兔出血症病毒单克隆抗体的制备及鉴定  

Preparation and identification of monoclonal antibodies against rabbit hemorrhagic disease virus

作　　者：刘怀然[1,2] 秦海斌[2,3] 刘家森[2] 单安山[1] 曲连东[2] 

机构地区：[1]东北农业大学动物科学技术学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所农业部实验动物质量监督检验测试中心,黑龙江哈尔滨150001 [3]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《中国兽医科学》2009年第2期145-149,共5页Chinese Veterinary Science

基　　金：黑龙江省博士后资金项目(LRB05-358);国家科技部科技基础性工作专项(2001DEB20055)

摘　　要：为建立兔出血症病毒（RHDV）的检测方法及为变异株监测做储备，用蔗糖密度梯度离心法纯化的RHDV免疫BALB／c小鼠，取其脾细胞与SP2／0细胞进行融合，以昆虫细胞一杆状病毒表达的重组VP60蛋白建立的间接ELISA法筛选抗RHDV单克隆抗体杂交瘤细胞，并对其主要生物学特性进行了鉴定。结果表明，筛选出6株单抗，它们分属于IgG1（AD4，AG10，DE2）、IgG2b（BC9，BE8）和IgM（BH3）亚类；这6株单抗杂交瘤细胞的平均染色体数为87～89条，将其冻存6个月后复苏，在体外传20代的过程中分泌的抗体效价变化不大，具有很好的稳定性；Western-blot及免疫荧光分析表明，单抗DE2、AG10识别线性表位，其余4株单抗识别构象型表位；竞争ELISA试验表明，这6株单抗可识别5种抗原表位。To develop a detection method for rabbit hemorrhagic disease virus（RHDV） and its variant strains,the spleen cells of BALB/c mice,immunized with RHDV purified from rabbits livers by sucrose density gradient centrifugation, were fused with SP2/0 cells. Hybridoma cells secreting monoclonal antibodies（McAbs） against RHDV were screened by indirect ELISA based on recombinant VP60 protein expressed in insect cells baculovirus system and the major characteristics of the MeAbs were examined. Six strains of hybridoma cells secreting McAbs of AD4,AG10,DE2,BC9, BE8, and BH3 were obtained after 3 cycles of cell fusion, and the subtypes of the McAbs belonged to IgG1 （AD4, AG10, DE2）, IgG2b（BC9, BES） and IgM（BH3） respectively. The hybridoma cells had the average chromosome numbers of 87 to 89 and had the stable antibody titer during 20 generations culture in vitro. Analyses using Western-blot and immunofluorescence assays indicated that McAbs DE2 and AG10 could recognize linearity epitopes and others recognize conformation epitopes of VP60 protein. Analysis using competitive ELISA assay revealed that the 6 strains of McAhs could recognize 5 types of RHDV antigen epitopes.

关 键 词：兔出血症病毒 单克隆抗体 抗原识别位点 

分 类 号：S852.659.6[农业科学—基础兽医学]