摘要
细菌双杂交系统是一种用于检测体内蛋白质互作的方法,该方法互补腺苷酸环化酶功能,通过检测细胞表达的β-半乳糖苷酶LacZ的活性,分析蛋白质互作能力。但在应用过程中,发现存在操作繁琐、灵敏度低、难实现高通量操作等缺陷。本研究目的是对原有细菌双杂交进行优化,建立一种操作方便、可批量操作、具有较高灵敏度和能够实现实时监测的细菌双杂交系统,本研究成功构建由lacZ启动子控制的luxCDABE的报告质粒pBBR-lacZ-luxCDABE,引入原有的检测系统,新的判读标准并不影响原有的判读标准,本文通过快速灵敏地冷光观察菌落或测定菌液冷光值即可判读结果,优化后的双杂交系统,阳性对照组冷光值为阴性对照组的几十(霍乱弧菌为宿主)至几百倍(大肠杆菌为宿主),阴阳性结果判读差异明显,不仅节约实验成本,提高工作效率,又实现了连续监测。该系统不仅以大肠杆菌为宿主获得成功应用,宿主范围也扩展到霍乱弧菌。该系统通过质粒将lacZ启动子的拷贝数提高,减弱了非腺苷三磷酸(cAMP)因素引起的lacZ启动子活性的干扰,无需繁琐耗时的实验操作,为大批量的蛋白质互作分析及互作蛋白质的筛选提供了有利工具。
The bacterial two-hybrid system is a method used to detect protein-protein interaction in vivo.The method is used to analyze protein-protein interaction by supplementing the adenylate cyclase function and detecting the activity ofβ-galactosidase.However,some deficiencies such as complex experimental operation,low sensitivity,infeasibility for the massive samples were identified.The purpose of this study is to optimize the original bacterial two-hybrid system,and to establish an optimized system with convenient operations,batch operation,high sensitivity and real-time monitoring.In this study,we successfully constructed the plasmid pBBR-lacZ-luxCDABE with the luxCDABE reporter gene controlled by the lacZ promoter,which were transformed into this system.The results could be obtained by clone observation or testing the luminescence value rapidly and sensitively.The values of the positive control were dozens(Vibrio cholerae as hosts)to hundreds of times(Escherichia coli as hosts)of those in negative control groups,and there were significant differences between these two groups.The optimized two-hybrid system could save experimental costs,enhance our operation effectiveness and enable real-time monitoring.It was not only successfully used in Escherichia coli as hosts to analyze the interaction of proteins,but also extended the host range to Vibrio cholerae(V.cholerae).The copy number of the lacZ promoter was increased through the plasmid pBBR-lacZ-luxCDABE and the interference from non cAMP factors was weakened.As a result,the sensitivity of the modified bacterial two-hybrid system was significantly improved.These simple experimental operations also provide a favorable tool for large-scale protein-protein interaction analysis and screening of interaction proteins.
作者
樊粉霞
赵文轩
阚飙
FAN Fen-Xia;ZHAO Wen-Xuan;KAN Biao(State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2021年第2期251-258,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(青年科学基金项目No.81501724)资助。
