Construction and prokaryotic expression of a recombinant immunotoxin IP-10-PE38KDEL

Objective To construct a new recombinant immunotoxin expression vector by fusing rattus IFN-γ-inducible protein gene and a truncated pseudomonas exotoxin A ramification(PE38KDEL)gene,and examine the expression of IP-10-PE38KDEL fusion protein in E coli BL21.Methords IP-10 gene was cloned by polymerase chain reaction(PCR)and the PE38KDEL gene was cleaved from a vector PRKL459K containing PE38KDEL by restriction endonucleases,then inserted to the prokaryotic expression vector pET-32a(+).The prokaryotic recombinant vector pET-32a(+)-IP-10-PE38KDEL was identified by PCR,restriction endonuclease digestion,and sequence analysis,and then transformed into E coli BL21 and induced by IPTG.The expressed product was obtained and the molecular weight and specificity of the protein were detected by SDS-PAGE and Western blotting.Results PCR,restriction endonuclease digestion,and sequence analysis revealed that the IP-10-PE38PE38KDEL fusion gene was cloned into the prokaryotic expression plasmid vector pET-32a(+)successfully.The IP-10-PE38PE38KDEL fusion protein was expressed in E coli BL21,which could react with the specific antibody.The molecular weight of the expression product was identical to the expected value.Conclusion A prokaryotic expression vector pET-32a(+)-IP-10-PE38KDEL is constructed successfully and the fusion protein is expressed stably in E coli BL21,which will provide the basis for the further study on function of the protein.