Prokaryotic Expression Of Chicken Macrophage Migration Inhibitory Factor And Anti-chMIF Ab Preparation

Macrophage migration inhibitory factor, abbreviated to MIF, is a conservative cytokine which plays important roles in immune regulation, inflammatory reactions, energy metabolism regulation, and so on. MIF has pleiotropic effects, it involves in congenital and acquired immunity, evokes other cytokine and chemokine secretion,and it can also interact with hypothalamus hypophysis adrenal gland axis and glucocorticoid, therefore influences cell proliferation, apoptosis, and phagocytosis. Obviously, MIF plays an important role in septicopyemia, infection, inflammation, autoimmune disease, transplant rejection, arteriosclerosis, cancer and its like. Relatively most research work focused on human and experimental animals, rather than farm animals, such as chicken. The purpose of our experiment is to get recombinant chicken MIF protein with prokaryotic expression vector, and prepare anti-chicken MIF polyclonal antibody.First of all, prokaryotic expression vector pEASY-E1-MIF was transfected into the expression-competence BL21(DE3) to get the transformants. Secondly, IPTG was used to induce the recombinant protein (MIF) expression. Next, Ni-NTA affinity column was used to purify the recombinant protein MIF. Finally, purified recombinant chicken MIF protein, as the antigen, was injected into rabbits to obtain anti-chicken MIF serum, and test the antiserum by western blot.The results were as follows:(1)The positive plasmid DNA sequencing verified that transformants contain the target gene, i.e., chicken MIF gene.;(2)Optimum condition of IPTG induction is to keep shaking at37℃for2.5h;(3)The SDS-PAGE showed that the purity of recombinant chicken MIF is good enough to be used to immunize animals;(4)The ELISA result manifested that the titer of rabbit anti-chicken MIF Ab reached1:12800;(5)Western blotting confirmed that rabbit anti-chicken MIF polyclonal antibodies work well.