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.
ABSTRACT
Objective:To construct a recombinant prokaryotic expression vector pQE30-hALRIP for further research.Method:PCR was used to amplify hALRIP codon region.The gene was inserted into pMD18-T vector and sequenced.Then the prokaryotic expression vector pQE30-hALRIP was constructed and identified. Results:Clonal recombinant vector pMD18- hALRIP and expression vector pQE30-hALRIP were constructed. DNA sequence analysis and restriction analysis showed both of the vectors were the same as the predicted results. Conclusion:Prokaryotic expression vector pQE30-hALRIP is successfully constructed.