VEGF165真核表达载体的构建与体外表达

Construction of Recombinant Plasmid pEGFP-VEGF165 in vitro and Expression in 293T Cells

目的:体外构建血管内皮生长因子(vascular endothelial growth factor,VEGF)165的荧光真核表达载体,并检测其在293T细胞中的表达。方法:应用RT-PCR方法从人单核细胞白血病细胞株THP-1中分离、扩增目的片段,单酶切克隆到真核表达载体pEGFP-C1中,构成重组质粒pEGFP-VEGF165,测序验证。通过脂质体介导质粒瞬时转染293T细胞,RT-PCR和免疫细胞化学检测VEGF165的表达。结果:通过RT-PCR获得576bp的cDNA片段,测序结果与基因库(Genbank)中序列完全一致。转染后经RT-PCR和免疫细胞化学检测证实转染重组质粒组有VEGF165蛋白表达,而转染空质粒组及未转染组没有检测到VEGF的表达。结论:本研究构建了人VEGF165荧光真核表达载体pEGFP-VEGF165,并能成功地在293T细胞中表达。

Objective： To construct fluorescent recombinant plasmid pEGFP-VEGF165 in vitro and to evaluate its expression in 293T cells. Methods： The target sequence of human VEGF165 was obtained and amplified from THP-1 cells by RT-PCR. The cDNA segment was cloned into a eukaryote plasmid pEGFP-C1 after restrictive endonucleases digestion. The pEGFP-VEGF165 was checked and verified by DNA sequence analysis. 293T cells were transiently transfected with pEGFP-VEGF165 by lipofectamine 2000 in vitro. RT-PCR and immunocytochemical analysis were employed to detect the expression of VEGF165. Results： The 576bp cDNA fragment was obtained by RT-PCR and cloned into pEGFP-C1. The recombinant plasmid pEGFP-VEGF165 was subjected to sequence analysis which indicated all nucleotides were identical to the human VEGF165 sequence provided by Genbank. Being transfected by Lipofectamine 2000, the expression of VEGF165 in 293T were detected. Conclusion： Recombinant fluorescent plasmid pEGFP-VEGF165 was constructed in vitro and expressed successfully in 293T cells.