人血管内皮细胞生长因子165重组腺病毒的构建及其在真核细胞中的表达

Construction of hVEGF165 Recombinant Adenovirus Vector and Its Expression in HEK293 Cells

目的:体外构建人血管内皮生长因子165(hVEGF165)的腺病毒表达载体,并检测其在HEK293细胞中的表达。方法:从重组质粒pcDNA3/hVEGF165中获得hVEGF165,经酶切及测序鉴定,将hVEGF165基因亚克隆到穿梭质粒pAdTrack-CMV,重组穿梭质粒经酶切线性化后,与pAEdasyl质粒在大肠杆菌BSJ183中进行同源重组,线性化后转染HEK293细胞进行包装扩增获取重组病毒上清,同时应用RT-PCR及ELISA法检测hVEGF165的表达。结果:目的基因的测序结果与人VEGF165序列(Genbank)相符。转染后经RT-PCR和ELISA检测证实转染重组质粒组hVEGF165 mRNA及蛋白表达,而转染空质粒组及未转染组没有检测到hVEGF165的表达。结论:本研究构建了人VEGF165重组腺病毒表达载体pAd-hVEGF165,并能成功地在HEK293细胞中表达。

Objective： To construct human vascular endothelial growth factor165（hVEGF165） recombinant adenovirus and investigate its expression in HEK293.Methods： The target gene of human VEGF165 was obtained from plasmid pcDNA3/hVEGF165 and was verified by DNA sequence analysis.The hVEGF165 gene was subcloned into shuttle vector pAdTrack-CMV.After identified with restriction enzymes,plasmid pAdTrack-hVEGF165 was linearized by digestion with restriction endonuclease PmeⅠ,and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination.After linearized by PacⅠ,the homologous recombinant adenovirus plasmid was transfected into HEK293 cells recombinant adenovirus.The high-level adenoviruses infected HEK293 cells and the expression of VEGF-1 by the transfected BMSC was examined with RT-PCR assay and ELISA.Results： The recombinant plasmid pAd-hVEGF165 was subjected to sequence analysis which indicated all nucleotides were identical to the human VEGF165 sequence provided by Genbank.After transfection,the expression of VEGF165 in HEK293 cell was detected.Conclusion： Combinant plasmid pAd-hVEGF165 was constructed in vitro and expressed successfully in HEK293 cells.