SV2A基因真核表达质粒构建及其在HEK293T细胞中的表达

目的构建鼠突触囊泡蛋白2A(SV2A)基因的真核表达质粒,并瞬时转染至人胚肾细胞(HEK293T)中,对其表达进行鉴定。方法以APP/PS1双转基因小鼠海马组织的cDNA为模板,扩增得到长2239 bp的SV2A基因编码序列,将此序列插入到真核表达载体p3×Flag-CMV-10多克隆位点区域中,得到真核表达质粒p3×Flag-CMV-10-SV2A,转化后挑取单克隆菌落经双酶切鉴定后送公司测序,将构建成功的重组质粒转染至HEK293T细胞中,利用蛋白质印迹法(Western blot)检测SV2A基因的表达情况。结果成功构建p3×Flag-CMV-10-SV2A重组质粒,并在转染至HEK293T细胞后,验证了相应蛋白表达。结论利用分子克隆技术成功构建了p3×Flag-CMV-10-SV2A真核表达质粒并在HEK293T细胞中正确表达,为后续实验奠定了基础。

Construction of eukaryotic expression plasmid of SV2A gene and its expression in HEK293T cells

Objective To construct the eukaryotic expression plasmid of mouse synaptic vesicular protein 2A(SV2A)gene and transfer it to human embryonic kidney cells(HEK293T)for identification.Methods The cDNA of APP/PS1 double transgenic mouse hippocampal tissue was used as template to amplify the 2239 bp long SV2A gene coding sequence,which was inserted into the eucaryotic expression vector p3×Flag-CMV-10 polyclonal site area,and the eucaryotic expression plasmid p3×Flag-CMV-10-SV2A was obtained.After transformation,the monoclonal colonies were selected and identified by double enzyme digestion and then sent to the company for sequencing,and the constructed recombinant plasmid was transformed into a successful one western blot was used to detect the expression of SV2A gene in HEK293T cells.Results The recombinant plasmid p3×Flag-CMV-10-SV2A was successfully constructed,and the expression of the protein was verified after transfection into HEK293T cells.Conclusion The eukaryotic expression plasmid p3×Flag-CMV-10-SV2A is successfully constructed by using molecular cloning technology and expressed in HEK293T cells,which lay a foundation for further experiments.