酵母双杂交文库筛选及N型钙离子通道与BART相互作用的初步分析  

Yeast Two-Hybrid Screening Assay and Preliminary Analysis of the Interaction Between N-type Calcium Channel and BART

作　　者：朱慧琴[1] 于盼盼[1] 林琳[1] 杭勤[1] 陆佩华[1] 黄立东[1] 

机构地区：[1]上海第二医科大学神经生物学实验室,上海市200025

出　　处：《医学分子生物学杂志》2006年第1期22-25,F0003,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.30370492)~~

摘　　要：目的应用酵母双杂交技术筛选N型钙离子通道相关蛋白,并对候选蛋白BART(binder of arltwo)进行初步分析。方法用N型钙离子通道α1亚基C端630个氨基酸残基肽段(Calcium channel630a-mino acids,CaCh630)作为探针,运用酵母双杂交技术筛选出与之相互作用的基因。通过PCR扩增并克隆候选基因BART,然后分别构建带有红色荧光蛋白标签的表达载体pDsRed-BART和带绿色荧光蛋白标签的pEGFP-CaCh630,共转染海马神经元后,用激光共聚焦扫描显微镜观察这两个外源蛋白在细胞中的定位情况。结果应用酵母双杂交技术筛选出6个相关基因,体外共转染试验发现BART和CaCh630分子在海马神经元内有共定位现象,进一步证明其可能存在相互作用。结论BART可能在维持钙离子通道细胞骨架中定位起重要作用。Objective To clone and identify the partners of C-terminus of the N-type calcium channel by using yeast two-hybrid screening assay （ YTHSA）. Methods A peptide with 630 amino acid residues from C-terminus of the N-type calcium channel was taken as a bait to acquire its partners, including BART and other candidates, via YTHSA. Then, the full-length BART gene was cloned into pDsRed2 with a red fluorescent tag, while the fragment of CaCh630 was subcloned into pEGFP with a green one. Both plasmids were co-transfected into hippncampus neurons, and the results were observed via co-localization using laser scanning con-focal microscope. Results Six relative proteins were found to interact with calcium channel molecule via YTHSA. BART, a candidate for partners, was studied, pDsRed-BART and pEGFP-CaCh630 were found to be co-localized in transfected neurons. Conclusion BART molecule might play an important role in stabilizing calcium channels on cytoskeleton.

关 键 词：酵母双杂交技术 钙离子通道 共定位 

分 类 号：Q189[生物学—神经生物学]