大黄鱼抗虫肽Pc-pis杀灭刺激隐核虫过程中细胞骨架损伤机理的研究

Cryptocaryonosis, as a Class II animal epidemic, has brought signi?cant economic losses to the marine fish culture industry in China. Fortunately, an antiparasitic peptide (Pc-pis) against Cryptocaryon irritans was first identified recently in our work. With first attention paid to scientific issues about cytoskeletal injury mechanism in our project, which arised from our previous observations on "membrane perforation" and " abnormality of ciliary movement" of C. irritans when exposed to Pc-pis, (1) the dynamic structure and molecular characteristics of cytoskeleton will be elucidated under cellular, subcellular and molecular level by using immunofluorescence technique and transcriptomic approach, which will help to provide a basis for the discovery of drug targets and specific antigen in C. irritans; (2) synergistic effects of Pc-pis and cytoskeleton-targeting drugs will be studied for the purpose of an effectively parasiticidal measure to solve the highly devastating cryptocaryoniosis by using antiparasitic polypeptide; (3) differentially expressed cytoskeletal proteins and genes will be further screened and identified with omics technology and subsequent computing.The results will help to elucidate the cytoskeletal injury mechanism of C. irritans at structural, physiological and molecular level and will offer theoretical support to the subsequent drug research and development of antiparasitic peptide.

刺激隐核虫病（cryptocaryoniosis）是严重影响我国海水鱼类养殖的二类动物疫病，本项目组从我国海水鱼类抗菌肽基因资源库中首次发现了1种能够杀灭刺激隐核虫的抗虫肽Pc-pis。针对Pc-pis杀虫过程中"膜穿孔"和"纤毛运动异常"所带来的细胞骨架损伤机理等科学问题，本申请拟（1）利用免疫荧光和转录组分析技术从细胞、亚细胞乃至分子水平上解析细胞骨架的动态结构及分子特征，为寻找抗虫药物作用靶标或特异性抗原奠定重要基础；（2）研究Pc-pis与破坏细胞骨架的特异性药物协同杀虫的效果，为解决刺激隐核虫病等重大疾病难题寻找1种高效的抗虫肽杀虫方式；（3）研究Pc-pis杀虫过程中细胞骨架动态结构的改变和膜内钙离子等生理指标的变化，进而利用组学技术筛选并鉴定差异表达的细胞骨架蛋白和基因，阐明Pc-pis损伤刺激隐核虫细胞骨架的的形态、生理及分子机理，为抗虫肽药物后续研发提供重要的理论支撑。

刺激隐核虫病是严重影响我国海水鱼类养殖的二类动物疫病，针对 Pc-pis 杀虫过程中“膜穿孔”和“纤毛运动异常”所带来的细胞骨架损伤机理等科学问题，项目组借助细胞学、组织学、分子生物学等多种手段，首先开展了刺激隐核虫细胞骨架的形态特征的分析，构建了刺激隐核虫的基因资源库，挖掘了细胞骨架相关基因；其次，分析了抗虫肽piscidin杀虫过程中的膜损伤和细胞骨架响应机制，开展了显微、超微的形态观察，探讨了基因表达谱及差异表达基因的分子响应。最后，开展了包括抗虫肽piscidin在内的多种杀虫剂对刺激隐核虫的毒理作用，明确了每种杀虫药物的细胞骨架损伤类型。.结果表明，（1）刺激隐核虫的幼虫外膜由3层膜组成，分别为外限制膜、外泡膜及内泡膜，由纤毛器的微管骨架和非纤毛器微官骨架支撑形成外膜结构，Lc-pis处理后，外膜的3层结构损毁，内泡膜裂解，细胞骨架受损。滋养体体表覆盖有单动基纤毛，胞咽周围双动基纤毛结构，微管样结构也在细胞核和线粒体中发现。（2）Pc-pis通过静电相互作用锚定在质膜上产生穿孔效应，环形孔的大小约2.4nm，继而进入胞质附着在细胞核膜，其所携带的正电荷对于Pc-pis的功能至关重要。（3）常用杀虫药物冰醋酸和硫酸铜均可造成了膜穿孔使细胞骨架造到严重破坏而福尔马林却不会形成穿孔，但对纤毛器的微管骨架造成严重损坏。（4）比较转录组分析表明，Lc-pis处理前后幼虫转录组中的上调基因远大于下调基因数，表明抗菌肽对幼虫体内基因表达的明显激活作用，涉及到大量微管微丝蛋白合成以及调节肌动与肌球蛋白等的运动等等，导致“纤毛运动异常”；而滋养体中细胞骨架相关基因，包括细胞色素氧化酶亚基、α微管亚基、β微管蛋白、ATP合成酶亚基等却显著下调，推测Lc-pis导致滋养体 “膜穿孔”抑制了滋养体的代谢活动与骨架蛋白合成等。研究结果不仅探索了抗虫肽piscidin杀灭刺激隐核虫的膜损伤及细胞骨架损伤机制，还为研发锚定细胞骨架的杀虫药物提供了重要思路。