Neuritin真核表达载体的构建及鉴定

Construction and Identification of Eukaryotic Expression Vector of Neuritin

Neuritin(CPG15)是一种神经营养因子,能促进神经突起生长,为了探讨Neuritin可能的作用机制,本研究构建了人类neuritin基因的真核表达载体。采用PCR技术扩增编码人neuritin基因cDNA全长序列,并纯化回收,酶切、连接入真核表达载体pcDNA3.1(-)A中,转化大肠杆菌DH5α,挑取阳性克隆pcDNA3.1(-)A-neuritin扩增后,提取质粒,进行双酶切鉴定以及DNA测序鉴定。结果显示,构建的重组质粒pcDNA3.1(-)A-neuritin含有450bp neuritin片段,且重组质粒所含的neuritin读框片段与GenBank NM 016588.2序列同源性100%。由此可知:成功构建了pcDNA3.1(-)A-neuritin真核表达载体,为进一步研究该基因的功能及作用机制奠定良好的实验基础。

Neuritin（CPG15） is a neurotrophic factor that can promote neurite growth.In order to explore possible mechanisms of Neuritin,we constructed the eukaryotic expression vector of human Neuritin in this study.Encoding full-length cDNA gene sequences of human neuritin was amplified by PCR,then this cDNA fragment was digested and ligated into pcDNA3.1（-） A of the eukaryotic expression vector.The constructs transformed into E.coli DH5α and positive clones were picked by pcDNA3.1（-） A-neuritin PCR amplification.This construct was confirmed by the double-enzyme digestion and DNA sequencing.The results showed that the recombinant plasmid pcDNA3.1（-） A-neuritin containing 450bp Neuritin fragment,In addition,the recombinant plasmid contained the open reading frame fragment of neuritin sequence and completely identical with GenBank NM 016588.2 sequence.It can be seen that we are successfully made construction of the pcDNA3.1（-） A-neuritin eukaryotic expression vector.This study is for further study of the mechanism of gene function and to lay a good experimental basis.