3种海水鱼piscidin基因特性及抗刺激隐核虫构效关系研究

Cryptocaryosis caused by the serious pathogen Cryptocaryon irritans was defined as the second class animal epidemic disease in 2009 in China, it has brought huge loss to the fishery industry since 1990s. Inspired by the well-known resistance of rabbitfish Siganus fuscescens to C. irritans and our newly discovered anti-parasitic peptide to this pathogen derived from large yellow croaker Pseudosciaena crocea, we have focused on the natural C. irritans-resistance piscidin from marine fish and intend to expatiate its relationship between the properties of this antiparasitic piscidin and their native resistance characters. To explore the mechanism of genetic determination on this natural C. irritans-resistance, we try to clarify the contribution of anti-parasitic piscidin of marine fish by analyses of their biological activities, physiological concentrations, as well as the immune response levels of piscidins among three species of marine fish (Siganus fuscescens, Sctaenops ocellata and P. crocea) and among individuals of large yellow croaker showing different resistance levels to C. irritans. This project will lay the theoretical basis to carry out C. irritans-resistant molecular breeding for improving the natural resistance level of marine fish to C. irritans. On the basis mentioned above, we will study the structure-properties relationship of piscidins obtained, which will serve as a basis for subsequent researches in molecular design and genetic engineering expression of a more efficient antiparasitic piscidin. This project will provide theoretical and technical support to develop efficient anti-parasitic chemicals from marine fish and solve the increasingly serious problem by cryptocaryosis.

刺激隐核虫病是严重威胁我国海水鱼养殖业健康发展的二类动物疫病，目前尚无有效的防治方法。本课题拟在课题组首次发现大黄鱼piscidin能裂解杀灭刺激隐核虫幼虫和滋养体的基础上，进一步研究眼斑拟石首鱼、褐蓝子鱼和大黄鱼等3种海水鱼piscidin基因结构和组织表达差异，揭示piscidin基因结构的多态性与抗刺激隐核虫活性之间的构效关系，筛选抗虫活性较高的piscidin模板，通过氨基酸残基替换等分子生物学技术对模板piscidin与抗虫活性相关的关键结构进行分子改造，构建具有抗虫活性高、结构稳定、使用安全的改造肽，建立改造肽的融合表达、蛋白纯化等体外重组技术，筛选出重组蛋白的最佳诱导表达条件。研究结果将为研发抗刺激隐核虫新型药物提供技术支撑，为解决日趋严重的海水养殖鱼类刺激隐核虫病防治难题提供理论依据。

刺激隐核虫病是严重威胁我国海水鱼养殖业健康发展的二类动物疫病！在发现大黄鱼piscidin能裂解杀灭刺激隐核虫幼虫和滋养体的基础上，项目提出深入研究大黄鱼、眼斑拟石首鱼和褐蓝子鱼piscidin基因结构和组织表达差异，分析其结构与抗虫活性的构效关系，构建其改造肽并探讨其活性、稳定性和安全性，为刺激隐核虫病的防治提供新途径。项目获得的主要研究内容与结果如下：.1.三种实验鱼piscidin的基因结构序列的分析：①获得大黄鱼和眼斑拟石首鱼的鳃、头肾等Piscidin-like 500~750bp的扩增产物，确定为piscidin基因蛋白编码区的基因序列，分别命名为Pc-pis和So-pis，上传NCBI数据库Genbank，登录号为KC357672 和JQ710935；②明确了Pc-pis和So-pis的信号肽比成熟肽结构优势域更为保守；③获得褐篮子鱼piscidin基因Sf-pis序列，其信号肽AA残基相比大黄鱼少而成熟肽AA残基较多，其序列结构与眼斑拟石首鱼比较相似。.2. 三种海水鱼类piscidin在关键组织结构特征的比较分析：①Pc-pis和So-pis在所有检测的鳃等10个组织都有表达，但在鳃中的表达量为其它组织的1100多倍；②在系统进化上Pc-pis和So-pis较为保守；③褐篮子鱼Sf-pis基因序列与Pc-pis、So-pis的同一性为96%，④三者皆为新型piscidin-like多肽，既有抗菌亦有抗虫的功能。.3. 大黄鱼Pc-pis-His改造肽活性分析: 构建了类肽Pc-pis-His，其比Pc-pis具有更强杀灭刺激隐核虫的功效，且具有较好的温盐和pH稳定性，并对养殖大黄鱼和眼斑拟石首鱼等具有很好的安全性。.4. 抗菌肽串联基因的构建与重组表达：以酶定向连接法合成了大黄鱼和眼斑拟石首鱼Pc-So-piscidin抗菌肽串联基因，添加SUMO标签蛋白，构建SUMO-PSP-PHT43表达载体，转化到Bacillus subtilis WB800N，成功对串联抗菌肽PSP进行诱导表达；抗菌实验显示，重组表达串联抗菌肽PSP具有更好的杀菌作用，可为水产疾病防治提供新型抗菌药物或饲料添加剂，结果已申报发明专利，公开号：CN106367429 A。.发表SCI源收录论文6篇；申请发明专利2项、授权实用新型专利1项；培养毕业博士生2名、硕士生一名。