大鼠Smac基因的克隆及其在心肌细胞H9C2中的表达

Cloning of rat Smac gene and its expression in cardiocyte H9C2

目的:克隆大鼠促凋亡基因Smac,构建真核表达载体pcDNA3.1+-rSmac,并在心肌细胞中表达。方法:应用逆转录聚合酶链式反应(RT-PCR)技术从大鼠肾组织中扩增到大鼠Smac基因的CDs片段,构建含Smac基因的真核表达载体pcDNA3.1+-rSmac,并将此重组质粒、空载体pcDNA3.1+、pcDNA3.1+-EGFP通过脂质体介导转染大鼠心肌细胞H9C2,荧光显微镜、流式细胞术间接检测转染效率,RT-PCR法检测外源基因的表达。结果:经测序目的基因序列与GenBank(NM_001008292)公布的结果一致,所构建的重组质粒pcDNA3.1+-rSmac经PCR和酶切鉴定所释放的片段大小与预期结果相符。荧光显微镜和流式细胞术检测,细胞转染有效,转染效率达38.36%。转染重组载体pcDNA3.1+-rSmac 48 h后,H9C2细胞Smac的mRNA水平明显升高。其中,对照组Smac/-βactin为1.19,空载体组Smac/-βactin为1.31,二者之间差异无显著性(P>0.05);转染pcDNA3.1+-rSmac组Smac/-βactin为1.67,与对照组比较差异具有显著性(P<0.01)。结论:克隆大鼠Smac基因成功,成功构建重组真核表达载体pcDNA3.1+-rSmac,并在转染后的心肌细胞中表达,为研究Smac基因在心肌细胞凋亡过程中的调控作用奠定基础。

Objective To clone the rat second mitochondria-derived activator of caspase （rSmac）, construct its eukaryotic expression vector, and express it in cardiocytes. Methods The rSmac CDs gene was amplified from the rat kidney tissue by reverse transcriptase-polymerase chain reaction （RT-PCR）. It was cloned into pcDNA3.1^＋ vector to construct the recombinant plasmid pcDNA3, l^＋ -rSmac. Then the recombinant plasmid pcDNA3. 1^＋ -rSmac, pcDNA3.1^＋ , and pcDNA3.1^＋ -EGFP were transfected into the rat myocardial cell line, H9C2 mediated by lipofectamine 2000. Among them, pcDNA3. 1^＋ -EGFP was used to detect the transfection efficacy indirectly through fluorescence microscoy （FM） and flow cytometry （FCM）. After transfection, RT-PCR method was used to detect the expression of exogenous Smac gene. Results The sequencing result indicated that the acquired sequence was in concordance with that published on GenBank. The recombinant plasmid pcDNA3. l^＋ -rSmac was idenfied by PCR and enzyme digesting, and the results were coincident with anticipation. The results detected by FM and FCM showed that the transfection was effective, and the efficacy was up to 38.36%. The mRNA level was increased after transfection with the recombinant plasmid pcDNA3.l^＋ -rSmac for 48 h, and the ratios of Smac/β-actin in control, vacant vector, and recombinant plasmid pcDNA3.l^＋ -rSmac group were 1.19, 1.31,1.67, respectively （P〉0.05, P〈0.01 vs control group）. Conclusion The rSmac gene is cloned and the recombinant plasmid pcDNA3.1^＋ -rSmac is constructed successfully. Moreover, it could be effectively expressed in transfectant H9C2, which establish fundament and benefit the further study on the mechanism of Smac in apoptosis of cardiocytes.