Lv Baolei, Li Ronggui, Li Guantian, Yu Huahua, Liu Song, Li Rongfeng
College of Life Sciences, Qingdao University, Qingdao, 266071, China.
CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China.
Toxicon. 2025 Nov;266:108537. doi: 10.1016/j.toxicon.2025.108537. Epub 2025 Aug 8.
Nemopilema nomurai, a large venomous jellyfish, caused numerous stinging incidents and even many fatal cases. The venom of N. nomurai contains various toxins, with hemolysin being one of the major components that play a crucial role in stinging. However, until now, the hemolysin has not been successfully isolated from N. nomurai, and its mechanism remains unclear. In this study, we established an improved method for preparing jellyfish toxins to facilitate subsequent purification. The SDS-PAGE profiles indicated that the crude toxin extracted using this improved method contained significantly fewer components than the traditional method. Moreover, the proteomic and toxicity analysis revealed that the refined extract still contained most of the key toxins including hemolysin, phospholipase, and other toxins. The hemolysin was then isolated from the refined jellyfish crude toxins using a two-step purification of gel filtration chromatography followed by anion-exchange chromatography. The molecular mass of this hemolysin was 45 kDa (NnTX-45) with an HC of approximately 30 μg/mL. Transmission electron microscopy observations revealed that NnTX-45 formed numerous pores, each with an inner diameter of 5.65 nm and an outer diameter of 13 nm approximately, on the erythrocyte membranes. Overall, our study successfully isolated and elucidated the preliminary hemolytic mechanism of the NnTX-45 from N. nomurai, which provides a highly purified toxin antigens for the development of jellyfish antivenom to treat this jellyfish sting in the future.
野村水母是一种大型有毒水母，引发了众多蜇人事件，甚至导致许多致命案例。野村水母的毒液含有多种毒素，其中溶血素是蜇人过程中起关键作用的主要成分之一。然而，迄今为止，溶血素尚未成功从野村水母中分离出来，其作用机制仍不清楚。在本研究中，我们建立了一种改进的水母毒素制备方法，以利于后续的纯化。SDS-PAGE图谱表明，用这种改进方法提取的粗毒素所含成分明显少于传统方法。此外，蛋白质组学和毒性分析显示，精制提取物仍含有包括溶血素、磷脂酶和其他毒素在内的大部分关键毒素。然后，通过凝胶过滤色谱法和阴离子交换色谱法两步纯化，从精制的水母粗毒素中分离出溶血素。这种溶血素的分子量为45 kDa（NnTX-45），半数溶血浓度约为30 μg/mL。透射电子显微镜观察显示，NnTX-45在红细胞膜上形成了许多孔，每个孔的内径约为5.65 nm，外径约为13 nm。总体而言，我们的研究成功分离并阐明了野村水母NnTX-45的初步溶血机制，这为未来开发治疗这种水母蜇伤的抗蛇毒血清提供了一种高度纯化的毒素抗原。
