基于BRET/Nanobit技术的探索性实验教学项目实践  

Practice of exploratory experimental teaching project based on BRET/Nanobit technology

作　　者：史影 郝振杰 SHI Ying;HAO Zhenjie(National Demonstration Center For Experimental Biology Education,College of Life Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学生命科学学院生物国家级实验教学示范中心,浙江杭州310058

出　　处：《实验技术与管理》2024年第9期194-198,共5页Experimental Technology and Management

基　　金：浙江省基础公益研究计划项目(TGC24C050003);国家自然科学基金项目(32070771)。

摘　　要：在科研转化型实践教学基础上,带领本科生探索建立了生物发光共振能量转移和蛋白质结构互补技术模型(BRET/Nanobit),并用以探讨GPCR介导的未知偏向性信号通路。把与课程相关的未知科学问题引入实际教学,对探索性实验项目的开展模式、实验路径、教学成效以及后续衔接方式进行了摸索。实践结果表明,科学问题引导型探索类实验能较大程度地激发学生的学习内驱力和深度思考,有望成为本科生实践教学和创新能力培养的重要内容。[Objective]Based on the research transformation experiments,we introduce scientific problem-oriented exploratory experiments,lead undergraduates to establish new technological models,BRET and Nanobit,and utilize this model to explore unknown GPCR-mediated bias signaling pathways.Through teaching practice,the feasible path and practical effect of such an exploratory project in undergraduate practical teaching and innovation ability training are discussed.[Methods]Rluc8 and GFP2 proteins were used to construct fusion expression plasmids with GPCR andβ-arrestin,respectively,and they were co-transfected into cells.When the GPCR is activated andβ-arrestin is recruited,BRET occurs due to the close proximity of the two proteins,ensuring that the specific fluorescence signal emitted by GFP2 can be detected.At the same time,Gα(i)-GFP2,Gα(s)-GFP2,and Gα(q)-GFP2 were constructed to explore the direct interaction between GPCR and different Gαisoforms.In addition,we constructed the Nanobit test system with interested GPCR,Gα(i)-lgbit,Gα(s)-lgbit,Gα(q)-lgbit,Gβ,and Gγ-smbit.After different concentrations of ligands were added to activate the GPCR,the G protein was activated;that is,the Gαsubunit was dissociated from the Gβγsubunit.Here,the luciferase activity was lost due to the separation of lgbit and smbit,and the fluorescence was reduced.The activation properties of ligands to GPCRS and the type of Gαsubunit that is activated were studied by analyzing the changes in fluorescence.[Results]The BRET curves of FPR1-Rluc8 andβ-arrestin1-GFP2,FPR1-Rluc8 andβ-arrestin2-GFP2,were obtained,along with the EC50 value and action window of the reaction,using the BRET experiment.The results showed that FPR1 interacts with bothβ-arrestin1 andβ-arrestin2,and the affinity withβ-arrestin2 is stronger than that withβ-arrestin1.This can dynamically reflect the recruitment process ofβ-arrestin1 andβ-arrestin2 after the reaction of formyl peptide on FPR1.In addition,the activation of Gαsubunits was obtained through the Na

关 键 词：探索性实验 G蛋白偶联受体 BRET Nanobit 

分 类 号：Q5-33[生物学—生物化学]