Molecular cloning and responsive expression of macrophage expressed gene from small abalone Haliotis diversicolor supertexta

Abstract

The complete cDNA sequence of macrophage expressed gene (sa Mpeg1), a perforin-like molecule, was isolated from small abalone ( Haliotis diversicolor supertexta) by homology cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA of sa Mpeg1 was 2781 bp, consisting of a 5′-terminal untranslated region (UTR) of 252 bp, a 3′-terminal UTR of 342 bp with a signal sequence TAA and a poly (A) tail, and an open reading frame of 2184 bp. The deduced protein (saMpeg1) was composed of 728 amino acids, and contains the cytolytic “helix-turn-helix” domain of perforin (residues 171–218), of which the alpha-helices are amphipathic as are those of perforin. A putative single transmembrane domain is located at residues 667–689, and a modified furin cleavage site (KRRRK; residues 689–693) immediately follows. The result of real time quantitative PCR showed that sa Mpeg1 was highly expressed at 8 h and 96 h post-injection of the Gram-negative bacterium Vibrio parahaemolyticus, but there was no change after TBT exposure. The structural similarity to mammalian perforin and the different gene expression level to bacterial infection and TBT exposure suggest that sa Mpeg1 may play a role in the immune response against microorganisms in small abalone.