Characterization and Functional Evaluation of <i>NK-lysin</i> from Clownfish (<i>Amphiprion ocellaris</i>)
Dapeng Yu,
Haohang Zhao,
Yiming Wen,
Tao Li,
Hongli Xia,
Zhiwen Wang,
Zhen Gan,
Liqun Xia,
Jianlin Chen,
Yishan Lu
Affiliations
Dapeng Yu
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Haohang Zhao
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Yiming Wen
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Tao Li
Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shenzhen 518000, China
Hongli Xia
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Zhiwen Wang
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Zhen Gan
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Liqun Xia
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Jianlin Chen
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
Yishan Lu
Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of NK-lysin was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of NK-lysin from the spleen of the healthy clownfish (Amphiprion ocellaris; AoNK-lysin) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the AoNK-lysin gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The AoNK-lysin gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of AoNK-lysin was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of A. ocellaris after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the Pichia pastoris expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly Streptococcus agalactiae, Streptococcus iniae, Salmonella typhi, Shigella sonnei, Pseudomonas aeruginosa, and Aeromonas caviae. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication.
