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

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 P2X 7 receptor (P2X 7 R). 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. • Recombinant Nk-lysin from Nile tilapia was effectively produced via Pichia pastoris expression system. • The recombinant inhibited obviously the growth of several pathogenic bacteria, especially Aeromonas hydrophila. • The recombinant promoted significantly the phagocytosis of monocytes and/or macrophages of fish. [ABSTRACT FROM AUTHOR]