pIRES2-GDNF-NT-3真核表达载体的构建与鉴定(英文)

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3 cDNA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双基因共表达载体。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 pIRES2-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 pIRES2-EGFP to generate the bi-cistronic eukaryotic expression plasmid pIRES2-GDNF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-GDNF-EGFP instead of EGFP creating plasmid pIRES2-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-3geneswereinsertedexpressionvectorpIRES2-EGFPcorrectly.Conclusion:TheGDNFandNT-3co-expressionplasmidissuccessfully constructed. It provides a novel expression system, which makes it possible to further study on the functions ofGDNFand NT-3 genes.