人朊蛋白基因真核表达载体的构建及鉴定  

Building and identification of prion gene eukaryotic expression vector of human

作　　者：张海英[1] 刘亦恒[2] 费小雯[1] 易西南[1] 吴多庆[2] 

机构地区：[1]海南医学院人体解剖学教研室,海口571101 [2]海南省海口市人民医院骨科中心,570208

出　　处：《重庆医学》2014年第19期2434-2436,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(81100246);海南省自然基金资助项目(811204);海口市重点科技计划项目(2013-52)

摘　　要：目的构建人朊蛋白基因(PRNP)真核表达重组质粒。方法从阿尔茨海默病(AD)患者外周血白细胞中提取总RNA,利用RT-PCR的方法扩增编码人PRNP的基因片段,应用基因重组技术将人PRNP基因片段克隆到真核表达载体pEGFP-N2中,经XhoⅠ及EcoRⅠ双酶切、单酶切及基因序列测定证实所构建的载体。结果 RT-PCR方法获得人PRNP的基因片段,限制性内切酶酶切分析、PCR法及序列测定证实为正确重组质粒,并且该重组载体能够在SH-SY5Y细胞中表达。结论构建的真核表达重组质粒pEGFP-N2-PRNP通过鉴定,结构正确,为后续研究PRNP的生物学功能奠定了基础。Objective To constructan eukaryotic expression recombinant plasmid named pEGFP-N2-PRNP. Methods Total RNA was extracted from alzheimer（AD） disease peripheral blood,and the PRNP gene was amplified by reverse transcription-poly- merase chain reaction（RT-PCR）. By using gene recombination technique,human PRNP cDNA was inserted into retrovira[ vector pEGFP-N2. The recombinant plasmid was identified by a pair of specified primers containing the restriction sites of Xho I and EcoR I Results The PRNP gene could be obtained by RT-PCR, the recombinant plasmid was identified by restriction endonuclease analysis,PCR and sequence analysis, and the expression vector pEGFP N2-PRNP, which could be stably expressed in SH-SYSY cells. Conclusion The recombinant plasmid pEGFP-N2-PRNP is constructed successfully,Which offers a basic for the further re search on PRNP biological fuction.

关 键 词：阿尔茨海默病 朊蛋白基因 pEGFP-N2真核表达载体 

分 类 号：R346[医药卫生—基础医学] R749.16