CN113559118B - 亚铁离子在防治鲤疱疹病毒Ⅱ型(CyHV2)感染中的应用 - Google Patents
亚铁离子在防治鲤疱疹病毒Ⅱ型(CyHV2)感染中的应用 Download PDFInfo
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Abstract
本发明公开了亚铁离子在防治鲤疱疹病毒Ⅱ型（cyprinid herpesvirus 2，CyHV2）感染中的应用，实验动物感染模型的建立显示，氯化亚铁能够对鲫鱼CyHV2感染起到显著的保护作用，结合qPCR实验结果发现氯化亚铁处理后病毒相关基因转录水平被显著抑制。因此，含有亚铁离子的化合物能够作为防治CyHV2的药物，并具有使用剂量无毒安全，保护效果佳的优点。
Description
技术领域
本发明属于水产养殖技术领域，具体涉及亚铁离子的医药用途，含有亚铁离子的化合物可用于预防和治疗鲤疱疹病毒Ⅱ型(CyHV2)感染引起的水产动物疾病。
背景技术
铁是生物体内重要的六大微量元素之一，是动植物正常生长和生活的必需元素。在生物体内，铁承担着多种生物学功能。一般地，铁在机体内以Fe2+/3+血红素形式存储，其中+2价亚铁离子的功能表现在许多方面。
亚铁离子作为血红蛋白的重要组成成分，负责携带氧及输送氧，实现红细胞的输氧功能。用于肌肉贮氧的肌红蛋白中均含有一个亚铁血红素，当肌肉运动时，它可以提供或补充血液输氧的不足；另外，亚铁离子能够与细胞内活性酶的代谢产物发生相互作用。细胞色素酶类在体内氧化还原过程中不可或缺，它是完成电子传递的关键角色，并在三羧酸循环过程中催化脱下的氢原子与由血红蛋白运输的氧合成水，以保证代谢。在这一氧化过程中所产生的过氧化氢等有害物质，能够被含铁的触媒所破坏而解毒。造血功能方面，铁影响脱氧核糖核苷酸合成及造血维生素的代谢。机体应对病原体入侵时，细胞内亚铁离子水平的升高能够通过催化氧自由基的形成，激活免疫因子NF-κB启动信号转导，促进局部炎症的产生，唤醒免疫反应，并防止感染的扩散。铁与酶之间的关键作用和其参与的造血功能决定着缺乏铁元素，会引起机体感染程度增加，白细胞功能异常，免疫屏障受损。亚铁离子在机体免疫过程中至关重要，但目前尚未有将外源亚铁离子应用于防治水生动物病毒病的实例。
鲤疱疹病毒Ⅱ型(cyprinid herpesvirus 2,CyHV2)是造成金鱼、鲫鱼等鲤科鱼类病毒性鳃出血症的病原，一旦暴发会引起养殖鱼类大规模死亡。其中尤以鲫作为中国重要的大宗淡水经济鱼类，疱疹病毒的流行每每带来巨大的经济损失。疱疹病毒病因其发病速度快，致死率高的特点，目前已被世界动物卫生组织(OIE)收入须重点检疫申报的疾病名录及我国“国家水生动物疫病监测”名录。目前，针对水生动物病毒病的防治手段十分有限，尽管针对鱼类疱疹病毒病原分离、组织病理观察、转录组分析等方面已开展一些研究，但研究人员尚未找到良好的防治方式。深入了解病原CyHV2，提出行之有效的防治手段是控制病毒暴发，减少经济损失是水产养殖业亟需解决的问题。
发明内容
本发明的目的在于提供含有亚铁离子的化合物在制备防治鲤疱疹病毒Ⅱ型(CyHV2)感染药物中的应用，FeCl2能够显著抑制鱼体免疫器官中病毒相关基因(包括ORF47，ORF48，ORF80)的表达，实验动物存活率模型证实FeCl2对CyHV2感染起到保护作用，含有亚铁离子的化合物能够用于制备防治CyHV2感染的药物。
为了实现上述目的，本发明采用以下技术方案：
含有亚铁离子的化合物在防治鲤疱疹病毒Ⅱ型(CyHV2)感染中的应用：在本发明的具体实施例中，感染CyHV2的鲫鱼在注射FeCl2后，因病毒感染造成的死亡率被显著降低，0.1mg/g FeCl2处理时死亡率为0，进一步检测鱼体中病毒相关基因扩增情况显示，FeCl2处理之后，鱼体免疫器官脾脏、头肾、体肾中病毒相关基因(包括ORF47，ORF48，ORF80)的表达量被显著抑制。同时，上述免疫器官中抗病毒免疫相关基因(包括caRIG-I A/B,caViperinA/B)表达量基本恢复正常水平。
本发明实施例中选取的鲫鱼是CyHV2的重要宿主，因此通过建立鲫鱼感染CyHV2的疾病模型，以及对病毒相关基因表达的检测，可知含有亚铁离子的化合物，如氯化亚铁(FeCl2)、硫酸亚铁(FeSO4)、磷酸亚铁(Fe3H16O16P2)、葡萄糖酸亚铁(C12H22O14Fe·2H2O)等适用于CyHV2感染的防治。含有亚铁离子的化合物作为预防或治疗CyHV2的药物，可以制成注射剂或口服剂，使用剂量低，成分单一，无毒副作用，没有药物残留的风险。
附图说明
图1为注射不同微量元素氯化物对感染CyHV2鲫鱼存活率的影响。
图2为注射FeCl2对感染CyHV2鲫鱼体内病毒基因表达水平的影响。
图3为注射FeCl2对感染CyHV2鲫鱼体内抗病毒免疫相关因子表达水平的影响。
具体实施方式
实施例1：FeCl2对鲫鱼感染CyHV2后致死率的影响
在35cm×23.5cm×36cm的水族箱中进行，每天换水并保持水温在22℃左右。5g左右的健康鲫鱼通过腹腔注射CyHV2病毒悬液建立人工感染模型，对照组腹腔注射病毒悬液250μl/尾与PBS 50μl/尾混合液，实验组使用病毒悬液250μl/尾按照总体积为300μl/尾分别稀释FeCl2溶液(0.12675g/ml)、氯化锰溶液(MnCl2，0.19791g/ml)、氯化铜溶液(CuCl2，0.17048g/ml)、氯化镁溶液(MgCl2，0.09521g/ml)、氯化钙溶液(CaCl2，0.111g/ml)至终浓度0.1mg/g(使用PBS补齐体积)，同时注射感染。感染后每天记录鲫鱼死亡情况，一直统计到15dpi(days post infection)，最终用Mantel-Cox分析鱼体存活率(图1)。该结果表明，对照组鲫鱼感染CyHV2后在第7天存活率为0，注射其余微量元素氯化物实验组鲫鱼全部死亡时间在1dpi至10dpi不等。而注射0.1mg/g FeCl2的鲫鱼未见死亡，存活率提高到100％，可见FeCl2对鲫鱼感染CyHV2有明显的保护效果。
实施例2：FeCl2对感染CyHV2鲫鱼体内病毒基因表达水平的影响
检测感染CyHV2之后的鱼体用0.1mg/g FeCl2处理后，鱼体中病毒相关基因的表达情况。按照实施例1所述方法分别处理对照组和FeCl2-CyHV2混合液组，每组鲫鱼分别腹腔注射300μl混合液，感染6天后用MS-222麻醉(0.1mg/ml)，解剖取脾脏(Spleen)、头肾(Headkidney)与体肾(Kidney)样品，使用TRIzol(Invitrogen)提取组织总RNA，由GoScript逆转录试剂盒(Promega)逆转录得到cDNA。通过qPCR检测病毒相关基因的表达情况(图2)(每组设置5个生物学重复)，结果表明：0.1mg/g FeCl2处理之后，鱼体免疫器官中病毒相关基因(包括ORF47，ORF48，ORF80)的表达显著下降。
实施例3：FeCl2对感染CyHV2鲫鱼抗病毒免疫相关基因表达的影响
进一步检测感染CyHV2之后的鱼体用0.1mg/g FeCl2处理后，抗病毒免疫基因的表达情况。5g左右的健康鲫鱼被分为PBS组，PBS-FeCl2混合液组，FeCl2-CyHV2混合液组和CyHV2病毒悬液组。其中，PBS组注射PBS 300μl/尾；PBS-FeCl2混合液组使用PBS稀释FeCl2溶液至0.1mg/g，总体积为300μl/尾；FeCl2-CyHV2混合液组使用CyHV2病毒悬液稀释FeCl2溶液至0.1mg/g；CyHV2病毒悬液组将CyHV2病毒悬液与FeCl2溶液等量体积的PBS混合；如上所述同时注射感染，感染6天后麻醉解剖取脾脏、头肾及体肾组织提取总RNA逆转录后进行qPCR检测(图3)。结果显示：CyHV2病毒悬液组鲫鱼脾脏、头肾、体肾中抗病毒免疫基因表达显著上调，加入FeCl2处理后相关基因表达基本恢复正常水平。
Claims (1)
1.FeCl2作为唯一活性成分在制备防治鲤疱疹病毒Ⅱ型感染的药物中的应用。
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