论文部分内容阅读
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 a fusion expression vector of human extracellular domain of human tumor endothelial marker 8 (hTEM8) with red fluorescent protein (RFP) and to express it in HEK293F cells. To further investigate the interaction between hTEM8 and its tumor angiogenesis The mechanism in the process lays the foundation for the experiment. Methods: RFP and hTEM8-N gene fragments were amplified by PCR using the plasmid pDsRed-Express-C1 and the recombinant plasmid pcDNA3.1 (+) - hTEM8 / Fc as templates. The fragments were inserted into the eukaryotic expression vector pcDNA3.1 (+ The recombinant expression vector pcDNA3.1 (+) - hTEM8-N-RFP was constructed and transfected into HEK293F cells. The expression of fusion protein in transfected cells was observed by fluorescence microscopy. The transfected cells were screened by G418. blot was used to detect the expression of hTEM8-N-RFP fusion protein in transfected cells. Results: The results of DNA sequencing and restriction enzyme digestion showed that the expression vector pcDNA3.1 (+) - hTEM8-N-RFP was successfully constructed and its sequence was correct. After transfection, HEK293F cells were observed by fluorescence microscope with red fluorescence. The monoclonal antibodies were screened by fluorescence microscope and stably transfected into the HEK293F cells. Fluorescent microscope was used to observe the expression of hTEM8-N-RFP Nuclear cell HEK293F expression was obtained. CONCLUSION: The eukaryotic expression vector pcDNA3.1 (+) - hTEM8-N-RFP was successfully constructed and expressed in HEK293F cells, which laid a good foundation for further study on the interaction protein and its physiological function of hTEM8.