草鱼呼肠孤病毒VP7蛋白单克隆抗体的制备与应用

PRODUCTION AND APPLICATION OF MONOCLONAL ANTIBODIES AGAINST VP7 CAPSID PROTEIN OF GRASS CARP REOVIRUS

草鱼呼肠孤病毒(Grass carp reovirus,GCRV)是导致该病的主要病原,研究将Ⅰ型草鱼呼肠孤病毒GCRV-873株的外衣壳蛋白VP7基因进行原核表达,获得高度纯化VP7重组蛋白,通过免疫BALB/c小鼠,首次制备筛选得到高效价单克隆抗体。结果显示,GCRV-I vp7基因可在原核表达系统中高效表达,主要以包涵体形式存在,大小约为40 kD。免疫小鼠后筛选到了5株IgG类型阳性杂交瘤细胞株,其中3株亚型为IgG1,2株亚型为IgG2a。Western Blot实验和直接免疫荧光实验显示,该抗体可特异识别GCRV-873,并且ELISA检测原核重组蛋白的效价高达204800,亲和常数为4.04×10^(9)。研究制备的VP7蛋白单克隆抗体,为GCRV-I病毒诊断技术开发及病毒感染机制的深入研究提供实验基础。

Grass carp reovirus(GCRV)poses great threat to the grass carp aquaculture industry and causes huge eco-nomic losses.Grass carp reovirus is the pathogen of this disease,which consists of three types,typeⅠ,ⅡandⅢ.GCRV-873 is the most well studied stain of type I GCRV,with its complete genome sequenced in the 1980s.It has a segmented double stranded RNA genome with 11 fragments,encoding 7 structural proteins.VP5 and VP7 proteins form virus capsid.In this study,the capsid protein VP7(GenBank:AF403396)of GCRV-873 strain was expressed in the E.coli cells.The cDNA fragment encoding the extracellular fragment of VP7 was cloned into pRSET-A vector and the resultant plasmid transformed into the E.coli BL21(DE3)cells for prokaryotic expression.The recombinant pro-tein was purified by size exclusion chromatography and used to immunize BALB/c mice for generation of monoclonal antibodies.It has been shown that the VP7 protein was highly expressed as inclusion bodies and the size was con-firmed to be approximately 40 kD by SDS-PAGE analysis.After immunization,five IgG positive hybridoma cell lines were obtained,among which three were IgG1 subtype and two were IgG2a subtype.Western blotting and direct im-munofluorescence analysis showed that the antibody could specifically recognize GCRV-I in the infected CIK cells,and the titer was high,reaching an affinity constant of 4.04×10^(9).The present study provides a practical approach for the development of diagnostic tools for detecting type I GCRV virus and in-depth investigation on the GCRV infection mechanism.