人血管生成素-1和血管内皮生长因子VEGF_(165)腺病毒载体构建

Cloning and construction of adenovi rus expressing human angiopoietin-1or vascular en dothelial growth factor

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。

AIM:We aimed to clone angiopoietin-1(Ang1)and vascular endothelial growth factor(VEGF)full-length DNAs of human origin and construct replication-deficient adenovirus encoding for either of these two genes which can be potentially served for c linical applications.METHODS:VEGF 165 and Ang1full-length cDNAs of human o rigin were amplified by RT-PCR,verified by sequencing,clo ned into a pShuttle-CMV vector,re-combined with a E1and E3regions doub le-deleted adenovirus,packaged in 293A cells,and purified by ultrac entrifugation.The titers of Ad-Ang1and Ad-VEGF 165 were determined by a tissue culture i nfectious dose 50 method.Expression of Ang1and VEGF 165 proteins in H9C2cardiac my-oblasts was examined by Western blot.To examine the protective properties of Ad-Ang1and Ad-VEGF 165 ,DNA fragmentation induced by H 2 O 2 was analyzed in H9C2cells 24hours after transfection.Ad-GFP served as a vehicle control.RESULTS :Sequencing analysis indicated that there is one base difference at site 1206(t)in Ang1compared with that of GeneBan k(c,U83508)although the coded amino acids are th e same (Ileucine).VEGF 165 cDNA sequence was same as that of GeneBank(AB021221).Western blot showed that protein lev els of Ang1and VEGF 165 were in-creased 3.53and 11.53fold respectively 24h after transfection as com-pared to control.Examination of DNA fragmentation suggested that Ang1and /or VEGF 165 significantly protected H9C2cells from H 2 O 2 induced apoptosis.CONCLUSIONS :The two constructed adenoviral vectors,Ad-Ang1and Ad-VEGF 165 ,functionally expressed target pro teins.We demonstrated,for the first time,th at the combined utilization of Ang1a nd VEGF 165 inhibited apoptosis,in addition to their angiogenesis properties.