| 栗炳南 李卫东 林俊堂 丰慧根.pIRES2-GDNF-NT-3 真核表达载体的构建与鉴定[J].,2014,14(31):6056-6061 |
| pIRES2-GDNF-NT-3 真核表达载体的构建与鉴定 |
| Construction and Identification of pIRES2-GDNF-NT-3 BicistronicEukaryotic Expression Vector |
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| DOI： |
| 中文关键词: 胶质细胞源性神经营养因子 神经营养素3 真核双表达载体 内部核糖体进入位点 |
| 英文关键词: GDNF NT-3 Bi-cistronic eukaryotic expression vector Internal ribosome entry site |
| 基金项目:河南省重大科技攻关项目(122101310100)；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) |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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