[Molecular design and construction of IL6D24-PE40KDEL, a novel recombinant interleukin6-pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemias expressing interleukin6 receptors].

Objective A novel recombinant interleukin6-Pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemic cells highly expressing IL6R has been molecularly designed and constructed in this study. Methods Firstly, REDLK at C-terminus of Pseudomonas exotoxin PE40 was replaced with KDEL using point mutatgenesis technology. Secondly, a cDNA encoding interleukin-6 devoid of N-terminal 24 amino acids[IL6D24] was fused to 5′ terminus of PE40KDEL DNA by the method of gene splicing by overlap extension, which could generate recombinant IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL fusion genes. Thirdly, recombinant fusion genes IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL were ligated into the EcoR I and Sma I cloning sites in the pBV220 plasmids respectively and then transformed into E.coli HB101 cells. The expressed recombinant exotoxin fusion proteins were purified to electrophoresis purity by Mono Q column chromatography. Its selectively killing was tested by the MTS colorimetric method using both U937 and CEM cells lines. Results Recombinant exotoxin fusion proteins IL6D24-PE40KDEL was expressed as a form of inclusion bodies at higher level of 40%～ of total proteins in bacterial cells. Western blot showed that the purified products could react specifically with IL6 monoclonal antibody and PEA antiserum, respectively. IL6D24-PE40KDEL was selectively cytotoxic to U937 cells expressing IL6R-positive with IC50 of 250 ng/ml, and but not CEM cells expressing IL6R-negative. Conclusions Two novel recombinant interleukin6-Pseudomonas exotoxin fusion proteins IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL have been successfully constructed and they can selectively kill the leukemic cells expressing highly IL6R.