Building and identification of prion gene eukaryotic expression vector of human / 重庆医学

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-poly-merase 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 a pair of specified primers containing the restriction sites of Xho Ⅰ and EcoRⅠ .Results The PRNP gene could be obtained by RT-PCR ,the recombinant plasmid was identified by restriction endonucle-ase 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 re-search on PRNP biological fuction .