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 VP4 protein of grass carp reovirus HZ08 strain

作　　者：曾伟伟[1] 王庆[1] 王英英[1] 石存斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔用药物创制重点实验室,广东省免疫技术重点实验室,广东广州510380

出　　处：《水产学报》2013年第3期450-456,共7页Journal of Fisheries of China

基　　金：国家科技支撑计划(2012BAD25B02);国家自然科学基金项目(31202026);公益性行业(农业)科研专项(200803013)

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,实验构建了能高效表达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,MAb)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κ链;间接ELISA试验证明,3株杂交瘤细胞分泌的MAb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∶720 000;IFA和Western-blotting结果显示,这株杂交瘤细胞分泌的MAb能够特异性识别GCRV-HZ08病毒粒子。本实验制备的MAb具有良好的生物学特性,为GCRV流行株血清学检测方法的建立及VP4蛋白相关功能研究奠定了基础。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 secrete monoclonal antibody （MAb） against VP4 protein using indirect ELISA. All the MAb were IgG1 subtype with Klight chain and could not react with GSRV, ISKNV, IHNV except GCRV-HZ08. The hybridoma cell line 2C2 was selected for MAb preparation, and the titers in cell culture medium of the ascetic fluids were up to 1 ： 720 000. The results of Western-blotting and IFA showed that the MAb could recognize the authenticVP4 protein of GCRV HZ08 particles. In present study, the MAb 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.

关 键 词：草鱼呼肠孤病毒 VP4蛋白 单克隆抗体 

分 类 号：Q511[生物学—生物化学] S917.1[农业科学—水产科学]