大鼠SLC7a8基因的克隆及真核表达载体的构建  

Cloning of rat SLC7a8 gene and construction of eukaryotic expression vector

作　　者：王本极[1] 黄晓燕[1] 林素[1] 周希[1] 戴晓春[1] 方俊旭[2] 杨德业[1] 

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

出　　处：《温州医学院学报》2011年第6期515-519,共5页Journal of Wenzhou Medical College

基　　金：温州市科技发展计划基金资助项目(S2002A132)

摘　　要：目的:克隆大鼠左旋氨基酸转运体(SLC7a8)基因cDNA的读码框(CDS),构建携带SLC7a8基因的重组真核表达载体,为进一步研究其在自发性高血压大鼠(spontaneously hypertensive rats,SHR)中的功能作准备。方法:从非高血压大鼠(wistar-kyoto,WKY)肾脏组织提取总RNA,通过RT-PCR得到总cDNA,PCR法扩增,产物连接pGEM-T Easy载体测序分析正确后,再以PCR方法扩增,将其连接入真核表达质粒pcDNA3.1(+)。以PCR、双酶切和测序鉴定正确后,将重组载体pcDNA3.1(+)-SLC7a8用脂质体包裹转染大鼠肾小管上皮细胞(NRK-52E),RT-PCR法及Western blotting法分别检测转染后SLC7a8 mRNA及蛋白表达水平。结果:成功克隆了大鼠SLC7a8基因cDNA,重组真核表达载体pcDNA3.1(+)-SLC7a8构建成功,且证实转染后肾小管上皮细胞的SLC7a8 mRNA及蛋白表达均明显高于空白对照组(P<0.05)及空质粒组(P<0.05)。结论:成功克隆了SLC7a8基因,并构建了真核表达载体pcDNA3.1(+)-SLC7a8,能够在NRK-52E细胞中获得有效过表达,这为进一步探讨SLC7a8基因的生物学功能奠定了基础。Objective： To clone rat SLC7a8 gene cDNA and construct its eukaryotic expression vector for its function study in SHR.Methods： Total RNA was extracted from rat kidney tissue.SLC7a8 cDNA was prepared by RT-PCR,and inserted into pGEM-T Easy vector and confirmed by sequence analysis.Then the pGEM-T Easy vector which contained SLC7a8 cDNA was also amplified by PCR,and cloned into pcDNA3.1（＋） plasmid.The recombinant plasmid was confirmed by PCR,double enzyme digestion analysis and DNA sequencing.Then the recombinant plasmid pcDNA3.1（＋）-SLC7a8 was transiently transfected into NRK-52E cells using Lipofectamin tm 2 000.The level of expression of SLC7a8 mRNA and protein was identified by RT-PCR and Western blotting after transfection.Results： The cDNA of SLC7a8 was cloned,and its recombinant eukaryotic expression vector was constructed successfully.Over-expression of SLC7a8 mRNA and protein was observed in NRK-52E cells transfected with SLC7a8 gene as compared to the blank control cells and the cells transfected with an empty vector（both P 0.05）.Conclusion： The pcDNA3.1（＋）-SLC7a8 expression vector was successfully constructed and over-expression of SLC7a8 gene in NRK-52E cells was obtained.The recombinant eukaryotic expres-sion vector will be used to investigate the biologic role of SLC7a8 in hypertension.

关 键 词：真核表达载体 SLC7a8 肾小管上皮细胞 基因 大鼠 克隆 

分 类 号：R544.1[医药卫生—心血管疾病] Q78[医药卫生—内科学]