Development of a new antibody for detecting natural killer enhancing factor (NKEF)‐like protein in infected salmonids

Abstract

The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.

Viral hemorrhagic septicaemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line

“…It has been reported before for a number of fish species, including rainbow trout, that the in vivo infection with VHSV up-regulated NKEF transcripts [27e29], and now this has been confirmed in vitro with RTS11 cells, however, the connection between cytotoxic activity and NKEF production in RTS11 remains uncertain because the effect that VHSV produce on NKEF protein expression was the opposite. This capacity to reduce the level of NKEF protein seems to be a feature of viruses, as this also has been observed with ISAV and IHNV infections [29,30], whereas LPS or parasites such as Caligus rogercresseyii increased the level of NKEF protein [30]. The mechanism through which viruses are capable of reducing protein synthesis of such molecules despite the induced up-regulation of transcripts remains unsolved and seems worthy of investigation.…”

“…NKEF protein expression by RTS11 was evaluated by both western blotting and flow cytometry through the use of monospecific polyclonal anti-epitope of NKEF antibody from O. mykiss [30]. For western blotting, cell lysates were prepared from control and VHSV-infected RTS11 cultures at 1, 4, 8, 11 and 14 days postinfection.…”

“…NKEF gene expression at the RNA level is increased in blood leukocytes by LPS, bacteria or vaccination in blood leukocytes [24e28]. Surprisingly, at protein level, NKEF has been shown to be down modulated in the liver of salmon upon infection with infectious haematopoietic necrosis virus (IHNV) [29] and in RTS11 cells upon exposure to infectious salmon anaemia virus (ISAV) [30].…”

Dietary administration of the probiotic SpPdp11: Effects on the intestinal microbiota and immune-related gene expression of farmed Solea senegalensis treated with oxytetracycline

“…This gene encodes the natural killer cell enhancing factor protein, which is included in peroxiredoxin, a family of antioxidant enzymes that protect cells against oxidative damage [74], cell death and tissue repair after injury [75]. The up-regulation of this gene has been reported by LPS treatment or pathogens such as bacteria, viruses and parasites [76][77][78] and suggests that the gene plays a primary role in the innate immune response against bacterial and viral agents [79]. The IGF-β gene encodes for the IGF-β protein, which is a potent inhibitor of apoptosis [80,81] and improves the outcome in the murine sepsis model [82] and hepatic bacterial clearance, likely due to decreased Kupffer cell apoptosis [83].…”

Immunomodulatory effect of cathelicidins in response to a β-glucan in intestinal epithelial cells from rainbow trout

“…For antibody production, animals were subcutaneously injected at 1, 7 and 14 days with 150 μg immunogen diluted 1:1 in FIS as a T helper cell activator, as previously described (Bethke et al, 2012) and 1:1 in Freund's adjuvant (Thermo). The antiserum was collected on day 18, centrifuged at 700 g for 10 min and the supernatant was stored at −20°C.…”