陈静妮,代礼平,雷燕,胡海浩,黄鉴涛,简纪常,闫秀英

• 1:广东海洋大学水产学院广东省水产经济动物病原生物学及流行病学重点实验室/水产经济动物病害控制广东普通高校重点实验室
• 2:广州利洋水产科技股份有限公司

摘要(Abstract)：

【目的】制备草鱼(Ctenopharyngodon idella)非特异性细胞毒性细胞受体蛋白-1 (NCCRP-1)和白细胞介素-10 (IL-10)的抗血清,探讨重组NCCRP-1和IL-10免疫在草鱼抗病毒感染过程中肾脏和肠病理学变化及对NCCRP-1和IL-10表达的影响。【方法】应用PCR扩增技术获得草鱼NCCRP-1和IL-10开放阅读框ORF,构建原核表达载体pET-NCCRP和pET-IL10;用表达的重组蛋白分别制备兔抗NCCRP-1和IL-10的抗血清,并采用ELISA方法测定抗血清的效价;分别用重组NCCRP-1和IL-10免疫草鱼,并用草鱼呼肠孤病毒(Grass carp reovirus,GCRV)GD108攻毒,感染7 d后制备草鱼肾脏和肠的病理切片,用定量PCR和western blot检测NCCRP-1和IL-10的表达。【结果】草鱼NCCRP-1和IL-10的ORF分别编码237个和179个氨基酸;草鱼NCCRP-1和IL-10的原核表达载体pET-NCCRP和pET-IL10在大肠杆菌(Escherichiacoli)BL21中成功诱导表达,最优表达条件分别是0.1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)37℃诱导4 h和1.0 mmol/L IPTG 37℃诱导6 h,western blot分析表明重组表达的蛋白为目的蛋白;制备兔抗NCCRP-1和IL-10抗血清的效价均高达1∶40 000,免疫和攻毒后,NCCRP-1免疫组草鱼肾脏与肠的病理变化较轻,且NCCRP-1在肾脏与肠中的表达量显著上调(P <0.05);IL-10免疫组草鱼肾脏与肠的病理变化较重,IL-10在肾脏与肠中的表达量变化不显著。【结论】外源性NCCRP-1免疫后增强了草鱼抵御GCRV-GD108感染的免疫应答,NCCRP-1对草鱼抗GCRVGD108感染效果显著;而外源性IL-10免疫后,其对草鱼抗GCRV GD108感染效果不显著。

关键词(KeyWords)： 草鱼;非特异性细胞毒性细胞受体-1;白细胞介素-10;表达;多克隆抗体;组织病理学

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金（31602199）;; 973项目（2009CB118704）

作者(Author): 陈静妮,代礼平,雷燕,胡海浩,黄鉴涛,简纪常,闫秀英

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