Functional characterization of a short peptidoglycan recognition protein, PGRP5 in grass carp Ctenopharyngodon idella

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella

“…Grass carp, Ctenopharyngodon idella, one of the most popular and economically important species in aquaculture industry of China, has received increasing attention in relation to its immune system (Chen et al, 2012(Chen et al, , 2010aXu et al, 2010;Xiao et al, 2010), and a short PGRP with amidase activity (gcPGRP5) has recently been characterized in this fish (Li et al, 2013). In the present study, a long PGRP, designated as gcPGRP6 was cloned from grass carp.…”

“…The expression of recombinant gcPGRP6 was induced in culture with IPTG, and the protein was purified by nickel resin chromatography for immunizing rabbit under denaturing conditions. The anti-serum against gcPGRP6 was produced as previously described (Li et al, 2013). The specificity of anti-gcPGRP6 antibody was evaluated by Western blotting using overexpressed gcPGRP6-GFP fusion protein.…”

“…36 h later, cells were stained for 5 min with Hoechst 33342 at a concentration of 4 lg/ml, before being observed under a fluorescent microscope (Zeiss) with an ultraviolet (UV) excitation filter. For endogenous localization of gcPGRP6, 2 Â 10 5 CIK cells were seeded per well in a six-well plate for one night, and immunofluorescence staining was proceeded using rabbit anti-gcPGRP6 antibody (1:500, v/v) according to the methods previously reported by Li et al (2013).…”

Expression and functional characterization of PGRP6 splice variants in grass carp Ctenopharyngodon idella

“…Cho et al (2005) reported that the antimicrobial effect of human PGRP-S against E. coli is synergistic with lysozyme, whereas human PGLYRP-1, PGLYRP-3, and PGLYRP-4 kill both Gram-positive and Gram-negative bacteria together with antibacterial peptides PLA2, α-and β-defensins . In our previous studies, it was shown that secretory gcPGRP6 and intracellular gcPGRP5 failed to induce the expression of antibacterial peptides Hepcidin and G-lysozyme (Li et al, 2013. However, the increase in the expression of Hepcidin and G-lysozyme was observed following the overexpression of gcPGRP6a, gcPGRP6b and gcPGRP6c variants, but not gcPGRP6d, an isoform encoding basically only the PGRP domain.…”

“…gcPGRP6 synthetic peptide (CGTKGFWDSIKSPKV) is specific for gcPGRP6 normal form, and gcPGRP6all synthetic peptide (CHRQATSTDCPGNAF) for all gcPGRP6 variants. For endogenous localization of gcPGRP variants, 2 × 10 5 CIK cells were seeded per well in a six-well plate overnight, and immunofluorescence staining was proceeded using rabbit antigcPGRP6 and anti-gcPGRP6all antibodies according to the methods previously reported by Li et al (2013).…”