携带人VEGF_(165)和ANG-1双基因的腺病毒载体的构建及目的基因的表达  

Construction and target gene expression of recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1

作　　者：张金康[1] 孟国林[1] 刘建[1] 胡蕴玉[1] 袁志[1] 段春光[1] 毕龙[1] 白峰[1] 黄鑫[1] 禚文昆[1] 李国臣[1] 

机构地区：[1]第四军医大学西京医院全军骨科研究所,西安710032

出　　处：《中国矫形外科杂志》2010年第4期312-315,共4页Orthopedic Journal of China

基　　金：国家自然科学基金(编号:30672142)

摘　　要：[目的]构建并鉴定携带人血管内皮细胞生长因子165(VEGF165)和血管生成素-1(ANG-1)双基因共表达重组腺病毒载体pAd-VIA。[方法]采用基因克隆技术克隆目的基因VEGF165、ANG-1基因,将得到的基因通过引入内部核糖体进入位点序列(IRES),亚克隆至含有报告基因EGFP的穿梭载体pTrack-CMV中,构建携带双基因的腺病毒穿梭载体pTrack-CMV-VIA。进而使用AdEasyTM腺病毒系统重组并包装腺病毒。通过绿色荧光蛋白(GFP)表达、酶联免疫吸附分析(ELISA)方法检测制备的腺病毒pAd-VIA感染的大鼠骨髓基质干细胞中外源基因VEGF165及Ang-1的表达。[结果]该重组腺病毒质粒经测序、酶切鉴定,证明基因序列正确。转染QBI-293A细胞后,可观察到GFP明显表达。重组合腺病毒载体pAd-VIA获得成功包装,扩增后病毒滴度为2×1010PFU/ml,大鼠骨髓基质干细胞转染48 h后,绿色荧光蛋白表达阳性,ELISA结果显示转染组在感染48 h后,培养细胞上清中的VEGF165浓度为(42.5±2.082)ng/105细胞,ANG-1浓度为(16.67±2.08)ng/105细胞,未转染组几乎未检测到外源基因的表达,有统计学差异(P<0.05)。[结论]成功构建携带人VEGF165、ANG-1双基因共表达重组腺病毒载体,为组织工程人工骨血管化的研究奠定基础。[Objective]To construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165（VEGF165） and angiogenin-1（Ang-1）,and to observe the expression of target gene after transfection.[Method]Molecular biologic techniques were used to clone the genes of VEGF165 and Ang-1,and PCR was used to amplify the DNA sequence of IRES（internal ribozyme entry site） from pIRES2-EGFP.The genes of VEGF165,IRES and Ang-1 were subcloned to pTrack-CMV one by one to get the plasmid named pTrack-CMV-VIA,which contained all the three genes.The linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1.Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA.The expression of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and enzyme linked immunosorbent assay（ELISA）.[Result]The recombinant adenovirus plasmid was consistent with that shown by sequencing and restriction endonucleases digestion.The recombinant pAd-VIA vector was packaged and amplified successfully in QBI-293A cells.The titer was 2×1010 PFU/ml after amplifying.High positive GFP was expressed in the field through fluorescence microscopy after being transfected by recombinant pAd-VIA.ELISA indicated the expression of the target genes.The difference between the tansfected and non-transfected group was significant（P〈0.05）.[Conclusion]The recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1 were successfully constructed,thus providing convenient gene transfer tools for constructing the new gene-modified artificial bone.

关 键 词：血管内皮细胞生长因子165 血管生成素-1 腺病毒载体 基因转染 

分 类 号：R68[医药卫生—骨科学] R346[医药卫生—外科学]