pIRES2-BDNF-VEGF165真核表达载体的构建与鉴定

背景：人脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)和血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)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-VEGF165并对其进行鉴定。方法：采用PCR的方法从人外周血单个核细胞的基因组DNA中获取人脑源性神经营养因子基因,然后将人脑源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建为pIRES2-BDNF-EGFP。人血管内皮生长因子165 cDNA片段是通过双PCR的方法从pIRES2-VEGF165-EGFP质粒中获取,接着将血管内皮生长因子165 cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中,最后构建成为含有即内部核糖体进入位点的pIRES2-BDNF-VEGF165双基因共表达载体。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,提取的人脑源性神经营养因子和血管内皮生长因子165均与基因库报道序列一致,片段长度分别为744 bp和576 bp。构建的pIRES2-BDNF-VEGF165双基因共表达载体经Eco RⅠ/Bam HⅠ切出BDNF条带,经BDNF/NotⅠ双酶切后可见 IRES- VEGF165基因片段,经 Eco RⅠ/NotⅠ双酶切后可见BDNF-IRES-VEGF165基因片段。RT-PCR 与Western-blot 方法检测显示,此载体转染后,HEK293 细胞均能表达人脑源性神经营养因子和血管内皮生长因子165 mRNA和蛋白。结果证实,实验成功构建了人脑源性神经营养因子和血管内皮生长因子165 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：

Construction and Identification of pIRES2-BDNF-VEGF165 bicistronic eukaryotic expression vector

BACKGROUND: Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF165) are essential genes for cell differentiation. 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.OBJECTIVE:To construct and identify pIRES2-BDNF-VEGF165 bicistronic eukaryotic expression vector.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 pIRES2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES2-BDNF-EGFP. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by double PCR. Next step was that VEGF165 cDNA fragment was cloned into the pIRES2BDNF-EGFP instead of EGFP to create a double gene co -expressing vector plasmid pIRES2-BDNF-VEGF165. Then, pIRES2-BDNF-VEGF165 was used to transfect HEK293 cells, and RT-PCR and western-blot assay were employed to test the co-expression of double genes.RESULTS AND CONCLUSION:BDNF and VEGF165 genes were cloned in this study. The DNA sequencing analysis demonstrated that the BDNF and VEGF165 were exactly consistent with the sequence recorded in the GenBank. The size of BDNF gene was 744 bp. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by PCR, and the size of VEGF165 gene was 576 bp. Enzyme digestion analysis indicated that BDNF and VEGF165 genes were inserted into the expression vector pIRES2-EGFP correctly and the BDNF and VEGF165 co-expression plasmid was successfully constructed. Then, by transfecting pIRES2-BDNF-VEGF165 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 VEGF165 genes.