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目的 :构建以鼠心肌轻链蛋白基因 (mlc- 2 v)为启动子、携带人血管内皮生长因子 h VEGF1 6 5基因的重组腺病毒载体 ,检测该重组腺病毒载体对心肌细胞转染的靶向性。 方法 :酶切腺病毒载体 PDC31 5自身启动子 CMV,构建腺病毒载体PDC31 7,分别接入 h VEGF1 6 5、mlc- 2 v基因 ,构建腺病毒载体 PDC- mlc- h VEGF1 6 5。鉴定正确后 ,将 PDC- mlc- h VEGF1 6 5与 L ipo-fectam ine共转染至 2 93细胞 ,经同源重组获得以 m lc- 2 v为启动子、携带 h VEGF1 6 5基因的重组腺病毒 Ad- m lc- h VEGF1 6 5,同步构建无靶向性的重组腺病毒 Ad- h VEGF1 6 5。扩增并测定滴度后 ,Ad- h VEGF1 6 5、Ad- mlc- h VEGF1 6 5分别转染体外培养的心肌细胞、骨骼肌细胞及平滑肌细胞 ,利用 EL ISA、Western印迹分析等方法检测 Ad- mlc- h VEGF1 6 5转染心肌细胞的靶向性。 结果 :构建的病毒正确 ,病毒滴度为 2 .8× 1 0 9pfu/ ml。转染 3d后 ,Ad- h VEGF1 6 5组在各培养细胞的上清液及细胞内均检测到 VEGF的表达 ,而 Ad- mlc- h VEGF1 6 5组仅在心肌细胞中有 VEGF的表达 ,且 Ad- m lc- h VEGF1 6 5组心肌细胞分泌的 VEGF要少于 Ad-h VEGF1 6 5组。结论 :成功构建了以 mlc- 2 v为启动子、携带人 VEGF基因的重组腺病毒 Ad- m lc- h
OBJECTIVE: To construct a recombinant adenovirus vector carrying murine VEGF gene (mlc-2 v) as promoter and human VEGF gene VEGF165 gene and to detect the effect of the recombinant adenovirus vector on cardiomyocytes transfected target Sexuality. Methods: The adenoviral vector PDC31 5 was digested with restriction endonuclease CMV to construct adenovirus vector PDC31 7. The recombinant adenoviruses were transfected into hVEG1 6 5 and mlc-2 v respectively to construct adenovirus vector PDC-mlc-h VEGF165. After identification correctly, PDC-mlc-h VEGF1 6 5 and L ipo-fectamine were cotransfected into 293 cells, and the recombination with m lc-2 v as promoter and carrying h VEGF1 65 gene was carried out by homologous recombination The recombinant adenovirus Ad-m lc-h VEGF165 was constructed and the recombinant adenovirus Ad-h VEGF1 65 was constructed. After amplification and determination of titer, Ad-h VEGF1 6 5 and Ad-mlc-h VEGF1 65 were transfected into cardiomyocytes, skeletal muscle cells and smooth muscle cells in vitro respectively. Targeting of mlc - h VEGF165 Transfected Cardiomyocytes. Results: The correct virus was constructed and the virus titer was 2.8 × 109pfu / ml. After transfection for 3 days, the expression of VEGF was detected in the supernatant and intracellular of Ad-h VEGF165 group, while the Ad-mlc-h VEGF165 group only expressed VEGF in cardiomyocytes, The VEGF secreted by Ad-m lc-h VEGF165 group was less than that of Ad-h VEGF165 group. Conclusion: The recombinant adenovirus Ad-m lc-h