S-Space College of Veterinary Medicine (수의과대학) Dept. of Veterinary Medicine (수의학과) Journal Papers (저널논문_수의학과)
Cloning, expression and bioassay of canine CTLA4Ig
- Shin, Il-Seob; Choi, Eun-Wha; Chung, Jin-Young; Hwang, Cheol Yong; Lee, Chang Woo; Youn, Hwa Young
- Issue Date
- Vet. Immunol. Immunopathol. 118, 12-18
- CTLA-4; Costimulatory pathway; Chimeric fusion protein; Protein expression; Dog; Mixed lymphocyte reaction
- Blockade of the B7:CD28 costimulatory pathway has been shown to inhibit humoral immunity, graft rejection, graft versus host disease and ameliorate autoimmune diseases. A soluble chimeric fusion protein, CTLA4Ig, binds to B7 with greater affinity than CD28 and blocks the binding of CD28 to B7. We describe the cloning and expression of canine CTLA4Ig, a recombinant chimeric fusion protein composed of the extracellular domain of canine CTLA-4 and the CH2–CH3 domains of canine immunoglobulin alpha constant region (IGHA) genes, linked via an immunologically inert flexible peptide. The recombinant CTLA4Ig protein of approximately 45 kDa molecular weight was expressed mainly as insoluble inclusion bodies in Escherichia coli. The protein was solubilized in denaturing buffer and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography followed by refolding. The yield was about 6 mg of recombinant CTLA4Ig per liter of culture. The purified protein was biologically active in one-way mixed lymphocyte reactions, demonstrating immunosuppressive activities in a dose-dependent manner. The findings suggest that recombinant canine CTLA4Ig protein could be valuable in assessing the function of CTLA-4 in the canine immune system and may be effective in autoimmune disease therapy.
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