抗鲤疱疹Ⅱ型病毒卵黄抗体制备及其功能鉴定  

Preparation and functional identification of egg yolk antibody against cyprinid herpesvirus 2

作　　者：寇海燕 郭培红 田丹阳 何稳 李坤 张婷婷 贡成良[1] 孙丙耀[1] 宋学宏[1] KOU Hai-yan;GUO Pei-hong;TIAN Dan-yang;HE Wen;LI Kun;ZHANG Ting-ting;GONG Cheng-liang;SUN Bing-yao;SONG Xue-hong(School of Biology and Basic Medical Sciences,Soochow University,Suzhou 215123,Jiangsu,China;Kunshan Best-Care Biological Technology Co.Ltd.,Kunshan 215345,Jiangsu,China)

机构地区：[1]苏州大学医学部基础医学与生物科学学院,江苏苏州215123 [2]昆山贝瑞康生物科技有限公司,江苏昆山215345

出　　处：《淡水渔业》2021年第1期65-71,共7页Freshwater Fisheries

基　　金：江苏省渔业科技类项目(Y2018-19)。

摘　　要：为制备抗鲤疱疹Ⅱ型病毒(CyHV-2)的卵黄抗体,探索防治异育银鲫(Carassius auratus gibelio)鳃出血病的新方法和途径,本研究利用原核表达系统产生具免疫原性的重组CyHV-2-ORF72衣壳蛋白,纯化后免疫蛋鸡;二次免疫后采用间接ELISA法抽检免疫蛋的特异性卵黄抗体(IgY)含量,收集抗体效价达到1∶8000的免疫蛋。将免疫蛋分成全蛋组与蛋黄组,采用微包膜和冷冻干燥技术,分别制成全蛋粉或蛋黄粉,利用Western blot和ELISA技术检测全蛋粉和蛋黄粉中的IgY特异性及效价。将蛋黄粉稀释液与CyHV-2混合后孵育鲤鱼上皮瘤细胞,进行抗体中和试验。以0.15%和0.30%的添加量分别将抗CyHV-2-ORF72的免疫全蛋粉拌入商品饲料制成功能性饲料,投喂已发病的两个池塘的异育银鲫进行生产性治疗试验。结果显示,全蛋粉和蛋黄粉中的IgY均能特异性地结合CyHV-2-ORF72衣壳蛋白,效价达到1∶8000~1∶16000;抗CyHV-2-ORF72的IgY与CyHV-2具有较好的亲和力,中和活性高于鼠抗ORF72血清;两个池塘异育银鲫成活率分别达到74.3%和87.6%,远高于对照池的28.3%和31.5%。综上所述,本研究制备的抗CyHV-2-ORF72卵黄抗体可特异性地中和CyHV-2病毒,具较显著的免疫保护作用。This experiment generated recombinant CyHV-2-ORF72 capsid protein with potential immunogenicity in prokaryotic expression system to produce egg yolk antibody against cyprinid herpesvirus 2(CyHV-2)and explore new methods and approaches to prevent and treat haemorrhagic gill disease of allogynogenetic crucian carp.The recombinant ORF72 protein was purified and used to immunize the laying hens.After the second immunization,random sampling was performed to determine the contents of specific yolk antibody(IgY)in immunized eggs by indirect ELISA.The immunized eggs with antibody titer reaching 1:8000 were harvested and divided into whole egg group and egg yolk group,and then the whole egg powder and egg yolk powder were prepared by freeze-drying technology after micro-encapsulation.The specificity and titer of IgY in the whole egg powder and egg yolk powder were then detected by using Western blot and ELISA techniques.Next,in vitro neutralization test was performed by incubating the CyHV-2-infected epithelioma papulosum cyprini cells with egg yolk powder diluents.In the full-scale therapeutic trial,the available feed was prepared by incorporating the whole egg powder with anti-CyHV-2-ORF72 antibody into a commercial feed with the addition of 0.15%and 0.30%,respectively,and fed to the sick fish in two ponds.The results showed that IgY in both whole egg powder and egg yolk powder could specifically bind to CyHV-2-ORF72 capsid protein,with titers ranging from 1:8000 to 1:16000.The anti-CyHV-2-ORF72 IgY had a high affinity with CyHV-2,and its neutralizing activity was higher than that of the mouse anti-ORF72 serum.Moreover,74.3%and 87.6%fish in the two experimental ponds survived,while only 28.3%and 31.5%fish in control ponds survived.Collectively,the anti-CyHV-2 egg yolk antibody prepared in this study shows the characteristics of virus neutralization and exhibits adequate immunoprotection against CyHV-2.

关 键 词：卵黄抗体 鲤疱疹Ⅱ型病毒 异育银鲫(Carassius auratus gibelio) 免疫保护 

分 类 号：S963.73[农业科学—水产养殖]