pcDNA3.1(+)-NRP1真核表达载体的构建与鉴定  

Construction and Identification of Eukaryotic Expression Vector pcDNA3.1(+)-NRP1

作　　者：刘培[1] 曹毅[1] 陈微[1] 刘改荣[1] 杨晓红[1] 陶茂灿[1] 罗宏宾[1] 

机构地区：[1]浙江中医药大学附属第一医院,杭州310006

出　　处：《浙江中医药大学学报》2012年第4期407-410,421,共5页Journal of Zhejiang Chinese Medical University

基　　金：浙江省科技厅面上项目基金(2009C33140)~~

摘　　要：[目的]从人角质形成细胞系HaCaT中克隆NRP1(Neuropilin-1)基因全长cDNA,构建含NRP1基因的重组真核表达载体,为下一步的NRP1基因功能研究奠定基础。[方法]采用RT-PCR法从人角质形成细胞系HaCaT中扩增NRP1基因全长cDNA,扩增产物通过TA克隆连接到pMD18-T载体进行测序鉴定,然后通过双酶切将全长cDNA克隆到真核表达载体pcDNA3.1(+),最后得到pcDNA3.1(+)-NRP1重组质粒。[结果]成功克隆NRP1基因全长cDNA,并成功构建了pcDNA3.1(+)-NRP1真核表达载体。[结论]pcDNA3.1(+)-NRP1真核表达载体的成功构建可以为NRP1基因功能的进一步研究及其临床基因治疗奠定实验基础。[Objective] To clone full-length cDNA of Neuropilin-1（NRP1） gene from Human Keratinocytes Cell line（HaCaT） and construct reco-mbinant eukaryotic expression vector pcDNA3.1（＋）-NRP1,and pave the road for deeply research on functions of NRP1.[Methods] The full-length cDNA of NRP1 was amplified from HaCaT cells by RT-PCR and then cloned into pMD18-T vector through TA base pairing.After identification by DNA sequencing,the plasmid pMD18-T-NRP1 was digested by restriction enzymes and the full-length cDNA of NRP1 was generated and cloned into the eukaryotic expression vector pcDNA3.1（＋）,then the recombinant plasmid pcDNA3.1（＋）-NRP1 was constructed.[Results] The full-length cDNA of NRP1 was successfully cloned,and the eukaryotic expression vector pcDNA3.1（＋）-NRP1 was successfully contructed.[Conclusion] The eukaryotic expression plasmid pcDNA3.1（＋）-NRP1 has been constructed successfully,which lays the foundation for further studies of biological functions and clinical gene therapy of NRP1.

关 键 词：HACAT细胞 NRP1 真核表达载体 

分 类 号：R331[医药卫生—人体生理学]