Production of Recombinant Chicken IgY-Fc and Evaluation of Its Transport Ability into Avian Egg Yolks

Abstract

Maternal immunoglobulin (Ig) Y, the avian counterpart of mammalian IgG, is efficiently transported into the yolks of maturing oocytes. We have previously shown that the Fc region plays a critical role in the IgY transport into avian egg yolks. The aim of this study was to produce recombinant IgY-Fc and to evaluate its transport ability into avian egg yolks. Two basic expression vectors were constructed: one mainly expressed three constant regions of chicken IgY heavy chain (Fcυ2-4) that contained three cysteine residues (C252, C340, C347) involved in inter-heavy chain disulfide bonds; and the other mainly expressed two constant regions (Fcυ3-4) that contained two cysteine residues (C340, C347). SDS-PAGE and Western blotting analyses indicated that both Fcυ2-4 and Fcυ3-4 were separated into multiple protein bands under non-reducing conditions, suggesting incomplete formation of inter-chain disulfide bonds. To prevent the incomplete disulfide bond formation, C340 and C347 were mutated individually to serine by site-directed mutagenesis. As expected, each of the mutated recombinant IgY-Fc produced a single protein band by SDS-PAGE analysis. When intravenously-injected into laying quail, the two mutants, Fcυ2-4C347S and Fcυ3-4C340S, were transported into the egg yolks at high levels, whereas the other two mutants, Fcυ2-4C340S and Fcυ3-4C347S, were transported into the egg yolks at lower levels. In conclusion, we have succeeded in producing a recombinant IgY-Fc retaining a high transport ability into the avian egg yolks; this protein will be provided as a useful tool for studying the mechanism of IgY transport into egg yolks.