【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。更多还原

【Abstract】 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-polymerase chain reaction(RT-PCR).By using gene recombination technique,human PRNP cDNA was inserted into retroviral vector pEGFP-N2.The recombinant plasmid was identified by apair of specified primers containing the restriction sites of XhoⅠ and EcoRⅠ.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-SY5Y cells.Conclusion The recombinant plasmid pEGFP-N2-PRNP is constructed successfully,Which offers a basic for the further research on PRNP biological fuction.更多还原