关键词: 胰岛-脑1基因, 真核表达载体, 转染

Abstract: Objective To construct and identify eukaryotic expression vector expressing Islet-brain 1(IB1) gene. Methods The total RNA was extracted from human insulinoma. IB1 gene was amplified by PCR from human IB1 cDNA library. The eukaryotic expression vector encoding IB1 was constructed by inserting the IB1 cDNA in the 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, and obtained the stably transfeced cell line. The inverted 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 showed that it contained 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 constructed successfully.

Key words: islet-brain 1 gene, eukaryotic expression vector, ransfection