Construction and prokaryotic expression of fusion gene IL3-PE38KDEL / 重庆医科大学学报

ABSTRACT

Objective:To construct a prokaryotic expression plasmid of PQE30-IL3-Linker-PE38KDEL and identify its recombinant protein expression.Methods:The IL3 and PE38KDEL gene were amplified by polymerase chain reaction(PCR) and cloned into the prokaryotic expression plasmid PQE30-Linker constructed after being sequenced.The recombinant vector confirmed by restriction endonucleases digestion,coenobium PCR,and DNA sequence analysis was transformed into E.coli SG13009.The expression of the protein was induced by IPTG.Relative molecular weight of the expression product was detected by SDS-PAGE.Finally,the fusion protein was examined by Western blot.Results:The results of restriction endonuclease digestion,coenobium PCR and DNA sequence analysis showed that the prokaryotic expression vector PQE30-IL3-Linker-PE38KDEL was constructed successfully.With induction of IPTG,the relative molecular weight of the expression product was identical to the expected value.The expressed 6?His-IL3-PE38KDEL fusion protein were identified at relative molecular mass of 57KD by Western blot with anti-His monoclonal antibody,showing the fusion protein expressed correctly.Conclusion:The fusion protein IL3-PE38KDEL is successfully constructed,which lays a solid foundation for the further research of protein purification and function.