基于Bt蛋白-昆虫受体识别机理的仿生亲和聚合物纳米颗粒的研制及其环境分析应用

Wide attention has been paid to the human health, environmental and ecological risk caused by the release of insecticidal crystal proteins from the transgenic bacillus thuringiensis (Bt) plants and biological insecticides. So, it is very urgent for us to develop a rapid, accurate, efficient and specific sample preparation method to monitor Bt proteins in environment and ecosystem. The study on insecticidal mechanism indicates that the binding between Bt protein and its receptor protein in target insects is specific, and the binding site is located on the epitope of Bt protein, namely loop 2, loop 3 or loopα-8 of domain II. The high specificity is attributed to the complementary multiple interaction between Bt protein epitope and receptor epitope. In this project, a series of polymer nanoparticles with biomimetic characteristics of the insect receptor protein are proposed to synthesize according to the hydrophobic, hydrophilic, positive charge and negative charge characteristics of the epitope of Bt proteins. The developed bioaffinity polymer nanoparticles are then used for the specific recognition, extraction, separation and purification of Bt proteins in environmental samples and transgenic Bt plants. This research can also provide an effective analytical method for the study of the environmental fate of Bt protein, and finally realize the monitoring of Bt protein in environment and ecosystem. An intensive research on molecular recognition mechanism is also intended to carry out by quartz crystal microbalance, phage display peptide library and recombinant protein technology. The result can help us to locate the real binding site between Bt proteins and the polymer nanoparticles, and thus illustrate the rationality and scientificity of developing bioaffinity polymer nanoparticles on the base of Bt protein-insect receptor recognition mechanism.

Bt蛋白引起的人类健康和环境生态风险问题要求我们对其实行严格、有效监控，而样品前处理方法的好坏是决定其分析监控成败的关键。Bt蛋白杀虫机理研究表明：Bt蛋白与靶标昆虫受体蛋白之间的结合位点位于Bt蛋白抗原表位，即其结构域 II的三个突环上，其特异性源于两者表位间互补的多重相互作用。本项目依据Bt蛋白抗原表位的疏水性、亲水性和电荷性特点，设计合成与其氨基酸序列特点相匹配的聚合物纳米颗粒，使其具有昆虫受体蛋白仿生特点，以用于环境样品和转基因作物中Bt蛋白的特异性识别、提取、分离和纯化，为Bt蛋白环境归趋研究奠定方法基础，最终实现对环境和生态系统中Bt蛋白的有效监控。运用石英晶体微天平、噬菌体展示肽库技术和蛋白质重组技术等手段深入探讨Bt蛋白在聚合物纳米颗粒表面的分子识别机理，准确定位其结合位点，以阐明基于Bt蛋白-昆虫受体识别机理设计合成仿生亲和聚合物纳米颗粒的合理性和科学性。

Bt蛋白引起的人类健康和环境生态风险问题要求我们对其实行严格、有效监控，而样品前处理方法的好坏是决定其分析监控成败的关键。研究表明，Bt蛋白与靶标昆虫受体蛋白之间的结合位点主要位于Bt蛋白抗原表位，即其结构域II的三个突环loop α8 280FRGSAQGIEGS290, loop 2 368RRPFNIGINNQQ379和loop 3 436FRSGFSNSSVSIIR449上，其特异性源于两者表位间互补的多重相互作用。鉴于此，本项目提出了一条构建聚合物仿生亲和配体的新途径，即模拟抗原-受体蛋白之间的识别机理，以抗原表位为作用“靶标”或“诱饵”定向合成和筛选聚合物纳米颗粒，使筛选出的聚合物纳米颗粒具有受体蛋白的仿生特点，从而实现目标抗原Bt蛋白的特异性识别和捕获。目前完成的工作主要包括：基于Bt Cry1Ac/Ab蛋白-烟草天蛾受体类钙粘蛋白Bt-R1识别机理——以Bt蛋白抗原表位368RRPFNIGINNQQ379为作用靶标——的仿生亲和聚合物纳米颗粒的研制及作用机理研究；Bt蛋白仿生亲和聚N-丙烯酰基甘氨酸、丙氨酸、缬氨酸和苯丙氨酸纳米颗粒的研制及作用机理研究；以烟草天蛾受体类钙粘蛋白结合位点865NITIHITDTNNK876代表的多肽为功能单体的Bt蛋白仿生亲和聚合物纳米颗粒的研制；以斜纹夜蛾受体氨肽酶N结合位点128HLHFHLP134代表的多肽为功能单体的Bt蛋白仿生亲和聚合物纳米颗粒的研制；基于聚合物仿生亲和吸附剂材料的Bt蛋白样品前处理方法的建立及在水和土壤分析中的应用研究；基于聚合物纳米颗粒“塑料”抗体的Bt蛋白酶联免疫试剂盒的研制及在转基因作物分析中的应用研究。该项目的实施为环境和转基因作物中Bt蛋白的特异性提取、分离、纯化和检测提供了可靠的分析方法，为转基因作物和生物农药中Bt蛋白的有效监控和环境归趋研究奠定了基础，同时也为其他蛋白类生物毒素的分子识别，分离纯化和吸附去除研究提供了理论依据和实践指导经验。