hTEM8-N-RFP融合蛋白真核表达载体的构建及在HEK293F细胞中表达

Construction of Eukaryotic Expressing Vector of the Extracellular Domain of hTEM8 Fused to RFP and Its Expression in HEK293F Cells

目的:构建人肿瘤内皮标志物8(hTEM8)胞外区(N端)与红色荧光蛋白(RFP)融合表达载体并在HEK293F细胞中表达,为进一步研究hTEM8的相互作用蛋白及其在肿瘤血管形成过程中的机制奠定实验基础。方法:以质粒pDsRed-Express-C1和重组质粒pcDNA3.1(+)-hTEM8/Fc为模板,PCR扩增RFP和hTEM8-N基因片段,先后插入真核表达载体pcDNA3.1(+)中,构建重组表达载体pcDNA3.1(+)-hTEM8-N-RFP,转染HEK293F细胞,通过荧光显微镜观察融合蛋白在转染细胞中的的表达,并用G418对转染的细胞进行加压筛选,Western blot检测hTEM8-N-RFP融合蛋白在转染细胞中的表达。结果:DNA测序、酶切鉴定的结果显示,表达载体pcDNA3.1(+)-hTEM8-N-RFP构建成功,且序列正确。转染后经荧光显微镜观察到HEK293F细胞中有红色荧光,经加压筛选单克隆后,在荧光显微镜下观察到稳定表达红色荧光的细胞株,Western blot检测到融合蛋白hTEM8-N-RFP在真核细胞HEK293F中获得表达。结论:成功构建了pcDNA3.1(+)-hTEM8-N-RFP真核表达载体,并在HEK293F细胞中表达,为后期研究hTEM8的相互作用蛋白和其生理功能奠定了良好的基础。

Objective： To construct eukaryotic expressing vector of the Extracellular Domain（N-terminus）of tumor endothelial marker 8（TEM8） Fused to red fluorescent protein（GFP） and expression in HEK293F cells,which will establish the research foundation for further studying the interaction of TEM8 and other proteins in cells and the function mechanism of TEM8 during tumor angiogenesis.Methods： RFP and TEM8-N gene was amplified by PCR using plasmid pDsRed-Express-C1 and recombinant plasmid pcDNA3.1（＋）-TEM8/Fc as templates,and inserted into eukaryotic expression vector pcDNA3.1（＋） in succession.HEK293F cells were transfected with the constructed recombinant plasmid pcDNA3.1（＋）-TEM8-N-RFP,and selected by 800 μg/mL G418.The expression of TEM8-N-RFP protein was verified by fluorescence microscopy and western-blot respectively.Results： PCR,restriction analysis and sequencing proved that the eukaryotic expression vector of TEM8-N-RFP recombinant fusion protein was constructed successfully.The red fluorescent protein could be observed in cells by fluorescence microscope after the transfection,and the stable express cell lines were received after selected by G418.Western blot results showed that the protein of TEM8-N-RFP was successfully expressed.Conclusions： Recombinant plasmid pcDNA3.1（＋）-TEM8-N-RFP was successfully constructed and expressed in HEK293T cells,which laid foundation for further research on the interaction of TEM8 and other proteins and its biological function in cells.