pIRES2-GDNF-NT-3真核表达载体的构建与鉴定

目的：采用一种简便和高效的方法构建双基因共表达载体pIRES2-GDNF-NT-3。方法：人胶质细胞源性神经营养因子和神经营养素3是采用PCR的方法从人外周血单个核细胞的基因组DNA中获取，将人胶质细胞源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建成为pIRES2-GDNF-EGFP．神经营养素3cDNA片段通过替换EGFP的方式插入到pIRES2-GDNF-EGFP中构建成为pIRES2-GDNF-NT-3双基因共表达栽体。结果：人胶质细胞源性神经营养因子和神经营养素3被克隆，通过测序和酶切鉴定的得知与基因库报道序列一致。结论：人神经生长因子和神经营养素3双基因真核表达载体成功构建，它提供了一个新的表达系统，为进一步研究双基因的功能奠定了基础。

Construction and Identification of pIRES2-GDNF-NT-3 Bicistronic Eukaryotic Expression Vector

Objective： Using a simple and efficient method to construct a bi-cistronic eukaryotic expression vector plRES2-GDNF-NT-3. Methods： GDNF and NT-3 genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. The GDNF cDNA fragment was inserted into the multiple cloning sites of plRES2-EGFP to generate the bi-cistronic eukaryotic expression plasmid plRES2-GDNF-EGFP. Then NT-3 cDNA fragment was cloned into the plRES2-GDNF-EGFP instead of EGFP creating plasmid plRES2-GDNF-NT-3. Results： GDNF and NT-3 genes were cloned; the DNA sequencing analysis demonstrated that the GDNF and NT-3 were exactly consistent with the sequence recorded in GenBank. Restriction analysis indicated that GDNF and NT-3 genes were inserted expression vector plRES2-EGFP correctly. Conclusions： The GDNF andNT-3 co-expression plasmid is successfully constructed. It provides a novel expression system, which makes it possible to further study on the functions of GDNF and NT-3 genes.