Molecular cloning, expression, and in silico analysis of a long type peptidoglycan recognition protein from half-smooth tongue sole, Cynoglossus semilaevis (Actinopterygii: Pleuronectiformes: Cynoglossidae)

Abstract

Background. Half-smooth tongue sole, Cynoglossus semilaevis Günther, 1873, a marine teleost, is an important aquaculture species of great economic value. In recent years, its farm production increase coincided, however, with the number of reported cases of bacterial diseases. Further understanding of its immune response to bacterial pathogens can provide more information on pathogenesis and how to prevent disease using immune-related strategy. Peptidoglycan (PGN) recognition proteins (PGRPs) play important roles in the innate immunity against bacterial infection. In the presently reported study, a long type PGRP in half-smooth tongue sole (csPGRP-L) was cloned, and its sequence features, PGN binding ability, and mRNA expressions in different tissues after bacterial infection were also analyzed. Materials and methods. The full length of csPGRP-L cDNA was obtained by RT-PCR and RACE-PCR method, and its sequence features were analyzed by multiple sequence alignment and phylogenetic tree. Meanwhile, its 3-D structure and PGN binding ability were analyzed by comparative modelling and molecular docking methods. Furthermore, the expressions of csPGRP-L in different tissues of healthy fish and fish infected with Streptococcus dysgalactiae were examined using quantitative real-time PCR method. Results. The full length of csPGRP-L cDNA was 1509 bp (GenBank accession No. HQ909441), with a 1446 bp of open reading frame (ORF) encoding 481 amino acids (aa), which possessed several conserved PGRP family features, e.g., a typical PGRP domain at its C-terminal, 3-D structure. Molecular docking showed that the csPGRP-L also possessed the PGN-binding ability. csPGRP-L was constitutive expressed in all the selected tissues from healthy fish and following S. dysgalactiae infection its expression was up-regulated in a tissue-specific expression pattern. Conclusion. The gene we cloned was exactly the homologue of vertebrates’ long type PGRP in half-smooth tongue sole which was confirmed by several analyses and the up-regulation of csPGRP-L after bacterial infection suggest that csPGRP-L plays important role in antibacterial and anti-infective action.