hBcl-2和hVEGF_(165)双基因共表达重组腺病毒载体的构建及鉴定

Construction and identification of recombinant adenovirus vector co-expressing hBcl-2 and hVEGF165 genes

目的构建hBcl-2和hVEGF165双基因共表达的重组腺病毒载体,为后续基因转染和动物实验提供实验基础。方法通过RT-PCR从A549细胞中分别获得hBcl-2和hVEGF165基因片段,并克隆到pMD-19T载体。将目的基因hBcl-2、IRES元件和hVEGF165依次定向克隆到腺病毒穿梭载体质粒pAd-Track-CMV上。线性化重组穿梭质粒后通过电穿孔转化含有腺病毒骨架质粒pAd-easy-1的感受态大肠杆菌BJ5183构建重组腺病毒载体质粒。脂质体转染线性化的重组腺病毒载体质粒于HEK293细胞以包装制备重组腺病毒。重组腺病毒经PCR鉴定之后进一步扩增、纯化。通过荧光计数确定病毒感染滴度。结果克隆的hBcl-2和hVEGF165基因测序正确,酶切重组穿梭载体质粒和腺病毒载体质粒得到特异酶切产物,重组腺病毒载体质粒转染HEK293细胞3 d后观察到GFP的表达,重组腺病毒的PCR得到hBcl-2和hVEGF165基因的PCR产物,滴度测定为5×109pfu/mL。结论成功构建制备了高滴度的hBcl-2和hVEGF165双基因共表达的重组腺病毒载体,为联合基因治疗心肌梗死的研究提供实验基础。

Objective To construct recombinant adenovirus vector co - expressing hBcl - 2 and hVEGF165 genes, so as to lay a foundation for the subsequent gene transfection and animal experiments. Methods The hBcl -2 and hVEGF165 gene fragments were obtained from A549 cells by RT - PCR and inserted into the pMD - 19T vector. The hBcl- 2 gene,IRES element and hVEGF165 gene were cloned into the shuttle plasmid pad -Track -CMV according to priority. The shuttle plasmid was linearized and transformed into the BJ5183 bactetia which carry the backbone vector pAd - easy - 1 for homologous recombination. The recombinant plasmid was linearized and transfected into HEK293 cells using Lipofectamine for packaging. The recombinant adenovirus were further amplified and purified after PCR identification. The final virus production was quantified with flurometry. Results The hBcl -2 and hVEGF165 genes were verified by gene sequencing. Both the recombinant shuttle plasmid and the recombinant adenovirus vector plasmid were verified by enzyme digestion. The expression of GFP was observed in HEK293 cells on the 3rd day after transfection. PCR showed that adenovirus carried the hBcl -2 and hVEGF165 genes. The titer was 5 × 10^9 pfu/mL. Conclusion We have successfully constructed a co - expressing recombinant adenovirus vector carrying hBcl - 2 and hVEGF165 genes , which lay the experimental foundation for the treatment of myocardial infarction using combined gene therapy.