EGFP标记的hVEGF_(165)重组腺病毒构建新方法及其相关特性的体外研究

A New Method for Construction of EGFP-Labled Recombinant Adenovirus Containing hVEGF_(165) and Its Property In Vitro

利用细菌内同源重组法快速构建增强型绿色荧光蛋白 (EGFP)标记的人血管内皮细胞生长因子 16 5(hVEGF165)重组腺病毒载体 ,并对其相关特性进行体外研究。首先将hVEGF165cDNA亚克隆到含EGFP基因的腺病毒穿梭质粒 pAdTrack CMV中 ,然后与腺病毒骨架质粒pAdEasy 1共同电击转化大肠杆菌BJ5183 ,同源重组获得重组腺病毒质粒 pAd EGFP/hVEGF165,鉴定后通过脂质体法转染HEK2 93细胞 ,产生重组腺病毒Ad EGFP/hVEGF165。通过体外试验观察病毒的形态、均一性和安全性 ,靶细胞的感染效率及hVEGF165的表达情况。结果显示 ,通过细菌内质粒间同源重组法在短期内成功地构建了复制缺陷型重组腺病毒载体Ad EGFP/hVEGF165;借助于EGFP的表达 ,直接在荧光显微镜下观测到靶细胞的感染效率和外源基因的表达。经纯化的病毒颗粒在电镜下成分均一 ,滴度可达 2× 10 12 pfu/ml。病毒感染Hela细胞后经多次传代未见细胞病变。以感染复数 (MOI) =10 0感染hUVEC可获得最大增殖刺激作用。同样浓度感染小鼠骨髓单个核细胞效率达 2 7.3% ,培养上清中hVEGF165蛋白含量达 1385± 332 pg/ 10 6cells。结论 :细菌内同源重组法构建腺病毒载体具有高效、省时、省力的特点 ,辅以EGFP为标记基因可以直观检测靶细胞感染和外源基因的表达情况。

By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labled recombinant adenovirus vector containing hVEGF 165 was generated quickly and its property was studied in vitro. First, hVEGF 165 coding sequence was subcloned into the shuttle plasmid pAdTrack CMV, then linearized and cotransferred with adenoviral backbone vector pAdEasy 1 into E.coli strain BJ 5183 . After positive kanamycin resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with PacⅠ and transfected into HEK 293 cells to assembly replication defective adenovirus Ad EGFP/hVEGF 165 . The further amplified recombinant adenoviruses were purified by CsCl banding at 32?000 rpm for 18 to 24 hours. Electron microscopy and PCR were performed for testing the recombinant adenovirus. The results showed that the purified particles were homogenous hexagon with a high titer up to 2×10 12 pfu/ml. An amplified band of 540 bp fragment demonstrated the successful insert of hVEGF 165 . Under fluorescence microscopy, the expression of EGFP was easily detected in HEK 293 and other target cells. The maximal stimulating effect on the proliferation of hUVEC was obtained when the given multiplicity of infection (MOI) of Ad EGFP/hVEGF 165 was 100. The rate of EGFP positive mouse bone marrow mononuclear cells analysed by flow cytometry was 27.3% after 24 hour incubation with Ad EGFP/hVEGF 165 (MOI=100), and the expression of hVEGF 165 protein in the conditioned medium was 1385±332 pg/10 6 cells. It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the prepared hightiter of Ad EGFP/hVEGF165 is an efficient helpful vector to transfer genes into target cells,all of which make the further in vivo experiments with VEGF165 possible.