CAJ | 학술논문

目的构建hBcl-2和hVEGF_(165)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hVEGF_(165)基因片段,并克隆到pMD-19T载体.将目的基因hBcl-2、IRES元件和hVEGF_(165)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hVEGF_(165)基因的PCR产物,滴度测定为5×109 pfu/mL.结论成功构建制备了高滴度的hBcl-2和hVEGF_(165)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.
목적 구건hBcl-2화hVEGF_(165)쌍기인공표체적중조선병독재체,위후속기인전염화동물실험제공실험기출.방법 통과RT-PCR종A549세포중분별획득hBcl-2화hVEGF_(165)기인편단,병극륭도pMD-19T재체.장목적 기인hBcl-2、IRES원건화hVEGF_(165)의차정향극륭도선병독천사재체질립pAd-Track-CMV상.선성화중조천사질립후통과전천공전화함유선병독골가질립pAd-easy-1적감수태대장간균BJ5183구건중조선병독재체질립.지질체전염선성화적중조선병독재체질립우HEK293세포이포장제비중조선병독.중조선병독경PCR감정지후진일보확증、순화.통과형광계수학정병독감염적도.결과 극륭적hBcl-2화hVEGF165기인측서정학,매절중조천사재체질립화선병독재체질립득도특이매절산물,중조선병독재체질립전염HEK293세포3 d후관찰도GFP적표체,중조선병독적PCR득도hBcl-2화hVEGF_(165)기인적PCR산물,적도측정위5×109 pfu/mL.결론 성공구건제비료고적도적hBcl-2화hVEGF_(165)쌍기인공표체적중조선병독재체,위연합기인치료심기경사적연구제공실험기출.
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.