Identification and expression analysis on bactericidal permeability-increasing protein/lipopolysaccharide-binding protein of blunt snout bream, Megalobrama amblycephala

摘要

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) belong to the lipid transfer protein/lipopolysaccharide-binding protein family and play a critical role in the innate immune response to Gram-negative bacteria. In the present study, a novel BPI/LBP from blunt snout bream, Megalobrama amblycephala (maBPI/LBP) was isolated by RACE techniques. The open reading frame (ORF) of maBPI/LBP gene encoded a polypeptide of 474 amino acids with a putative 18-aa hydrophobic signal peptide. Structurally, the maBPI/LBP showed highly similar to those of BPI/LBPs from invertebrate and teleost, LBPs and BPIs from mammal, which contained an N-terminal BPI/LBP/CETP domain BPI1 with a LPS-binding domain, a C-terminal BPI/LBP/CETP domain BPI2, and proline-rich domain. The homologous identities of deduced amino acid sequences displayed that the maBPI/LBP possessed significant similarity (96.61% and 90.07%) with those of grass carp and common carp, respectively. The recombinant protein of maBPI/LBP showed effectively kill Gram-negative bacteria. The maBPI/LBP gene was expressed in a wide range of normal tested tissues, with the highest expression levels in the kidney. The experiments revealed that the mRNA expression of maBPI/LBP in spleen considerably up-regulated from 2 h to 8 h post LPS stimulation, and peaked rapidly at 2 h (7.40-fold, P 0.05), which confirmed that maBPI/LBP was the absolute sensitive to LPS stimulation. Furthermore, the level of maBPI/LBP mRNA expression reached the maximum for a second time at 24 h after LPS stimulation. These results suggested that maBPI/LBP was a constitutive and inducible acute-phase protein contributing to the host immune defense against pathogenic bacterial infection in M. amblycephala. This study will further our understanding of the function of BPI/LBP and the molecular mechanism of innate immunity in teleost. (C) 2015 Elsevier Ltd. All rights reserved.