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目的 :构建人胰岛素样生长因子 - 1(hIGF - 1)真核表达质粒pcDNA3.1(- ) /hIGF - 1并通过转染成肌细胞来验证其生物活性。方法 :应用DNA重组方法将编码hIGF - 1cDNA克隆于真核表达载体pcDNA3.1(- )中 ,构建pcDNA3.1(- ) /hIGF - 1真核表达质粒 ;应用阳性脂质体介导的基因转染技术 ,将真核表达质粒pcDNA3.1(- ) /hIGF - 1瞬时转染C2C12成肌细胞中 ;采用RT -PCR及免疫组化染色等方法检测hIGF - 1基因在成肌细胞中的表达情况 ;应用MTT法检测转染后细胞上清液中hIGF - 1的生物活性。结果 :将构建好的真核表达质粒pcDNA3.1(- ) /hIGF - 1转染成肌细胞后 ,hIGF - 1mRNA及蛋白表达水平明显增高 ;转染后的成肌细胞培养上清液具有促使成纤维细胞增殖的生物活性。结论 :成功构建了真核表达质粒pcDNA3.1(- ) /hIGF - 1,其转染成肌细胞后可获得较高水平hIGF - 1蛋白的表达 ,所表达出hIGF - 1蛋白具有生物学活性。
OBJECTIVE: To construct the eukaryotic expression vector pcDNA3.1 (-) / hIGF - 1 of human insulin - like growth factor - 1 (hIGF - 1) and verify its biological activity by transfection into myoblasts. Methods: The hIGF - 1 cDNA was cloned into the eukaryotic expression vector pcDNA3.1 (-) by DNA recombination to construct the eukaryotic expression plasmid pcDNA3.1 (-) / hIGF - 1. The positive liposome - mediated gene Transfection technique was used to transiently transfect pcDNA3.1 (-) / hIGF - 1 into C2C12 myoblasts. RT - PCR and immunohistochemistry were used to detect the expression of hIGF - 1 in myoblasts The biological activity of hIGF - 1 in the cell supernatant was detected by MTT assay. Results: The expression of hIGF - 1 mRNA and protein was significantly increased after transfection of the constructed eukaryotic expression plasmid pcDNA3.1 (-) / hIGF - 1 into myoblasts. The supernatant of myoblasts transfected with Biological Activity of Fibroblast Proliferation. CONCLUSION: The eukaryotic expression plasmid pcDNA3.1 (-) / hIGF - 1 was successfully constructed and transfected into myoblasts to obtain a higher level of hIGF - 1 protein. The expressed hIGF - 1 protein has biological activity .