文章摘要
曾伟伟,王 庆,王英英,石存斌,吴淑勤.草鱼呼肠孤病毒HZ08株VP4蛋白单克隆抗体的制备及鉴定[J].水产学报,2013,37(3):450~456
草鱼呼肠孤病毒HZ08株VP4蛋白单克隆抗体的制备及鉴定
Preparation and identification of monoclonal antibody against grass carp reovirus HZ-08 VP4 protein
投稿时间:2012-11-03  修订日期:2012-12-24
DOI:DOI:10.3724/SP.J.1231.2013.38410
中文关键词: 草鱼呼肠孤病毒  VP4蛋白  单克隆抗体
英文关键词: grass crap reovirus virus  VP4 protein  monoclonal antibody
基金项目:国家自然科学基金(31202026);农业公益性行业科研专项(200803013)
作者单位E-mail
曾伟伟 中国水产科学研究院珠江水产研究所 zww8810303@163.com 
王 庆 中国水产科学研究院珠江水产研究所  
王英英 中国水产科学研究院珠江水产研究所  
石存斌 中国水产科学研究院珠江水产研究所  
吴淑勤 中国水产科学研究院珠江水产研究所 wushuqin021@21cn.com 
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中文摘要:
      为了建立针对草鱼呼肠孤病毒(GCRV)流行株的血清学检测方法,本研究构建了能高效表达GCRV HZ08株主要衣壳蛋白VP4的重组表达载体pET32a-S6,利用纯化的VP4重组蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/0细胞融合,经过克隆和筛选,获得3株能稳定分泌抗VP4重组蛋白单克隆抗体(Monoclonal Antibody,McAb)的杂交瘤细胞,分别命名为2C2、2F3和5E5。抗体经亚型鉴定均为IgG1,轻链为kappa 链;间接ELISA试验证明,三株杂交瘤细胞分泌的McAb可特异性识别GCRV-HZ08,与GSRV,ISKNV,IHNV均无交叉反应。选择2C2作为腹水生产细胞株,免疫小鼠后腹水ELISA效价为1:720000;IFA和Western blot 结果显示,这株杂交瘤细胞分泌的McAb能够特异性识别GCRV-HZ08病毒粒子。本研究制备的McAb具有良好的生物学特性,为GCRV流行株检测方法的建立及VP4蛋白相关功能研究奠定了基础。
英文摘要:
      Grass carp reovirus (GCRV) has been considered as the most pathogenic agent and poses a significant threat to grass carp culture. A virulent reovirus strain, HZ08, was isolated from diseased grass carp in Zhejiang province, and has been proven to be epidemic strain in China. To development serological methods for detecting prevalent GCRV, the gene encoded for major outer capsid VP4 was selected clone to plasmid pET32a( ) and the purified recombinant VP4 protein was injected into BALB/c mice through subcutaneous route. The spleen cells from immuned BALB/c mice were fused with SP2/0 myeloma cells, and three hybridoma cell lines, designated as 2C2, 2F3 and 5E5 respectively, were screened out to be able to secret monoclonal antibody (McAb) against VP4 protein using indirect ELISA. All the McAb were IgG1 subtype with κlight chain and could not react with GSRV, ISKNV, IHNV except GCRV-HZ08. The hybridoma cell line 2C2 was selected for McAb preparation, and the titers in cell culture medium of the ascetic fluids were up to 1:720,000. The results of western blot and IFA showed that the McAb could recognize the authenticVP4 protein of GCRV HZ08 particles. In present study, the McAb against VP4 of GCRV HZ08 was successfully prepared, which laid a foundation of developing a rapid determination method for GCRV and further study of structures and functions of VP4 protein.
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