肿瘤内皮标志物8同型物Ⅰ基因真核表达载体的构建及其在HEK293F细胞中的表达

Construction of TEM8 IsoformⅠExpressing Vector and its Expression in Human Embryonic Kidney 293F Cells

目的:构建肿瘤内皮标志物8(TEM8)基因真核表达载体,实现TEM8在HEK293F细胞中的外源表达。方法:用PCR技术扩增TEM8基因,经限制性酶切、连接、转化,插入pcDNA3.1(+)-EGFP真核表达载体,并通过脂质体将TEM8表达质粒转染至HEK293F细胞中,Western印迹检测TEM8的表达。结果:PCR扩增得到TEM8基因,构建了真核表达载体pcDNA3.1(+)-TEM8-EGFP并转染HEK293F细胞,经G418加压筛选及有限稀释法得到生长性状良好、表达效率高的单克隆细胞系TEM8-EGFP/HEK293F;Western印迹证明过表达细胞系TEM8-EGFP/HEK293F显著表达TEM8蛋白。结论:构建了表达TEM8的重组HEK293F工程细胞系TEM8-EGFP/HEK293F,为进一步研究TEM8的生理功能奠定了基础。

Objective： To construct the eukaryotic expression vector of tumor endothelial marker 8（TEM8） gene and express it in HEK293F cells. Methods： TEM8 gene was amplified by PCR and inserted into eukaryotic ex-pression vector pcDNA3.1（＋）-EGFP after a series of digestion, ligation, transformation. Then, HEK293F cells were transfected with the constructed recombinant plasmid by liposome-mediated transfection, and the expression of TEM8 was detected by Western blotting. Results： TEM8 gene was amplified by PCR, and the eukaryotic expres-sion vector pcDNA3.1（＋）-TEM8-EGFP was successfully constructed and transfected into HEK293F cells. The TEM8/HEK293F cells, resulting from G418 screening and limiting dilution assay, displayed characters of favorable growth and high-level expression. Western blotting showed that the TEM8/HEK293F cells expressed TEM8 protein markedly. Conclusion： Recombinant HEK293 cell line expressing TEM8, named TEM8-EGFP/HEK293F, was con-structed, which laid the foundation for further study on the physiological function of TEM8.