The Construction Of Humanized ScFv And Its Recombinant Immunotoxin Against CML Cells And Identification Of The Biological Activity

Objective: Chronic myeloid leukemia （CML） is a hematologicmalignancy of indefinite proliferation of hematopoietic progenitor cells.Drug resistance and relapse are the two problems of CML currently. Withthe development of recombinant DNA technology, immunotoxin, especiallythe recombinant immunotoxin, plays a more and more important role intumor immunotherapy. Various immunotoxin reagents have been used inclinical trials. In our study, we prepared an anti-CML cells hscFv, fused itwith ETA’ to prepare a rIT, then identified their prokaryotic expression,purification, and preliminary study of its biological activity, aiming toprovide an efficient and selective approach for the targeted therapy ofCML.Methods:1. The hscFv gene fragments were amplified by PCR,linked it into the prokaryotic expression pET32a（+） and transformed intothe expression strain E.coli BL21（DE3）, then analyzed by SDS-PAGE andwestern blot. The fusion protein was purified by Ni²⁺-NTA. Its biological activity was demonstrated by CM-ELISA and FCM.2. The hscFv gene was ligated into pWW20vector containing ETA’gene to form the hscFv-ETA’ gene which was sub-cloned into thepET32a（+） expression vector. The antigen binding activity of hscFv-ETA’was charactered as hscFv, and the targeted killing effect on CML cells orpatients cells was identified by MTT and apoptosis assays.Results: The amplified hscFv and hscFv-ETA’ gene fragment wereidentified by restriction enzyme digestion and DNA sequencing analysis.The hscFv and hscFv-ETA’ gene were successfully expressed in E. coli,showed by SDS-PAGE analysis, and the relative molecular mass （Mr） was46kDa or88kDa identified by western blot, respectively. The antigenbinding activity was detected by CM-ELISA and FCM. The targeted killingeffect of hscFv-ETA’ on CML cells or patients cells was revealed by MTTand cell apoptosis assay.Conclusion: The hscFv and hscFv-ETA’ fusion protein aresuccessfully constructed and expressed in E. coli expression system.Theretains antigen recognition characteristics and the targeted killing effectwere proved, which will provide a basis for the further study on animalmodel or clinical evaluation．...