血管内皮细胞生长因子真核表达质粒的构建及其在心肌细胞中的表达

目的 :构建血管内皮细胞生长因子 （ VEGF1 65）真核表达质粒 pc DNA3 .1（ -） /h VEGF1 65,并通过转染心肌细胞来验证其生物活性。方法 :应用 DNA重组方法 ,将编码 h VEGF1 65全长 c DNA克隆于真核表达载体 pc DNA3 .1（ -）中 ,构建 pc DNA3 .1（ -） /h VEGF1 65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 pc D-NA3 .1（ -） /h VEGF1 65瞬时转染培养的大鼠心肌细胞中 ;采用 RT-PCR,ELISA,Western blot及免疫组化染色等方法检测 VEGF1 65基因在心肌细胞中的表达情况 ;应用 MTT法检测转染后细胞上清液中 VEGF1 65的生物活性。结果 :将构建好的真核表达质粒 pc DNA3 .1（ -） /h VEGF1 65转染心肌细胞后 ,VEGF m RNA及蛋白表达水平明显增高 ,转染后的心肌细胞培养上清液具有促使内皮细胞增殖的生物活性。结论 :成功构建了真核表达质粒 pc DNA3 .1（ -） /h VEGF1 65,其转染心肌细胞后可获得较高水平 VEGF蛋白的表达 ,所表达出 VEGF蛋白具有生物学活性

Construction of eukaryotic expression plasmid of VEGF165 gene and its expression in myocardial cells

AIM:To construct a eukaryotic expression vector of human vascular endothelial growth factor(VEGF 165 ) gene, and to investigate the transfection and expression of pcDNA3.1( )/hVEGF 165 eukaryotic expression plasmid in myocardial cells. METHODS: pcDNA3.1( )/hVEGF 165 eukaryotic expression plasmid was constructed. Primarily cultured rat myocardial cells were transiently transfected with LipofectAMINE2000. RT PCR, ELISA, Western blot and immunohistochemical methods were used to detect the expression of VEGF gene and MTT to detect the biological activity of the conditioned medium after the transfection. RESULTS: There were significant increases of VEGF mRNA and protein in the myocardial cells transfected with pcDNA3.1( )/hVEGF 165 . The conditioned medium after the transfection showed the biological activity to stimulate the proliferation of endothelial cells. CONCLUSION: The pcDNA3.1( )/VEGF 165 , a eukaryotic expression plasmid for hVEGF 165 gene is constructed. High levels of VEGF mRNA and protein expression can be obtained in the myocardial cells transfected with pcDNA3.1( )/hVEGF 165 eukaryotic expression plamid. The expressed protein has the biological activity.