反义重组腺病毒质粒pAdxsi-GFP-hVEGF的构建与鉴定

目的构建反义重组腺病毒质粒pAdxsi-GFP-hVEGF。方法从人外周血细胞中提取总RNA,RT-PCR法反转录合成人cDNA文库。设计带有酶切位点的引物,调取VEGF目的基因,插入pShuttle-GFP-CMV穿梭载体。经鉴定序列正确后,将VEGF目的基因构建至亚克隆重组腺病毒质粒pAdxsi中。结果成功构建反义重组腺病毒质粒pAdxsi-GFP-hVEGF,经酶切鉴定,目的基因分子量与预计的相同,测序插入载体部位正确。结论反义重组腺病毒质粒pAdxsi-GFP-hVEGF构建正确、稳定,为进一步抑制人VEGF基因的表达和体内外实验打下了良好的基础。

Construction and identification of the Adenoviral vector pAdxsi-GFP-hVEGF

Objective To construct the Adenoviral vector pAdxsi-GFP-hVEGF.Methods Total RNA was isolated from blood cells and cDNA was constructed by RT-PCR,a pair of primers were synthesized with restriction site to get human VEGF cDNA fragments,and VEGF fragments were inserted into the shuttle vector pShuttle-GFP-CMV.After the sequence was confirmed,the human VEGF fragments were subcloned into Adenoviral vector pAdxsi-GFP-hVEGF.Results Adenoviral vector pAdxsi-GFP-hVEGF was successfully constructed,and the molecular ...