K_(ATP)通道Kir6.2亚基点突变体Kir6.2AAA在大鼠心肌细胞中的表达  

The expression of K_(ATP) channel mutant subunit Kir6.2AAA in rat cardiomyocytes

作　　者：张晓晖[1] 魏春山[1] 林坚[1] 熊益群[1] 徐超英[1] 刘心亮[1] 穆桂萍[1] 徐绍钢[1] 刘文赫[1] 

机构地区：[1]广州中医药大学深圳附属医院中心实验室,广东深圳518033

出　　处：《细胞与分子免疫学杂志》2014年第4期391-395,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学青年基金(81101450)

摘　　要：目的制备含K ATP通道亚基点突变的重组腺病毒,并将其在大鼠心肌细胞中表达。方法针对Kir6.2位点的引物,利用Overlap PCR的方法定点突变Kir6.2的GFG氨基酸变成AAA,并将其克隆到pShuttle载体中进行序列分析,经PmeⅠ线性化、连接到腺病毒表达载体pAdEasy-1中,将pAdEasy-1包装进脂质体、转染入大鼠原代心肌细胞,并利用反转录PCR和Western blot法进行检测。结果成功制备了携带大鼠Kir6.2AAA及EGFP基因的重组腺病毒,病毒的滴度为2.64×1011VP/mL。荧光显微镜下可见Kir6.2AAA重组体腺病毒感染后的大鼠心肌细胞表达EGFP而发出绿色荧光,反转录PCR证实重组腺病毒载体Ad-Kir6.2AAA感染的心肌细胞中Kir6.2AAA的表达显著上调,Western blot法证明Kir6.2AAA在大鼠心肌细胞中过表达。结论成功构建了携带EGFP基因的Kir6.2AAA重组腺病毒载体并将其在大鼠心肌细胞中正确表达。Objective To construct a recombinant adenovirus vector carrying KATP channel mutant subunit Kir6.2AAA and express it in rat cardiomyocytes. Methods Based on the primers for Kir6.2 subunits, Kir6.2 GFG amino acids were site-directed mutated into AAA by means of overlap PCR. PCR products were cloned into pShuttle vector for sequence analysis. After Pine I linearization, it was transformed into adenovirus expression vector pAdEasy-1. Then the pAdEasy-1 was packaged into liposome and transfected into primary cultured rat cardiomyocytes. The expression of Kir6. 2AAA was confirmed by reverse transcription PCR（RT-PCR） and Western blotting. Results The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully, and the virus titer was 2.64 x 1011 VP/mL. After infected by the recombinant adenovirus expressing Kir6.2AAA, rat cardiomyocytes expressed EGFP and emitted green fluorescence under a fluorescence microscope. RT-PCR demonstrated that the expression of Kir6.2AAA was significantly up-regulated in the infected cardiomyocytes, and Western blotting also proved the over-expression of Kir6.2AAA in the cardiomyocytes. Conclusion The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully and expressed correctly in rat cardiomyocytes.

关 键 词：Kir6 2基因 点突变 腺病毒载体 

分 类 号：R392.33[医药卫生—免疫学] Q786[医药卫生—基础医学]