人脑源性神经营养因子与神经营养素3真核双表达载体的构建与鉴定.docVIP

人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定 栗炳南，李卫东，林俊堂，丰慧根 新乡医学院生命科学技术学院，河南省新乡市? 453003 Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3 Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China 摘要? 背景：脑源性神经营养因子(brain-derivedneurotrophic factor，BDNF)和神经营养 素3 (Neurotrophines-3，NT-3)在细胞分化过程中有重要作用。病毒载体临床应用存在安全隐患，利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。目的：构建双基因共表达载体pIRES2-BDNF-NT-3并对其进行鉴定。方法:?BDNF和NT-3基因核心序列是通过直接PCR的方法从外周血单个核细胞的基因组DNA中获取。然后将BDNF的cDNA片段插入到pIRES2-EGFP多克隆位点构建pIRES2-BDNF-EGFP载体，随后将NT-3 cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中，最后构建成为含有内部核糖体进入位点(IRES)的pIRES2-BDNF-NT-3双基因共表达载体。实验通过双酶切和DNA测序方法对其鉴定，将重组的双基因共表达载体感染HEK293细胞，利用RT-PCR与 Western-blot方法检测双基因的表达。结果与结论：DNA测序显示，提取的BDNF和NT-3均与基因库报道序列一致。构建的pIRES2-BDNF-NT-3双基因共表达载体经Eco RⅠ/Bam HⅠ切出BDNF条带，经Bam HⅠ/NotⅠ双酶切后切出IRES-NT-3片段，经Eco RⅠ/NotⅠ双酶切后切出BDNF-IRES-NT-3片段。RT-PCR与Western-blot方法检测显示，此载体转染后的HEK293细胞均能表达BDNF和NT-3 mRNA和蛋白。结果证实，实验成功采用IRES序列构建了能分别表达的BDNF和NT-3双基因真核表达载体。 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 全文链接： 关键词?：?组织构建；?组织工程；?脑源性神经营养因子；?神经营养素3；?真核双表达载体；?转染；?双PCR； ?? Abstract： BACKGROUND:?Human brain-derived neurotrophic factor (BDNF) and neurotrophine-3 (NT-3) are essential genes for cell differentiation. Viral vector has been used numerously in clinical practice, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question.OBJECTIVE:?To construct and identify pIRES2-BDNF-NT-3 bicistronic eukaryotic expression vector.METHODS:?BDNF and NT-3 genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. The BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES2-BDNF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-BDNF-EGFP, instead of EGFP, to