Molecular cloning and characterization of the lipopolysaccharide and β-1, 3-glucan binding protein in Chinese mitten crab (Eriocheir sinensis)

Abstract

The lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a pattern recognition protein which is fundamental for the innate immune response of crustaceans. A partial cDNA produced by the random sequencing of cDNA clones from a haemocyte cDNA library of Eriocheir sinensis showed similarity to the LGBP gene of the Chinese shrimp (Fenneropenaeus chinensis). The full-length cDNA was subsequently cloned and sequenced by the technique of rapid amplification of cDNA ends (RACE). The E. sinensis LGBP gene (designated as Es-LGBP) was 1236 bases long and was capable of encoding a polypeptide of 362 amino acids showing significant similarity to homologous genes in shrimp. The crab LGBP deduced amino acid sequence carrying conserved features of this gene family including a potential recognition motif for beta-1, 3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp)cell adhesion sites. Real-time quantitative reverse transcription-PCR (RT-PCR) analysis showed that LGBP gene expresses in haemocytes, hepatopancreas, muscles, gills, stomachs, and intestines with the highest level in haemocytes and the lowest in the stomach. The LGBP gene expression is up-regulated upon bacterial infection and the binding of lipopolysaccharide and beta-1, 3-glucan to LGBP could induce a series of immune reactions. The temporal expression of the LGBP gene after bacterial challenge was measured by real-time quantitative RT-PCR. The result demonstrated that the LGBP gene expression in crab was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 and 24 h. Our data suggest that the crab LGBP is an inducible acute-phase protein that could be critical in crab immunity.