South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, 510300, Guangzhou, Guangdong Province, China; Guangdong Provincial Engineer Technology Research Center of Marine Biological Seed Industry, Guangzhou, Guangdong Province, China.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, 510300, Guangzhou, Guangdong Province, China.
Dev Comp Immunol. 2020 Jun;107:103658. doi: 10.1016/j.dci.2020.103658. Epub 2020 Feb 19.
NK-lysin is an important part of the innate immune defence system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, NK-lysin from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length NK-lysin cDNA was 731 bp, which comprised a 5'-UTR of 63 bp, an ORF of 444 bp, and a 3'-UTR of 224 bp, and encoded 147 amino acids; NK-lysin consisted of a conserved saposin B domain and six conserved cysteines that formed three pairs of disulfide bonds. The genomic organization of NK-lysin was also determined and the gene consisted of four introns and five exons. The predicted promoter region of ToNK-lysin contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that ToNK-lysin was ubiquitously expressed in all examined tissues; the highest mRNA levels were observed in the skin, kidney and intestine, while the lowest expression level was detected in the stomach. After P. damselae stimulation, the expression level of NK-lysin mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant NK-lysin expressed in pGEX-6P-1 was approximately 37 kDa. The purified recombinant protein showed antibacterial activity against gram-positive and gram-negative bacteria. The results indicate that golden pompano NK-lysin has potential antimicrobial roles in fish innate immunity.
NK 溶素是先天免疫防御系统的重要组成部分，在抵抗病原微生物入侵方面发挥着重要作用。本研究对卵形鲳鲹（Trachinotus ovatus）NK 溶素进行了鉴定，并研究了其对鳗弧菌（Photobacterium damselae）的表达情况。NK 溶素全长 cDNA 为 731bp，包含 63bp 的 5'-UTR、444bp 的 ORF 和 224bp 的 3'-UTR，编码 147 个氨基酸；NK 溶素包含一个保守的 Saposin B 结构域和六个保守的半胱氨酸，形成三对二硫键。还确定了 NK 溶素的基因组结构，该基因由四个内含子和五个外显子组成。预测的 ToNK-lysin 启动子区域包含几个潜在的转录因子结合位点。定量实时（qRT-PCR）分析表明，ToNK-lysin 在所有检测组织中均广泛表达；在皮肤、肾脏和肠道中观察到最高的 mRNA 水平，而在胃中检测到最低的表达水平。在鳗弧菌刺激后，卵形鲳鲹各组织中 NK-溶素 mRNA 的表达水平显著上调。此外，SDS-PAGE 显示在 pGEX-6P-1 中表达的重组 NK-溶素的分子质量约为 37kDa。纯化的重组蛋白对革兰氏阳性和革兰氏阴性细菌均具有抗菌活性。结果表明，卵形鲳鲹 NK 溶素在鱼类先天免疫中具有潜在的抗菌作用。
