A Study On Carrier Construction,Heterologous Expression And Purification Of Fusion Protein ABD-Fc-IL-2 And Biological Activity In Vitro And Pharmacodynamics In Vivo

Objective:The SP-ABD-Fc-IL-2 fragment was constructed and inserted into the vector plasmid pcDNA3.1(+)by double enzyme digestion and ligation.The recombinant plasmid was then transfected into CHO-S cells for heterologous expression,and the fusion protein was purified by r Protein A beads affinity chromatography.The proliferation activity of the fusion protein on CTLL-2 cells and the interaction with HSA protein were analyzed to verify its biological activity in vitro.The fusion protein was further injected into mice to analyze its half-life pharmacodynamics in vivo.Methods:1.The recombinant gene SP-ABD-Fc-IL-2 was constructed,and ligated into the vector pcDNA3.1(+)after double digestion and ligation.The recombinant plasmid was verified by double enzyme digestion and Sanger sequencing.2.pcDNA3.1(+)/SP-ABD-Fc-IL-2 plasmid was transfected into CHO-S cells,and the proteins in supernatant were collected and purified by affinity chromatography using r Protein A beads.3.Band-analysis of fusion proteins ABD-Fc-IL-2 and Fc-IL-2 was performed by SDS-PAGE and Western blot.4.MTT colorimetry was used to determine the effect of the fusion protein ABD-Fc-IL-2 and Fc-IL-2 on the proliferation of CTLL-2 cells,and its biological activity was determined.5.The interaction between ABD fragment in the fusion protein and HSA protein was analyzed by Ni-Pull down-Western blot.6.The two fusion proteins were injected into mice respectively,and the serum at each time point was collected.The concentration of the two fusion proteins in the serum was calculated and analyzed by ELISA double antibody sandwich method,and the pharmacodynamics of the half-life was studied.Results:1.The results of double enzyme digestion and Sanger sequencing of plasmid pcDNA3.1(+)/sp-ABD-Fc-IL-2 showed that the plasmid has been constructed successfully.2.SDS-PAGE and Western blot analysis of the fusion protein ABD-Fc-IL-2 and Fc-IL-2 showed that the fusion proteins could be expressed in CHO-S cells,and the proteins purified by r Protein A affinity chromatography were of high purity.3.MTT colorimetric analysis showed that the fusion proteins ABD-Fc-IL-2 and Fc-IL-2 promoted the proliferation of CTLL-2 cells and retained the biological activity of IL-2.4.Ni-Pull down-Western blot analysis showed that the interaction between ABD fragment and HSA protein was not affected in the fusion protein ABD-Fc-IL-2.5.The concentration of fusion protein in serum of mice was quantitatively analyzed by double antibody sandwich ELISA method,and the analysis data showed that the half-life of fusion protein ABD-Fc-IL-2 was longer than that of Fc-IL-2.Conclusion:1.The plasmid pcDNA3.1(+)/SP-ABD-Fc-IL-2 was successfully constructed.2.The purified ABD-Fc-IL-2 fusion protein has biological activity in vitro.3.The fusion protein ABD-FC-IL-2 prolonged the plasma half-life.