Study on the expression of Nk-lysin from Nile tilapia (Oreochromis niloticus) in Pichia pastoris and its biological function

Abstract

Nk-lysin, the compound of the natural killer cells (NK) and cytotoxic T lymphocytes (CTL), is an effective antimicrobial peptide that not only presents pathogen-killing activity but also plays an important role in immunomodulatory effect. To obtain large quantity of Nk-lysin from Nile tilapia (Oreochromis niloticus) for biological assays, a 366 bp cDNA sequence encoding the mature peptide of Nk-lysin was cloned into a secretion plasmid of pPIC9K, and a large-scale of the recombinant Nk-lysin was effectively produced via Pichia pastoris expression system. Antibacterial assay shown that the recombinant Nk-lysin obviously inhibited the growth of several pathogenic bacteria, including Vibrio cholerae (MIC = 31.25 μg/mL), Pseudomonas aeruginosa (MIC = 31.25 μg/mL), and Aeromonas hydrophila (MIC = 7.81 μg/mL) which is the main pathogen of aquatic fish, including Nile tilapia. Further studies revealed that recombinant Nk-lysin significantly enhanced the phagocytic activity of monocytes/macrophages (MO/MΦ) on microspheres (0.5 and 1.0 μm beads) and bioparticles (Streptococcus agalactiae and Aeromonas hydrophila), and reactive oxygen species (ROS), these activities might be mediated by the P2X7 receptor (P2X7R). Moreover, the increased expressions of pro-inflammatory factor in il-12 and ifn-γ genes and the decreased expressions of anti-inflammatory factor in il-10 and tgf-β genes were observed in MO/MΦ after recombinant Nk-lysin treatment significantly. Meanwhile, the expression of tlr9, myd88, jak1, and nf-κb genes were up-regulated significantly in Nk-lysin-treated MO/MΦ. Taken together, these results indicate that recombinant Nk-lysin of Nile tilapia processes the capacities on antibacterial and immunomodulation, and might be a promising antibiotic substitute. Furthermore, applying P. pastoris expression system to product bioactive Nk-lysin of Nile tilapia could be sufficient to meet the requirements for both research and aquacultural application.