Salusin-a基因真核表达载体的构建及在HEK293细胞中的表达

目的:研究增强型绿色荧光蛋白真核表达载体Salusin-a(pEG-FP-Salusin-a)的构建及在人胚胎肾细胞系293细胞(HEK293)中的表达。方法:提取人单核细胞系THP-1细胞Salusin-a的mRNA,逆转录多聚酶链反应(RT-PCR)扩增Salusin-a基因,克隆入pEGFP-N3载体,双酶切、菌落PCR及测序鉴定pEGFP-Salusin-a质粒。用脂质体转染pEGFP-Salusin-a入HEK293细胞,分为转染细胞组、质粒对照组和空白对照组,检测分析各组荧光蛋白和Salusin-a基因的表达。结果:成功构建pEGFP-Salusin-a质粒,并转染HEK293,细胞转染效率86%,转染细胞组和质粒对照组绿色荧光蛋白的表达明显高于空白对照组(P0.05);转染细胞组Salusin-amRNA的表达明显高于质粒对照组和空白对照组(P0.05)。结论:重组pEGFP-Salusin-a质粒能转染HEK293细胞并表达Salusin-a,为Salusin-a基因功能的进一步研究提供了实验基础。

Research on Salusin-a Gene of Eukaryon Expression Vector Constuction and Expression in HEK293 Cells

Objective:To study the eukaryotic green fluorescent protein expression vector Salusin-a(pEGFP-Salusin-a)plasmid construction and its expression in HEK293 cells.Method:We amplified the Salusin-a gene by RT-PCR,and cloned into multiple clone sites of pEGFP-N3 vector.The pEGFP-Salusin-a recombinant plasmids were digested by restriction enzyme,bacterial-colony PCR and sequencing were used for identifying.The efficiency of pEGFP-Salusin-a transfected into HEK293 cells by liposome was checked.The HEK293 cell were then randomly divided into three groups:control group,empty plasmid transfection group,and recombinant plasmid tansfection group.The Salusin-a gene expression in HEK293 cells was analysised by fluorescence intensity and RT-PCR.Results:The pEGFP-Salusin-a plasmid was successfully constructed.The efficiency of pEGFP-Salusin-a transfected into HEK293 cells was about 86%.And we found that both fluorescence intensity and Salusin-a mRNA of empty plasmid transfection group and recombinant plasmid tansfection group were significant higher than the control group.Conclusion:The pEGFP-Salusin-a can be transfected into HEK293 cells and expressed,which provide a experimental base to research the Salusin-a gene function.