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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 explore the feasibility of constructing adenovirus vector carrying hIGF-Ⅰ gene. Methods The coding sequence of hIGF-Ⅰ gene was cloned from pcDNA3.1-hIGF-Ⅰ plasmid, digested and ligated into pAdshuttle-CMV. The recombinant plasmid pAdshuttle-CMV-hIGF- This was transformed into Escherichia coli BJ5183 containing the adenovirus backbone vector pAdeasy-1 and subjected to homologous recombination to obtain recombinant pAdeasy-1-IGF-I. After identification, pAdEasy-1-IGF-Ⅰ was transfected into human embryonic kidney cells (Ad293 cells) to obtain recombinant adenovirus (rAd-hIGF-Ⅰ) containing hIGF-Ⅰ gene. After re-identification, Ad293 cells repeatedly infected with freeze-thaw amplification adenovirus, 50% Tissue Culture Infection (TCID50) detection of virus titer. The virus particles were infected with green monkey kidney fibroblasts (COS-7 cells), the expression of green fluorescent protein (GFP) was observed by fluorescence microscopy, the expression of hIGF-Ⅰ gene at mRNA level by RT-PCR, Protein expression. Results The recombinant adenovirus vector rAd-hIGF-Ⅰ containing hIGF-Ⅰ gene was successfully constructed and transfected into COS-7 cells. The expression of green fluorescent protein (EGFP) was detected. At the same time, a 318 bp band was amplified by RT-PCR. kDa. The results showed that rAd-hIGF-Ⅰ could be successfully transfected into COS-7 cells and the hIGF-Ⅰ gene could be successfully expressed in COS-7 cells. Conclusion The recombinant adenovirus rAd-hIGF-Ⅰ containing hIGF-Ⅰ gene was successfully constructed and proved to be effective transfection of COS-7 cells, which provided an efficient gene vector for the further improvement of tissue-engineered cartilage.