Expression And Purification Of Recombinant Protein Of Human CTLA4 Extracellular Domain In Yeast GS115 And Study Of Its Bio-activities In Vitro And In Vivo

Backgroud: The activation of antigen-specific T cells plays a vital role in the initiation and development of some immune-related diseases, such as transplant rejection and allergic diseases. Effective activation of antigen-specific T cells not only requires the first signal transduction through T-cell receptor(TCR) binding with peptide-MHC complex on the antigen presenting cell (APC), but also needs the second signal, termed costimulation. Costimulation critical to the degree and consequence of T cell activation is provided by interaction between soluble factors or cell-surface molecules on the T cell and on the APC. It has been demonstrated that B7-CD28 pathway is a prominent costimulation pathway which leads full T cell activation, induces differentiation and regulates the production of cytokines. Lacking and blocking of B7-CD28 pathway will result in blockage of the T cell activation and proliferation. T cell will be apopotosis, clone depletion and /or anergy, in turn inducing an antigen-specific tolerance. CTLA4 (Cytotoxic T lymphocyte associated antigen 4), a prominent negative costimulator, can competitively bind with B7, and block B7-CD28 pathway and T cell activation by producing inhibitory singals. Recombinated human CTLA4Ig fusion protein had been used experimentally and clinically in prevention and treatment of transplant rejection, autoimmune diseases and allergic diseases, and positive curiative results were obtained. CTLA4Ig fusion protein was expressed by genetically fusing the DNA fragment encoding 1-125 amino acid residues of CTLA4 extracellular domain with DNA fragment encoding an IgG Fc region. Itsinfunctional region is the extracellular domain of CTLA4. The sequence of CTLA4 extracellular domain determines its space construction and binding specific with its ligand. Without heterologous DNA, the function of protein of CTLA4 extracellular domain will be more identical to that of natural CTLA4 theoretically. Meanwhile, because the fragment of CTLA4 extracellular domain is only 400bp, its expression will be more stable than that of CTLAIg fusion protein( 1.2kb). Yeast GS115, one kind of Pichia pastoris, has been used to express varied heterologous proteins. GS115 can grow in a high density and produce heterologous proteins by secretion, which are in a high-level and easy to be purified. Hence, yeast GS115 was used to express the target protein in this study.Objection: To obtain the biological activated recombianated protein of huaman CTLA4 extracellular domain(CTLA4e), and to explore its effects on allergic diseases and transplant rejection.Methods: 1.Expression and purification of recombinant human CTLA4 extracellular domain: a 400bp DNA fragment of CTLA4 extracellular domain was obtained from the pCTLA4/Ig plasmid with genetical technique. Then, this DNA fragment was inserted into pPIC9 plasmid to construct the single copy plasmid of yeast expression system (pPIC9-CTLA4125). Furthermore, the multi-copy plasmid (pPIC9K-CTLA4125) was constructed. After the plasmid was assayed for DNA sequence, it was transformed into GS115 by electroporation. The stable expression yeast was obtained by screening and was induced to ferment by methanol. After ultrafiltration and rough separation with ammonium sulfate and anion-exchange chromatography, the recombination protein of CTLA4 extracelluar domain was rectified from the ferment supernatants. The expression of target protein was identified with Dot-ELISA, SDS-PAGE and Western blotting.2. Inspection for the biological activities of recombination protein of?CTLA4 extracellular domain in vitro: We investigated the role of CTLA4e in T cell proliferation of MLR with 3H-TdR incorporation.3. Investigation for the biological activities of recombination protein of CTLA4 extracellular domain in vivo: First, we observed the effects of CTLA4e on allergic rhinitis in mice. After sensitized and challenged by ovalbumin(OVA), 60 mice were equally divided into allergic rhinitis group(OVA group), Saline group and CTLAe group(200 U g/mice,...