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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
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)
