中国组织工程研究Issue(50)：8719-8728,10.DOI:10.3969/j.issn.2095-4344.2013.50.016
pIRES 2-BDNF-VEGF 165真核表达载体的构建与鉴定
Construction and Identification of pIRES 2-BDNF-VEGF 165 bicistronic eukaryotic expression vector
摘要
Abstract
BACKGROUND:Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation. Virus mediated method has been used numerously in researches, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question. <br> OBJECTIVE:To construct and identify pIRES 2-BDNF-VEGF 165 bicistronic eukaryotic expression vector. <br> METHODS:BDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then, the BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES 2-BDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by double PCR. Next step was that VEGF 165 cDNA fragment was cloned into the pIRES2BDNF-EGFP instead of EGFP to create a double gene co-expressing vector plasmid pIRES 2-BDNF-VEGF 165 . Then, pIRES 2-BDNF-VEGF 165 was used to transfect HEK293 cells, and RT-PCR and western-blot assay were employed to test the co-expression of double genes. <br> RESULTS AND CONCLUSION:BDNF and VEGF 165 genes were cloned in this study. The DNA sequencing analysis demonstrated that the BDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of BDNF gene was 744 bp. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by PCR, and the size of VEGF 165 gene was 576 bp. Enzyme digestion analysis indicated that BDNF and VEGF 165 genes were inserted into the expression vector pIRES 2-EGFP correctly and the BDNF and VEGF165 co-expression plasmid was successful y constructed. Then, by transfecting pIRES 2-BDNF-VEGF 165 into HEK293 cells, double genes were expressed at the mRNA and protein level. It provides a novel expression system, which enables further study on the functions of BDNF and VEGF 165 genes.关键词
组织构建/组织构建基础实验/脑源性神经营养因子/血管内皮生长因子165/真核双表达载体/内部核糖体进入位点/转染/双PCR/省级基金分类
医药卫生引用本文复制引用
栗炳南,李卫东,林俊堂,丰慧根..pIRES 2-BDNF-VEGF 165真核表达载体的构建与鉴定[J].中国组织工程研究,2013,(50):8719-8728,10.基金项目
实验由新乡医学院重点领域招标课题(ZD2011-16)；河南省教育厅科学技术研究重点项目(13A180850)共同资助@@@@the Tender Subject of Key Research Areas of Xinxiang Medical Uuniversity in 2011, No. ZD2011-16 （ZD2011-16）
Key Projects in Scientific Research of Henan Provincial Education Department, No.13A180850 （）
