Expression Of The Extracellular Domain Of CD47 Protein And Its Role In Jurkat Cells

Malignancy process will produce a series of cytological and genetic mutations, leading to activation of oncogenes, inactivation of tumor suppressor gene. Most of the malignant cells have some common features, including a strong proliferative activity, tissue invasion and metastasis, poorly regulated replicative potential, sustained angiogenesis, evasion of cell death by a variety of pathways and so on. Recent studies have shown that tumor cells can escape immunosurveillance through a number of special mechanisms. CD47 as an anti-phagocytosis signaling molecules plays an important role in tumor immunesurveillance. The tumor cells can inhibit the phagocytosis by up-regulating the expression of CD47. In the research of different leukemia, they found that many types of leukemia cells and leukemia stem cells have a higher levels of CD47 expression than normal cells. The ligation of CD47 and its receptor SIRPα(signal regulatory proteinα)on phagocytic cells could inhibit signal transmission and prevent phagocytosis of tumor cells. The CD47-SIRPαinteraction can be therapeutically targeted with a monoclonal blocking antibody against CD47, which enables phagocytosis of tumor cells . This has been certified in acute myeloid leukemia (AML), bladder cancer, non-Hodgkin's lymphoma (NHL) ,and acute lymphocytic leukemia cells in vitro and in vivo, and the specific molecular mechanism of CD47-SIRPαsignaling pathway remains to be further studied.The high expression of CD47 on tumor cells have to bind to the receptors SIRPαon macrophages to paly the function. Therefore, we expressed the extracellular domain of human CD47 which contains the binding sites in the prokaryotic expression system to play a "occupying "function, and compete with CD47 on tumor cells surface, thereby blocking the CD47-SIRPαsignaling pathway to promote phagocytosis of tumor cells,similar with the function of CD47 monoclonal antibody. Through this approach we hope to further study the role and the molecular mechanisms of CD47 in leukemia treatment and other malignant tumor targeted therapy,which has an important significance.For these goals,we did the following works:1. Prokaryotic expression of soluble extracellular domain of CD47 protein. Using the human lymph node as a template to amplify the cDNA of CD47 extracellular molecules, after the sequencing, the fragment will be inserted into the prokaryotic expression vector pET32a (+), access to get the expression vector pET32a-hCD47ext. After transformed into E. coli BL21, protein expression induced with IPTG will be used to induce the protein expression. Then optimizing the expression conditions, and use the best expression conditions of the target protein to expand the expression system. The protein was successfully refolded and purified using dialysis and affinity chromatography. Use the SDS-PAGE and Western blot to analyse the protein expression and correctness of the protein.2. The binding of soluble protein TrxHis-hCD47ext to its receptor SIRPα. Adding the CD47 extracellular protein to the culture supernatants of macrophages, and then use the anti-His antibodies to detect the indirect fluorescence intensity of the cell surface by Flow cytometry,which verifies whether TrxHis-hCD47ext could bind to SIRPα.3. The role of TrxHis-hCD47ext in human acute T cell lymphoblastic leukemia(T-ALL) cell line Jurkat. To verify whether the soluble extracellular domain of CD47 protein is able to block the CD47-SIRPαsignaling pathway, thereby promoting macrophage phagocytosis of leukemia cells, we labeled Jurkat cells by Dio and co-culture with macrophages, the TrxHis-hCD47ext was added to the total culture supernatant,and then use the fluorescence microscopy to observe the changes of macrophages phagocytosis, and to verify the activity and function of TrxHis-hCD47ext.Conclusion:1. Implement the soluble expression of extracellular domain of human CD47 protein in a prokaryotic expression system, achieved the pure TrxHis-hCD47ext protein.2. Verified that the extracellular domain of human CD47 protein couid bind to the receptors SIRPαon macrophages effectiveiy.3.Using the Jurkat cells and macrophages co-culture methods we verified the activaty of TrxHis-hCD47ext protein and show that this protein could promote macrophage phagocytosis of Jurkat cells.