Expression Of The Recombinant SIRPα-Fc Fusion Protein And Its Activity Evaluation

Objective:Signal regulatory proteinα(SIRPα), a transmembrane protein belonging to the immunoglobulin superfamily, its extracellular ligand is CD47.The signal interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of tumor cells by macrophages. Blocking antibodies against CD47, recombinant SIRPα protein, CD47 extracellular protein have been shown to promote phagocytosis of tumor cells by macrophages. After researching the molecular mechanism of the antibodies against CD47, recombinant SIRPα protein and CD47 extracellular protein in the process of tumor treatment. In this paper, we try to express a recombinant SIRPα-Fc fusion protein in prokaryotic system to effectively interrupt the interaction between SIRPα and CD47. To obtain recombinant SIRPα-Fc fusion protein, two histidine is added at the amino terminus of the recombinant protein SIRPα and Fc fragment is inserted at the carboxyl terminus of the recombinant protein. Therefore, this protein is easier to purify,and it can bind the Fc receptor on the macrophage surface when it is capable of binding CD47 on the surface of tumor cells, macrophages can hold tumor cells more tightly, so the ability of macrophages to clear tumor cells will be more enhanced in theory, while the ability of macrophages to swallow normal cells will be diminished through this mechanism. In addition, this protein is soluble, and it has an advantage of small molecular weight. Finally, the aim of this study to lays a foundation for further research the molecular mechanism of the recombinant SIRPα-Fc in the process of tumor treatment, it is of great significance for the treatment of malignant tumor.Method:(1) Prokaryotic expression of recombinant SIRPα-Fc fusion protein. To synthesis of recombinant DNA that is inserted two histidine at the amino terminus of the recombinant protein SIRPα which has been reported and the transformed Fc fragment was inserted into the carboxyl terminus of SIRPα.Then the restriction endonuclease NdeI site is designed at the 5,end and the restriction endonuclease sites SailI is designed at the 3,end. Full synthesized gene fragments after digested by NdeI and SalI, then connected to the Pmal-p5 X vector which was digested by the same enzymes. Finally, the recombinant Pmal-p5X-SIRPα-Fc expression vector was constructed. And the expression vector was transformed into Escherichia coli Dh5α, after the plasmid amplification was identified correctly, the expression vector was transformed into Escherichia coli BL21(DE3), protein expression in Escherichia coli induced with IPTG will be used induced the protein expression. Then optimizing the expression conditions of the target protein to expand the expression system. The protein was successfully obtained and purified using dialysis and affinity chromatography. Usethe SDS-PAGE and Western blot to identify the expression of the protein and correctness of protein.(2)Tested the activity of the recombinant SIRPα-Fc fusion protein in vitro. To verify whether the recombinant SIRPα-Fc fusion protein is able to block the CD47-SIRPα signaling pathway, thereby promoting macrophage phagocytosis of tumor cells. Tumor cells were labeled by transfection technology and co-culture with macrophages, then the recombinant SIRPα-Fc fusion protein was added to the total culture supernatant, and after two hours incubation, using fluorescence microscope to observe the changes ofmacrophages phagocytosis, and to verify the activity and function of SIRPα-Fc fusion protein.Result:(1) Implement the soluble expression of recombinant SIRPα-Fc fusion protein in a prokaryotic system, achieved the pure SIRPα-Fc fusion protein.(2) The activity of the recombinant SIRPα-Fc fusion protein was verified through the tumor cells and macrophages co-culture method. And we found the phagocytic index of macrophage SIRPα group, SIRPα-Fc fusion protein group were significantly higher than PBS group, but there was no significant difference between SIRPα group and SIRPα-Fc fusion protein group. And the control and treatment groups in the phagocytic index were lower than reported in the literature data.Conclusion: Because macrophage phagocytosis index is lower than the data reported in the literature. However, it could not be further studied because of time and financial constraints. Therefore, in order to verify whether the recombinant SIRPα-Fc fusion protein is able to block the CD47-SIRPα signaling pathway, and whether can further promote macrophage phagocytosis of tumor cells than SIRPα. We will optimize the experimental conditions and experimental methods, choose different types of tumor cell lines in vitro, and establish animal models of disease in vivo.