Genetic Engineering Expression And Preliminary Function Of Immunodegradable Regulatory SCTLA - 4 And SPD - L2 - SCTLA - 4 Fusion Proteins

CTLA-4, also know as CD 152, is a T-cell surface molecule that displays a high degree of similarity to CD28. CTLA-4 is associated with negative regulation of T-cell activation in the antigen-presenting process. PD-L2 is the 5th member of the B7 family, the interaction between PD-L2 and PD-1 could dampen the TCR-MHC signal pathway. Since both CTLA-4 and PD-L2 could down regulate pathogenic T cell proliferation, these two molecules can be developed as therapeutic agents in the application for organ transplantation and auto-immune diseases.Large scale fermentation and purification of His-sCTLA-4 form Engineered Pichia pastors strain was conducted to get sufficient protein, the Immunosuppressive function of His-sCTLA-4 was then proved in rat Rheumatoid arthritis model. In order to get entirely native sCD152 without His-tag, we engineered a new sCTLA-4 expressing pichia patrors strain. Howerver, the subsequent purification steps are difficult to follow due to low expression level and tagless. To solve this problem, we designed another Prokaryotic vector, pET-sCTLA-4, which contains a His-tag followed by a thrombin cleavage sites, then sCTLA-4 was successfully purified form E.coli. During mice allogeneic mixed lymphocyte reaction and antigen specific lymphocyte transformation tests, when sCTLA-4 was added at a final concentration of 10ng/μ the Inhibitory rate reaches 69% and 50% respectively, which shows that sCTLA-4 can supress cellular immune responses significantly in vitro.For the purpose of develop a potential new drug for acute rejection in organ transplanate and autoimmune disease, we designed a new Immunosuppressive molecule-sPD-L2-sCTLA-4-by Bioinformatics and Immunology principle, then we constructed pET-sPD-L2-sCTLA-4 plasmid and purificated the recombinant protein form E.coli BL21 system. In mice allogeneic mixed lymphocyte reaction and antigen specific lymphocyte transformation tests, we found that sCTLA-4-sPD-L2 fusion protein had a remarkably inhibited the lymphocte activation, this result has provied a stong evidence for the immunosuppressive effect of sCTLA-4-sPD-L2 fusion protein. Our work has lay a foundation for the future immunosuppress drug development.