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目的:克隆人血管内皮生长因子(vascularendothelialgrowthfactor165,VEGF165)和血管生成素-1(angiopoietin-1)的全长编码基因,构建表达该基因的复制缺陷型腺病毒载体Ad-Ang1和Ad-VEGF165。方法:通过RT-PCR方法克隆人VEGF165和Ang1全长编码基因。Ad-VEGF165和Ad-Ang1通过同源重组方法构建。将Ad-VEGF165和/或Ad-Ang1转染大鼠胚胎心脏成肌细胞(H9C2)24h后,Westernblot方法分析VEGF165和Ang1蛋白表达量;核酸电泳分析细胞基因组降解检测凋亡水平。结果:测序显示VEGF165序列与基因库序列相同;Ang1序列与基因库序列存在一个碱基的差异,但编码氨基酸无改变。VEGF165和Ang1蛋白表达分别为对照组的11.65倍和3.53倍。Ad-VEGF165和/或Ad-Ang1转染后的H9C2细胞对过氧化氢诱导的细胞凋亡具有明显的抵抗能力。结论:所构建的Ad-Ang1和Ad-VEGF165能有效转染心脏成肌细胞,表达出功能性目的蛋白,具有抗细胞凋亡作用。
OBJECTIVE: To clone full-length coding genes of human vascular endothelial growth factor (VEGF165) and angiopoietin-1 and construct replication-deficient adenovirus vectors Ad-Ang1 and Ad-VEGF165. Methods: Human VEGF165 and Ang1 full-length genes were cloned by RT-PCR. Ad-VEGF165 and Ad-Angl were constructed by homologous recombination methods. After transfection of Ad-VEGF165 and / or Ad-Ang1 into rat embryonic cardiac myoblasts (H9C2) for 24 h, the expression of VEGF165 and Ang1 protein was analyzed by Western blotting. The level of apoptosis was detected by nucleic acid electrophoresis. Results: Sequencing showed that the sequence of VEGF165 was the same as that of gene bank. There was one base difference between Ang1 sequence and gene bank sequence, but no change of coding amino acid. VEGF165 and Ang1 protein expression were 11.65 times and 3.53 times of the control group respectively. H9C2 cells transfected with Ad-VEGF165 and / or Ad-Angl had a significant resistance to hydrogen peroxide-induced apoptosis. CONCLUSION: The constructed Ad-Ang1 and Ad-VEGF165 can effectively transfect cardiac myoblasts and express functional target proteins with anti-apoptotic effects.