Construction and prokaryotic expression of a recombinant immunotoxin fused with mouse macrophage inflammatory protein-1α and truncated diphtheria toxin

Objiective To construct a new recombinant immunotoxin expression vector by fusing mouse macrophage inflammatory protein-1α gene and a truncated diphtheria toxin(DT390) gene,and examine the expression of mMIP-1α-DT390 fusion protein in Escherichia coli.Methods mMIP-1α cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction(RT-PCR),and the DT390 gene was cloned from a eukaryotic expression vector Srα-DT390 by PCR.The obtained genes were digested by restriction endonucleases,and then inserted to the expression plasmid pET-32a(+) to produce a recombinant vector pET-32a(+)-mMIP-1α-DT390.The confirmed vector was transformed to E.coli BL21(DE3) and induced by IPTG,and then the expressed protein was obtained and detected by SDS-PAGE and Western-blotting.Results The new recombinant immunotoxin expression vector PET-32a(+)-mMIP-1α-DT390 was constructed successfully.The mMIP-1α-DT390 fusion protein was expressed in E.coli BL21(DE3),and the molecular weight of the fusion protein was identical to the expected value.Furthermore,the protein could reacted with the specific antibody against mMIP-1α and DT390,respectively.Conclusion pET-32a(+)-mMIP-1α-DT390 recombinant immunotoxin expression vector may have some potential value in clinical application.