Construction and identification of eukaryotic expression vector expressing Islet-brain 1 gene / 基础医学与临床

ABSTRACT

Objective To construct and to identify eukaryotic expression vector expressing Islet-brain 1(IB1) gene.Methods Total RNA was extracted from human insulinoma.IB1 gene was amplified by PCR from human IB1cDNA library.The eukaryotic expression vector encoding IB1 was constructed by inserting the IB1 cDNA into EcoR I/Kpn I sites of the pEGFP-N1 vector with the green fluorescent.The construct was transfected into RINm5F cell line,screened by G418.The phase contrast fluorescence microscope,flow cytometer,and Western blot were used to identify the recombinant plasmid and transfeced cell line.Results The RT-PCR products for IB1(AA1-280)generated from human insulinoma was 840 bp.Sequence analysis proved the same sequence as published in Gen-Bank.Two bands showed that pEGFP-N1 vector encoding IB1 digested by EcoR I or Kpn I.Western blot showed IB1 gene was expressed in RINm5F cells.Conclusion The recombinant prokaryotic expression plasmid pEGFP-N1-IB1 has been successfully constructed.