Prokaryotic expression, purification, and refolding of leukocyte cell-derived chemotaxin 2 and its effect on gene expression of head kidney-derived macrophages of a teleost fish, ayu (Plecoglossus altivelis)

LECT2 improves the outcomes in ayu with Vibrio anguillarum infection via monocytes/macrophages

“…Here, we found that rPaLECT2 treatment led to increased peritoneal monocytes/macrophages in both uninfected and infected ayu at 12 hpi. In our previous work we showed that mouse macrophages are the major targets of LECT2 in vivo [6], and rPaLECT2 could enhance the functions of ayu head kidney-derived monocytes/macrophages in vitro [7,25]. Therefore, we hypothesized that monocytes/macrophages may also mediate the effect of LECT2 on the survival rate of bacteria-infected ayu.…”

“…rPaLECT2 with bioactivity was produced as previously reported [25]. rPaLECT2 in 100 ml saline (0, 0.005, 0.05 or 0.5 mg/g body weight) was injected intraperitoneally (ip) to ayu immediately after infection ip with 1.2 Â 10 4 colony forming units (CFU) V. anguillarum per fish.…”

Molecular characterization of mudskipper (Boleophthalmus pectinirostris) hypoxia-inducible factor-1α (HIF-1α) and analysis of its function in monocytes/macrophages

“…Mudskipper kidney-derived MO/MФ were isolated as previously described [25]. In brief, kidney leukocyte-enriched fractions were obtained using Ficoll-Paque PREMIUM (GE healthcare, New Jersey, USA) density gradient centrifugation.…”

Molecular characterization of a transmembrane C-type lectin receptor gene from ayu (Plecoglossus altivelis) and its effect on the recognition of different bacteria by monocytes/macrophages

“…The amplicons with the expected size were digested by BamH I and EcoR I, and subsequently inserted into the pET28a(+) vector. The recombinant plasmid pET28a-PaCTLRC was constructed and transformed into Escherichia coli BL21 (DE3) for over-expression.rPaCTLRC was expressed as an inclusion body and was purified as previously reported (Zhang et al, 2011). In brief, the IPTG-induced cultures were harvested by centrifugation at 10,000 × g for 10 min at room temperature, and the cell pellet was resuspended in 20 ml sonication buffer (50 mM KH 2 PO 4 , 1 mM EDTA, and 300 mM KCl, pH 8.0) for 10 min.…”