携带hIGF-Ⅰ基因的腺病毒载体构建

Construction of recombinant adenovirus vector containing hIGF-Ⅰ gene

目的探讨构建携带hIGF-Ⅰ基因腺病毒载体的可行性。方法从pcDNA3.1-hIGF-Ⅰ质粒中克隆出hIGF-Ⅰ基因的编码序列,酶切、连接后,插入腺病毒系统中的穿梭载体pAdshuttle-CMV,获得重组质粒pAdshuttle-CMV-hIGF-Ⅰ,将其转化含有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183,进行同源重组,获得重组子pAdeasy-1-IGF-Ⅰ。鉴定后,将pAdeasy-1-IGF-Ⅰ转染人胚肾细胞(Ad293细胞),获得含hIGF-Ⅰ基因的重组腺病毒(rAd-hIGF-Ⅰ)。再鉴定后,Ad293细胞反复感染冻融扩增腺病毒,50%组织培养感染量法(TCID50)检测病毒滴度。将病毒颗粒感染绿猴肾成纤维细胞(COS-7细胞),荧光显微镜观测绿色荧光蛋白(GFP)的表达,RT-PCR分析hIGF-Ⅰ基因在mRNA水平的表达,Western blot分析hIGF-Ⅰ在蛋白水平的表达。结果成功构建出含有hIGF-Ⅰ基因的腺病毒载体rAd-hIGF-Ⅰ,转染COS-7细胞后发现绿色荧光蛋白表达,同时RT-PCR扩增出318 bp的目的条带,Western blot得到7.6kDa大小的目的蛋白,证明了腺病毒载体rAd-hIGF-Ⅰ能成功转染COS-7细胞,hIGF-Ⅰ基因能在其中成功表达。结论成功构建出含有hIGF-Ⅰ基因的腺病毒载体rAd-hIGF-Ⅰ,并且证实其能对COS-7细胞进行有效转染,为组织工程软骨的进一步改良提供了高效的基因载体。

Objective To construct the recombinant adenovirus vector containing hIGF-Ⅰ gene and observe the expression in COS-7 cells.Methods The hIGF-Ⅰ gene coding sequence was cloned into pAdshuttle-CMV plasmid to get pAdshuttle-CMV-hIGF-Ⅰ,which would be transformed into competent E.Coli BJ5183 carrying backbone plasmid pAdeasy-1 already.The homologous recombinant adenoviral plasmid was delivered into Ad293 cells after verification.The identified recombinant adenovirus(rAd-hIGFⅠ)was amplified in Ad293 cells.Viral par...