝胰腺中SCOSs, IL-6和 IL-1β的mRNA表达比肌肉中的强。肌肉中mT-2s增强STAT3磷酸化但是抑制STAT1的磷酸化。 肝胰腺中mT-2s对JAK/STAT 信号通路的增强作用比肌肉中的显著。有关磷酸化水平，T-2显著诱导JAK1 和 STAT2的磷酸化，而肌肉、肝胰腺中mT-2s和T-2-GluA分别诱导JAK3、JAK2、STAT1的磷酸化。总体来说，T-2 和 mT-2s均有细胞毒性，但是T-2对RAW264.7中JAK/STAT 信号通路免疫分子毒性比对虾中mT-2s的强。研究成果共发表论文13篇，其中SCI收录11篇；培养研究生6名。