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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 prepare a recombinant adenovirus containing K ATP channel subunit point mutation and to express it in rat cardiomyocytes. Methods The primer of Kir6.2 was used to detect the GFG amino acid of Kir6.2 by Overlap PCR and then cloned into pShuttle vector for sequence analysis. After linearized by PmeⅠ, it was ligated into the adenovirus expression vector In pAdEasy-1, pAdEasy-1 was packaged into liposomes and transfected into rat primary cardiomyocytes. Reverse transcription PCR and Western blot were used to detect pAdEasy-1. Results The recombinant adenovirus carrying rat Kir6.2AAA and EGFP gene was successfully prepared. The virus titer was 2.64 × 1011VP / mL. The fluorescence of Kir6.2AAA recombinant adenovirus-infected rat cardiomyocytes expressed EGFP under fluorescence microscope, and green fluorescence was detected. Reverse transcriptase PCR confirmed the expression of Kir6.2AAA in Ad-Kir6.2AAA-infected cardiomyocytes Western blot showed that Kir6.2AAA was overexpressed in rat cardiomyocytes. Conclusion Kir6.2AAA recombinant adenovirus vector carrying EGFP gene was successfully constructed and correctly expressed in rat cardiomyocytes.