CAJ | 학술논문

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。
목적：구건종류내피표지물8（TEM8）기인진핵표체재체，실현TEM8재HEK293F세포중적외원표체。방법：용PCR기술확증TEM8기인，경한제성매절、련접、전화，삽입pcDNA3.1（+）-EGFP진핵표체재체，병통과지질체장TEM8표체질립전염지HEK293F세포중，Western인적검측TEM8적표체。결과：PCR확증득도TEM8기인，구건료진핵표체재체pcDNA3.1（+）-TEM8-EGFP병전염HEK293F세포，경G418가압사선급유한희석법득도생장성상량호、표체효솔고적단극륭세포계TEM8-EGFP/HEK293F；Western인적증명과표체세포계TEM8-EGFP/HEK293F현저표체TEM8단백。결론：구건료표체TEM8적중조HEK293F공정세포계TEM8-EGFP/HEK293F，위진일보연구TEM8적생리공능전정료기출。
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.