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

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。

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

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.