The Renaturation,purrificationand Identification Of Il3-pe38kdel ,A Fusion Toxin Consisting Of A Truncated Pseudomonas Exotoxin A (pe38kdel) Linked To Human Interleukin 3 (il3)

Acute myelogenous leukemia(AML) is organized the most common malignant blood disease. Present study indicated that Leukemia Stem Cells(LSC) are primitive rare population of AML cells capable of creating and mainting the leukemia clone.Due to a predominantly G0 cell-cycle status, LSCs may not be responsive to conventional chemotherapeutic agents, compared with leukemia blasts. So it is key searching a method of killing these LSCs. The present studies show that LSCs also have some features that are not found in norma; HSCs ,such asαhain of Interleukin-3 recepter(IL-3R) ,CD123. Our objective in this study was to construct a new recombinant immunotoxin with specific killing activity to LSCs targetly.Objective: To obtain IL3-PE38KDEL,a new recombinant immunotoxin consisting of a truncated pseudomonas exotoxin A (PE38KDEL) linked to human interleukin 3 (IL3) in E.coli SG13009, with high purity and normal bioactivity . Methods: the recombinant plasmid PQE30-IL-3-PE38KDEL,was transformed into E.coli SG13009.After 1PTG induction,the expressed protein products were verified by mass spectrometry and Western blotting. purified by Ni2+-NTA agarose affinity chromatography , refolding and it was identified.Rerults: The recombinant immunotoxin Was expressed in E coli SG13009 clone strain containing PQE30-IL-3-PE38KDEL induced by IPTG in the form of inclusion body. SDS-PAGE analysis revealed that expression level of the product was up to 17.56% of the total bacterial proteins. It was a protein band about Mr abave 54 900 in gel,which could be identified by PEA of anti- pseudomonas exotoxin A monoclonal antibody with Western blotting technique. The highly purified recombinant protein by Ni2+-NTA agarose affinity chromatography and refolding . Mass spectrometry demonstrated that the recombinant protein was the recombinant immunotoxin consisting of a truncated pseudomonas exotoxin A linked to human interleukin 3.Conclusion: IL3-PE38KDEL was successfully expressed in E.coli.A suitable protocol for purification of the recombinant protein was set up.After purification, pure protein of the expressed IL3-PE38KDEL was got. It laid a solid foundation for the further reseatch of function.