Cloning, expression and bioassay of canine CTLA4Ig.
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 45kDa 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 6mg 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.
Shin
Il-Seob
IS
Department of Veterinary Internal Medicine, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, San 56-1, Sillim 9-dong, Gwanak-gu, Seoul 151-742, South Korea.
Choi
Eun-Wha
EW
Chung
Jin-Young
JY
Hwang
Cheol-Yong
CY
Lee
Chang-Woo
CW
Youn
Hwa-Young
HY
D000069594:Abatacept; D000818:Animals; D001681:Biological Assay; D049109:Cell Proliferation; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D018796:Immunoconjugates / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D007070:Immunoglobulin A / Q000235:genetics*; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects
D018076:DNA, Complementary; D018796:Immunoconjugates; D007070:Immunoglobulin A; D007127:Immunoglobulin Constant Regions; D011993:Recombinant Fusion Proteins; D000069594:Abatacept