人类G蛋白偶联鲜味受体T1R1/T1R3纳米-辣根过氧化物酶电化学型传感器的研制

G protein coupled receptors (GPCRs) constitute a receptor superfamily, which can perform various biofunctions, especially the nutrient sensing. GPCRs have extremely huge diversity in extracellular domains, but precise conservative in the intracytoplasmic domains for G protein signal amplification system. In this project, we plan to fabricate the nanometer gold - sulfur cordierite-chitosan-horseradish peroxidase as signal amplifier system, on the basis of nano gold sol. coupling to GPCR-multiple thiols of T1R1 + 3 intracellular domains expressed and separated from the hT1R1/T1R3 transgenic human embryonic kidney cells (HEK239). While the interactions between receptors and their ligands take place, the changes of electrochemical signals will be amplified by the horseradish peroxidase instead of the G protein signal amplifier system. This can be used for the quantitative determination of receptor-ligand interaction. The stages of fabrications will be characterized by transmission electron microscopy (TEM), atomic force microscope (AFM) and UV–visible spectral scanning, cyclic voltammograms and ac impedance methods. With this, the novel electrochemical type nano gold double membrane human taste sensors will be developed. Then the biosensor can be used for quantitative determination of umami substances, such as monosodium glutamate (MSG), aspartic acid, inosinic acid (IMP) and guanosine monophosphate (GMP); and central metabolic metabolic intermediates, such as α-ketone glutaric acid, pyruvic acid, lactate and others. The interaction parameters and kinetics of these substances with T1R1, T1R3, and their heterodimers can be obtained, which would help to investigate the interaction regulation of umami substances, including amino acids, peptides, IMP and GMP, with their corresponding targets. The nano biosensor can also be used as a high sensitive and specific quantitative method to explore biological functions of the umami substances. The establishment and perfection of this biosensor may provide a generic technology platform for investigating the numerous GPCRs and the laws of the ligand-receptor interactions.

本项目拟在纳米金-硫堇-壳聚糖-辣根过氧化物酶信号放大系统的基础上，通过纳米金溶胶偶联经人胚肾细胞(HEK239细胞)表达得到的人类鲜味G蛋白偶联受体-T1R1+3胞内结构域的多个巯基，从而放大其胞外结构域与配基之间的相互作用，替代G蛋白信号放大系统，实现受体-配基互作的定量化测定。利用光谱扫描、原子力显微镜、循环伏安及交流阻抗等方法表征组装的各阶段，研制出电化学型双层纳米金人类鲜味传感器。将该电极用于谷氨酸单钠盐、天冬氨酸、肌苷酸和鸟苷酸等鲜味物质，α-酮戊二酸、丙酮酸、乳酸等中心代谢途径中间化合物的定量测定；建立这些味觉组分或代谢中间化合物与hT1R1/T1R3及其异二聚体的互作参数和动力学，为进一步研究鲜味物质（包括氨基酸、肽类、IMP和GMP）与味觉受体作用规律，进行生物功能评价等提供高敏感、特异性、定量化检测方法。该技术的建立将有望为GPCR-配基互作规律的研究提供通用的技术平台

本项目在纳米金-硫堇-壳聚糖-辣根过氧化物酶信号放大系统的基础上，通过纳米金溶胶偶联经人胚肾细胞(HEK239细胞)表达得到的人类鲜味G蛋白偶联受体-T1R1+3胞内结构域的多个巯基，从而放大了胞外结构域与配基之间的相互作用，替代细胞内G蛋白信号放大系统，成功研制出电化学型纳米金双膜人类鲜味传感器，用于谷氨酸单钠盐、天冬氨酸、肌苷酸和鸟苷酸等鲜味物质，α-酮戊二酸、丙酮酸、乳酸等中心代谢途径中间化合物的定量测定;建立了这些味觉组分或代谢中间化合物与hT1R1/T1R3及其异二聚体的互作参数和动力学，进一步将这些受体自组装到相应受体天然缺陷型武昌鱼、牛、猫等动物味蕾组织上，和相应纳米金受体传感器进行了比较分析，其皮尔逊相关系数达到0.93以上，说明这些hGPCRs通过自组装到相应天然受体缺陷型味蕾组织上能够很好地工作。该技术的建立将有望为GPCR-配基互作规律的研究提供通用的技术平台。本项目共发表论文学术论文21篇，其中SCI收录论文15篇；EI收录4篇，按照中科院2019年JCR期刊分区：I区5篇，II区3篇。IF=10.257论文2篇，IF=7以上2篇，SCI总影响因子达到67.634。申请发明专利3项；获得授权发明专利2项。