In Vitro Expression Of Chicken CD40L And In Vivo Characterization Of Its Biological Activity

Objictive:This project was designed to express the bivalent Chicken-CD40L as an adjuvant candidate using yeast Pichia pastoris expression system and then assess its adjuvant activity with Newcastle Lasota vaccine in chicken.Methods:The chicken-CD40L gene was codon-optimized according to the Pichia pastoris preferred codon. To facilitate the downstream purification,6×His tag (CAC CAC CAC CAC CAC CAC) was added at the C-terminus. The DNA construct was linearized and transformed into Pichia Pastoris by electric shock. The transformants were verified by PCR. The expressed and purified bivalent chicken-CD40L protein was confirmed by SDS-PAGE and Weastern blot analysis. The bivalent chicken-CD40L protein was captured using Ni-NTA Resin and then fine purified using anion exchange resin. We found that the expressed and purified bivalent chicken CD40L protein was glycosylated via DTT and PNGase-F analysis. The in vivo adjuvant function was assessed with the Newcastle Lasota vaccine in chickens.Results:1) The recombinant plasmid pwPICZalpha-bi-chicken-CD40L was successfully constructed.2) The DNA construct was transformed into yeast Pichia Pastoris and verified by PCR.3) The bivalent chicken CD40L was expressed and purified through two-step purification strategies.4) The expressed bivalent chicken CD40L was glycosylated and verified via PNGase-F analysis.5) The bivalent chicken CD40L did not show significant adjuvant function when assessed with Newcastle disease Lasota vaccine together in chicken. The adjuvant activity was analyzed using antibody titer, Superoxide Dismutase(SOD), Gluthathione Peroxidase(GSH-PX) and Malondialdehyde(MDA).Conclusion:We have successfully constructed and expressed the bivalent chicken CD40L using yeast Pichia Pastoris expression system. We did not observe significant adjuvant activity of the bivalent chicken CD40L with Newcastle Lasota vaccine at the tested dose of 1 ?g or 0.1 ?g per chicken.