Construction And Verification Of The Daintain/AIF-1 Bait Plasmid For The Yeast Two-hybrid System

The yeast two-hybrid system is a powerful and sensitive molecular genetic approach for studying protein-protein interactions in vivo. Over the seventeen or so years since the first report of the system by Fields and Song, it has been modified in numerous ways and has been widely adapted for the study of cell biology, oncology, proteomics and so on. Daintain was a bioactive macrophage factor discovered by Chen ZW in 1994 and was first isolated from porcine intestine. It is a 146-aa residue polypeptide and the amino acid sequence is highly identical to that of rat and human allograft inflammatory factor-1(AIF-1), so we named it Daintain/AIF-1. In 1997 Chen ZW reported that Daintain/AIF-1 exerted a dual influence on insulin secretion in vivo at different peptide concentrations and a particularly dense accumulation of Daintain/AIF-1-immunoreactive macrophages was observed in the insulitis affecting the pancreatic islets of prediabetic BB rats, suggested that Daintain/AIF1 may have a role in connection with the pathogenesis of insulin-dependent diabetes mellitus. Daintain/AIF-1 has been detected in the red pulp of spleen, dendritic cells of the gut, brain microglia and testes, and its expression has been associated with many immune system diseases such as diabetes, pancreatitis and cranial neuritis.In view of the association of the Daintain/AIF-1 with autoimmune reactions, we intend to use the yeast two-hybrid technique to define the proteins that interact with Daintain/AIF-1 in human pancreas. To construct the bait plasmid,the full length Daintain/AIF-1 gene from the vector pcDNA3.1(-)-AIF-1 was subcloned into the bait vector pGBKT7 of yeast two-hybrid system GAL4. After verified by DNA sequencing, the recombinant plasmids pGBKT7-AIF-1 were subsequently transferred into yeast AH109,their expression product showed to be nontoxic to AH109 cells,and did not activate the reporter genes LacZ and HIS3 of the GAL4 system. These results suggest that the bait plasmid pGBKT7-AIF-1 is qualified for the following cDNA library screening. So we draw a conclusion that the yeast two-hybrid GAL4 system can be utilized to study Daintain/AIF-1-interacting proteins.