关键词: 基因表达, 载体, 增强型绿色荧光蛋白, 心肌细胞

Abstract: To construct rat calcineurin catalytic subunit α(CnA)/enhanced green fluorescent protein EGFP gene coexpression plasmid for exploring the effect of calcineurin on the myocardium apoptosis induced by ischemia-reperfusion . Methods Total RNA was isolated from the heart of the adult Wistar rat, and CnA CDS segment of approximate 1 590 bp size was amplified by reverse transcription PCR method. CnA cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-CnA. CnA cDNA segment excised from plasmid T-CnA was ligated with pShuttle2 which had inserted IRES-EGFP segment into before. The DNA of the recombinant plasmid was extracted and was identified by double digesting with Nhe Ⅰ and Not Ⅰ. The right clone was named pShuttle2-CnA-IRES-EGFP. Results Sequencing result verified that the PCR product of CnA gene was identical to GenBank(NM_017041). 1% agarose electrophoresis showed the bands of recombinant plasmid pShuttle2-CnA-IRES-EGFP digested by Nhe Ⅰ and Not Ⅰ were in the right range corresponding with expectation (1 590 bp and 5 240 bp). Conclusion Recombinant eukaryotic expression plasmid carrying rat CnA cDNA as well as a report gene-EGFP gene is successfully constructed in this experiment.

Key words: gene expression, vector, enhanced green fluorescent protein, cardiomyocytes