大鼠CnA/EGFP基因共表达的真核表达质粒的构建  

Construction of rat CnA/EGFP recombinant eukaryotic gene coexpression plasmid

作　　者：沈小梅[1] 张巨艳[2] 成蓓[1] 

机构地区：[1]华中科技大学同济医学院协和医院心血管内科,湖北武汉430022 [2]山西医科大学第一附属医院老年病科,山西太原030001

出　　处：《吉林大学学报（医学版）》2007年第1期25-28,共4页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30170378)

摘　　要：目的:构建大鼠钙调磷酸酶A亚基(CnA)α基因/EGFP共表达的真核表达质粒,为研究钙调磷酸酶基因在缺血缺氧再灌注诱导的心肌细胞凋亡中的作用提供生物表达系统。方法:RT-PCR方法扩增大鼠CnA基因(约1 590 bp),克隆至T载体,经酶切鉴定和DNA测序证实CnA序列正确后,再用内切酶将CnA从T-CnA质粒中切出,与已插入报告基因IRES-EGFP基因片段的重组pShuttle2载体(pShuttle2-IRES-EGFP)连接,构建CnA/EGFP共表达的真核表达质粒pShuttle2-CnA-IRES-EGFP。结果:DNA测序结果显示扩增的CnA DNA序列与相应的GenBank(NM_017041)所公布的序列完全相同;NheⅠ和NotⅠ双酶切重组质粒pShuttle2-CnA-IRES-EGFP,1%的琼脂糖凝胶电泳分析可见大小分别为1 590和5 240 bp两条片段,与预期值相符。结论:成功构建了以增强型绿色荧光蛋白(EGFP)标记的大鼠CnA基因的真核表达质粒。Objective 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 eDNA 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.

关 键 词：钙调磷酸酶 基因表达 载体 增强型绿色荧光蛋白 心肌细胞 

分 类 号：Q781[生物学—分子生物学] Q782