hVEGF_(165)四环素调控腺病毒载体的构建及其在大鼠成肌细胞表达

Construction of tetracycline-regulative adenoviral vector containing human vascular endothelial growth factor 165 gene and its expression in rat myoblast

目的构建表达人血管内皮生长因子165基因(hVEGF165)的四环素调控重组腺病毒,并测定其在原代培养大鼠成肌细胞的表达水平。方法亚克隆hVEGF165全长cDNA到穿梭质粒pTREShuttle2,获得四环素可调控的目的基因表达盒;亚克隆表达盒到BDAdeno-XTMViralDNA,获得重组腺病毒质粒;重组质粒转染HEK293细胞以包装产生具有感染能力的重组腺病毒颗粒AdTRE.VEGF165。hVEGF165重组腺病毒和Tetoff调节病毒共感染原代培养的成肌细胞,酶联免疫法检测目的基因的表达。结果成功构建了能表达hVEGF165的四环素调控的重组腺病毒载体。hVEGF165重组腺病毒(20pfu/cell)和Tetoff调控病毒(20pfu/cell)共感染骨骼肌成肌细胞后5d,ELISA检测显示培养上清中VEGF165蛋白浓度显著高于未感染的细胞培养上清[(14.80±1.15ng/ml)×105/(cell·24h)vs(1.39±0.36ng/ml)×105/(cell·24h),P<0.01)]。结论四环素调控表达的腺病毒系统可高效地转导VEGF基因进入哺乳动物细胞;原代培养的骨骼肌成肌细胞感染AdTRE.VEGF165后可高水平地分泌VEGF165蛋白。

AIM To construct tetracycline （Tet）-regulatable recombinant adenovirus containing human vascular endothelial growth factor 165 gene （hVEGF165 ）, and to detect its expression in myoblast. METHODS A Tet-responsive mammalian expression cassette, which could be regulated by tetracycline, was made by subcloning the full-length cDNA encoding hVEGF165into pTRE-Shuttle2 vector. The expression cassette was then subcloned into the BD Adeno-XTM Viral DNA to form an adenoviral plasmid. Next, the recombinant plasmid was packaged into infectious recombinant adenoviral particles by transfecting HEK 293 cells. Primary rat skeletal myoblasts were infected with both the hVEGF165 recombinant adenovirus and Tet-off regulatory virus. Expression was tested by enzyme-linked immunosorbent assay. RESULTS We successfully constructed a tetracycline regulatable recombinant adenoviruses vector containing the cDNA for human VEGF165, a secreted endothelial cell specific angiogenic growth factor. The primary skeletal myoblasts infected with AdTRE. VEGF165 （20 pfu/cell） and Tet-off regulatory virus （ 20 pfu/cell） demonstrated higher VEGF165 protein secretion into the supernatant compared with uninfected group[ （14.80±1.15 ng/ml）×10^5/（cell·24 h） vs （ 1.39 ± 0. 36 ng/ml） × 10^5/（cell· 24h）, P 〈0.01 ] , as measured by ELISA at 5 d after infection. CONCLUSION Tet-regulatable adenoviral expression systems are used to deliver gene of VEGF into mammalian cells efficiently. The primary skeletal myoblasts infected with AdTRE. VEGF165 secretes VEGF165 protein at high levels.