NR4A1基因的克隆及真核表达载体的构建  

Cloning of the mouse NR4A1 gene and construction of its eukaryotic expression vector

作　　者：王良国[1,2] 黄晓燕[1,2] 林素[1,2] 潘嘉林[1,2] 戴晓春[1,2] 王本极[1,2] 吴漪浩[1,2] 杨德业[1,2] 

机构地区：[1]温州医学院附属第一医院心内科 [2]温州医学院心血管生物和基因研究所,浙江温州325000

出　　处：《温州医学院学报》2013年第4期232-236,共5页Journal of Wenzhou Medical College

基　　金：浙江省自然科学基金资助项目(Y2110112);温州市科技局科研基金资助项目(Y20100016)

摘　　要：目的 :克隆小鼠孤儿核受体(NR4A1)基因全长cDNA的读码框,构建携带NR4A1基因的重组真核表达载体,为研究其在心肌细胞中的功能做准备。方法:从Pax-8敲除的小鼠心肌组织中提取总RNA,通过逆转录得到总cDNA,利用逆转录PCR法扩增,将目的产物与pGEM-T Easy载体连接,测序分析正确后,再以相同PCR方法扩增,将其与真核表达载体pIERS2-ZsGreen1连接,构建重组质粒。经限制性酶切及测序鉴定后,利用Lipofectamine2000将重组载体pIERS2-ZsGreen1-NR4A1转染到H9C2心肌细胞。实验分三组:①实验组(PZ-NR4A1组):细胞转染1.2μg pIERS2-ZsGreen1-NR4A1重组载体;②阴性对照组(NC组):细胞转染1.2μg pIERS2-ZsGreen1空载体;③空白对照组(BC组):给予的常规培养液,未做其他处理。并用RT-PCR法和Western blotting法检测转染后NR4A1 mRNA和蛋白的表达。结果:成功构建了小鼠pIERS2-ZsGreen1-NR4A1真核表达载体。转染重组载体到细胞后,相比BC组(1.00±0.00)和NC组(0.99±0.16),PZ-NR4A1组(2.62±0.21)NR4A1 mRNA表达水平明显升高(P<0.05),而BC组和NC组mRNA表达水平差异无统计学意义(P>0.05)。PZ-NR4A1组(0.72±0.11)NR4A1蛋白的表达量与BC组(0.17±0.07)和NC组(0.21±0.08)相比,PZ-NR4A1组蛋白表达量明显升高(P<0.05),而BC组和NC组蛋白表达量差异无统计学意义(P>0.05)。结论:通过基因重组技术,成功克隆了NR4A1基因,并构建了真核表达载体pIERS2-ZsGreen1-NR4A1,且能够在H9C2心肌细胞中获得有效过表达,这为进一步探讨NR4A1基因的生物学功能及机制奠定了基础。Objective： To clone mouse orphan nuclear receptors （NR4A1） full-length cDNA and construct its recombinant eukaryotic expression vector for the preparation to study its function in cardiomyocytes. Methods： The total RNA from Pax-8 knockout mice cardiac tissue was extracted. The cDNA was amplified by semi- quantitative PCR before inserted into pGEM-T Easy vector and confirmed by sequence analysis. The pGEM-T Easy vector which contained NR4A1 cDNA was amplified by PCR. The pIERS2-ZsGreenl vector which con- tained NR4A1 cDNA was cloned and constructed. The recombinant vector pIERS2-ZsGreenl-NR4A1 was transfected into H9C2 cardiomyocytes by Lipofectamine2000. The level of expression of NR4A1 mRNA and protein was assayed with RT-PCR and Western blotting. The experiment was divided into three groups： the PZ- NR4A1 group was transfected with 1.2 lag pIERS2-ZsGreenl-NR4A1 recombinant vector, the NC group was transfected with 1.2 lag pIERS2-ZsGreenl empty vector, the BC group was given the conventional common medium without other treatment. Then using RT-PCR method and Westren blotting method to assay the expres- sion of NR4A1 mRNA and protein after transfection. Results： The cDNA of NR4A1 was cloned, and its recombinant eukaryotic expression vector was constructed successfully. After the transfection, the level of expression of NR4A1 mRNA and protein was significantly higher than the level of the BC group （P〈0.05） and NC group （P〈0.05）, while there was no significantly difference between the two groups （P〉0.05）. Conclusion： Through gene recombination technology, the pIERS2-ZsGreen 1-NR4A 1 expression vector is successfully con-structed and effective expressed in H9C2 cardiomyocytes, which can further explore the biological function and mechanism of the NR4A1 gene in cardiomyocytes apoptosis.

关 键 词：NR4A1 真核表达载体 心肌细胞 基因 克隆 

分 类 号：R363.1[医药卫生—病理学]