CFP荧光蛋白文库构建及FRET技术筛选钙调素结合蛋白  

Construction of CFP-labeled Protein Library and Screening Calmodulin Binding Proteins by FRET Technology

作　　者：孟凡力[1,2] 关珊[1] 刘晓进[1] 李朝军[3] 

机构地区：[1]南京师范大学生命科学学院,南京210023 [2]南京医科大学基础医学院,南京211166 [3]南京模式动物研究所,江苏省分子医学生物技术重点实验室南京大学医学院,南京210093

出　　处：《中国细胞生物学学报》2013年第12期1779-1785,共7页Chinese Journal of Cell Biology

摘　　要：钙调素(Calmodulin,CaM)是细胞内Ca2+信号的主要受体,能够与靶蛋白相互结合调节靶蛋白的活性,在细胞增殖、分化、凋亡、迁移等过程中都起着重要作用。荧光共振能量转移(f luorescence resonance energy transfer,FRET)技术是目前研究蛋白质相互作用比较成熟的方法之一。作者通过Cre-loxP位点特异性重组技术构建了带有CFP荧光蛋白标记的文库,与YFP-CaM共同转染HEK293细胞,应用荧光共振能量转移技术(FRET)进行检测,挑取发生FRET作用的单个细胞,并进行单细胞PCR检测。由此扩增出的片段通过测序和蛋白序列数据库NCBI进行序列比对后,筛选出与CaM产生相互作用的蛋白。目前,已经通过这种方法成功地筛选到了一些与CaM相结合的蛋白,从而为进一步研究CaM蛋白在生理环境下的作用提供有利条件。Calmodulin （CAM）, the main receptor for intracellular Ca2＋ signals, regulates the activity of its target proteins by interacting with them and plays an important role in the cell proliferation, differentiation, apop- tosis, migration, etc. Fluorescence resonance energy transfer （FRET） technology is one of the mature methods for studying proteins interactions. By applying Cre-loxP site-specific recombination technology we constructed CFP- labeled library, cotransfected HEK293 cells with YFP-CaM plasmids, and used fluorescence resonance energy transfer （FRET） technology to detect the protein interaction. We picked up the cells which generated FRET and applied single-cell PCR detection. By sequencing the PCR products and comparing them with the database NCBI, we screened the unknown proteins which interacted with CaM. Taken together, we have found some CaM binding proteins through the construction of CFP-labeled protein library and applying the FRET technology. Our study provides the conditions for further study of CaM protein in physical environment.

关 键 词：钙调素 CRE-LOXP 荧光共振能量转移 单细胞PCR 

分 类 号：Q51[生物学—生物化学]