【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。更多还原

【Abstract】 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.更多还原