Zhang Xiaohui, Wei Chunshan, Lin Jian, Xiong Yiqun, Xu Chaoying, Liu Xinliang, Mu Guiping, Xu Shaogang, Liu Wenhe
Central Laboratory, Shenzhen Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518033, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Apr;30(4):391-5.
To construct a recombinant adenovirus vector carrying KATP; channel mutant subunit Kir6.2AAA and express it in rat cardiomyocytes.
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 Pme 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.
The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully, and the virus titer was 2.64×10(11); 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.
The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully and expressed correctly in rat cardiomyocytes.
构建携带KATP通道突变亚基Kir6.2AAA的重组腺病毒载体，并在大鼠心肌细胞中表达。
根据Kir6.2亚基的引物，通过重叠PCR将Kir6.2 GFG氨基酸定点突变为AAA。将PCR产物克隆到pShuttle载体中进行序列分析。经Pme I线性化后，转化到腺病毒表达载体pAdEasy-1中。然后将pAdEasy-1包装成脂质体并转染到原代培养的大鼠心肌细胞中。通过逆转录PCR（RT-PCR）和蛋白质免疫印迹法证实Kir6.2AAA的表达。
成功构建了携带基因片段Kir6.2AAA和EGFP的重组腺病毒，病毒滴度为2.64×10(11) VP/mL。用表达Kir6.2AAA的重组腺病毒感染后，大鼠心肌细胞表达EGFP并在荧光显微镜下发出绿色荧光。RT-PCR显示感染的心肌细胞中Kir6.2AAA的表达显著上调，蛋白质免疫印迹法也证实心肌细胞中Kir6.2AAA过表达。
成功构建了携带基因片段Kir6.2AAA和EGFP的重组腺病毒，并在大鼠心肌细胞中正确表达。
