Expression, Purification Of The IL3-PE38KDEL In E.coil And Identification Of Its Biological Activity In Vitro

Acute myelogenous leukemia (AML) is organized the most common malignant blood disease. Present study indicated that Leukemia Stem Cells (LSCs) are primitive rare population of AML cells capable of creating and mainting the leakemic clone. Due to a predominantly G0 cell-cycle status, LSCs may not be responsive to conventional chemotherapeutic agents, compared with leukemia blasts. The present studies show that LSCs also have some features that are not found in normal HSCs, such asαchain of Interleukin-3 Recepter (IL3-Rα). So the IL3-Rαchain is supposed to be an important therapy target. Our objective in this study was to construct a new recombinant immunotoxin IL3-PE38KDEL with specific killing activity to LSCs targetly.Objective: Our experiments are designed to construct prokaryotic expression vector of PQE30-IL3-Linker-PE38KDEL and purify the recombinant protein by ultrasound with Ni2+-NTA resin, denatarat and renaturat the fusion protein which is of benefit to us to better understand the function of IL3-PE38KDEL. As well, we will investigate the function of the purified recombinant protein on the invasion of LSCs by MTT, make use of TF-1 human erythroleukemia cell-line which have the special phenotype CD123. These results may give us some very important information to further study.Methods:1. Prokaryotic expression and identification of recombinant plasmid PQE30-IL3-Linker-PE38KDEL: The recombinant vector IL3-PE38KDEL was identified with DNA sequencing and endonucleases. Then transform the recombinant vector into Escherichia coli SG13009. The target protein was expressed in the host bacteria under the induction of IPTG, and was identified by 15% SDS-PAGE. The expressed product was identified by Westernblot. 2. The fusion protein was purified from the supernatant of cells broken by ultrasound with Ni2+-NTA resin. Identify the recombinant protein IL3-PE38KDEL biological activity in vitro: After purification and gradient dialysis renaturation, the effection of the recombinant protein IL3-PE38KDEL on the invasion of TF-1 cells and dose-dependent manner were investigated by MTT experiment.Results: 1. Recombinant protein IL3-PE38KDEL was successfully constructed. The fusion gene insered into pQE30 plasmid was confirmed by restriction enzyme analysis and DNA sequencing. Recombinant IL3-PE38KDEL protein can be expression efficiently in E.coli.SG13009. Confirmed by SDS-PAGE, the molecular weight of the recombinant protein is 57kDa. The results of Westernblot indicated that the fusion protein could bind specially to the anti-6His serum. After purification by Ni2+-NTA resin, the purity of fusion protein IL3-PE8KDEL is more than 90%. 2. After gradient dialysis renaturation, we got more purify fusion protein. The purified fusion protein was able to inhibit the invasion of TF-1 cells in a dose-dependent manner. We also found that the concentration of the recombinant IL3-PE38KDEL protein for 200μg/ml, restraint ability reached to high peak, the cell invasion of 83% were suppressed, and restrains rate when concentration went up to 500μg/ml to top, is 85%.Conclusions: We have successfully constructed recombinant pQE30-IL3-Linker-PE38KDEL plasmid by utilizing prokaryotic expression vector pQE30 and the recombinant IL3-PE38KDEL protein has been expressed in E.coil.SG13009. And the targeted protein was purified by affinity chromatography. 2. Considered together, these results provided a strong indication that the purified fusion protein was able to inhibit the invasion of TF-1 cells in a dose-dependent manner. And statistical analysis showed that it has statistical significance.This result showed that we had gained active recombinant protein IL3-PE38KDEL by prokaryotic expression system. It was also first time to find that the concentration of the recombinant fusion protein for 200μg/ml, cultivanted for 72 hours, restraint ability reached to high peak, the cell invasion of 83% were suppressed, and restrains rate when concentration went up to 500μg/ml to top, is 85%. The results of this study should therefore be generalizable to the fusion protein effect the activity of NF-κB in AML stem cell and have important implications for establishment of animal model.