草鱼NCCRP-1和IL-10的表达、抗体制备及组织病理变化  

Expression,Antibody Preparation of NCCRP-1 and IL-10 of Grass Carp,and Histopathological Changes

作　　者：陈静妮 代礼平[1] 雷燕 胡海浩 黄鉴涛 简纪常[1] 闫秀英[1] CHEN Jing-ni;DAI Li-ping;LEI Yan;HU Hai-hao;HUANG Jian-tao;JIAN Ji-chang;YAN Xiu-ying(Guangdong Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,and Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Fisheries College of Guangdong Ocean University,Zhanjiang 524088,China;Guangzhou Liyang Aqua-Technology Co.Ltd,Guangzhou 510515,China)

机构地区：[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,广东湛江524088 [2]广州利洋水产科技股份有限公司,广东广州510515

出　　处：《广东海洋大学学报》2021年第5期10-18,共9页Journal of Guangdong Ocean University

基　　金：国家自然科学基金(31602199);973项目(2009CB118704)。

摘　　要：【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(Escherichia coli)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∶40000,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GCRV GD108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。【Objective】To prepare antibodies against non-specific cytotoxic cell receptor protein 1(NCCRP-1)and interleukin-10(IL-10),and study the pathological changes and NCCRP-1 and IL-10 gene expression in the kidney and intestine of the recombinant NCCRP-1 and IL-10 immunized grass carp Ctenopharyngodon idella after they were challenged by GCRV.【Methods】The open reading frame(ORF)of the grass carp NCCRP-1 and IL-10 gene were acquired by PCR.Prokaryotic expression plasmids pET-NCCRP and pET-IL10 were constructed followed by recombinant protein production.Antibodies against NCCRP-1 and IL-10 were prepared respectively with the expressed proteins in rabbits.Titers of antibodies were characterized by ELISA method.Grass carp were immunized with the recombinant proteins NCCRP-1 and IL-10 respectively and challenged with grass carp reovirus(GCRV)GD108.Pathological sections of kidney and intestine of were performed 7 days after challenge,and the expression of NCCRP-1 and IL-10 were detected by real-time quantitative PCR and western blot.【Result】The ORFs of NCCRP-1 and IL-10 encoded 237 and 179 amino acids respectively.Prokaryotic expression vectors of pET-NCCRP and pET-IL10 were constructed successfully.Target fusion proteins of NCRP-1 and IL-10 were expressed in E.coli BL21,and the optimal expression conditions were 0.1 mmol/L isopropyl-β-D-thiogalactopyranoside(IPTG)at 37℃for 4 hours and 1.0 mmol/L IPTG at 37℃for 6 hours.Western blot results confirmed that the expressed fusion proteins were the targeted protein.The titers of antibodies against NCCRP-1 and IL-10 were 1∶40000.After immunization and challenge,results of histopathology of kidney and intestine of grass carp and expression of NCCRP-1 and IL-10 showed that pathological changes of grass carp immunized with NCCRP-1 was mild,and expression of NCCRP-1 was up-regulated significantly;Pathological changes of grass carp immunized with IL-10 were serious,and the expression of IL-10 was unchanged.【Conclusion】After immunization with exogenous NCCRP-1,imm

关 键 词：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 

分 类 号：S943.112[农业科学—水产养殖] Q78[农业科学—水产科学]