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目的构建VEGFA基因重组真核表达载体pEGFP-VEGFA,并检测其在人胚胎肾细胞(HEK293T)中的表达。方法以人脐血单核细胞的RNA为模板,采用RT-PCR技术扩增VEGFA基因,并将其定向插入真核表达载体pEGFP-Nl中,构建重组真核表达载体pEGFP-VEGFA。经限制性核酸内切酶BglII和SalI酶切和DNA测序鉴定后,用脂质体法将pEGFP-VEGFA转染HEK293T细胞,转染后24 h和48 h采用荧光显微镜观察pEGFP-VEGFA的表达。结果 pEGFP-VEGFA被双酶切为4 697 bp和1 251 bp两条条带,测序结果证实VEGFA序列与GenBank公布的VEGFA mRNA序列完全一致。在经转染的HEK293T细胞内观察到较强的绿色荧光,表明pEGFP-VEGFA成功转染HEK293T细胞,并在其中得到了表达。结论成功构建重组真核表达载体pEGFP-VEGFA,为进一步研究VEGFA基因的生物学功能奠定了基础。
Objective To construct a recombinant eukaryotic expression vector pEGFP-VEGFA containing VEGFA gene and detect its expression in human embryonic kidney (HEK293T) cells. Methods The RNA of human umbilical cord blood mononuclear cells was used as template to amplify VEGFA gene by RT-PCR and inserted into eukaryotic expression vector pEGFP-Nl to construct recombinant eukaryotic expression vector pEGFP-VEGFA. After restriction endonuclease BglII and SalI digestion and DNA sequencing, pEGFP-VEGFA was transfected into HEK293T cells by lipofectamine. The expression of pEGFP-VEGFA was observed by fluorescence microscope 24 h and 48 h after transfection. Results Two bands of 4 697 bp and 1 251 bp were digested by pEGFP-VEGFA. The results of sequencing confirmed that the VEGFA sequence was identical to the VEGFA mRNA sequence published in GenBank. Strong green fluorescence was observed in transfected HEK293T cells, indicating that pEGFP-VEGFA was successfully transfected into HEK293T cells and was expressed therein. Conclusion The recombinant eukaryotic expression vector pEGFP-VEGFA was successfully constructed and laid the foundation for the further study of the biological function of VEGFA gene.