摘要
为了更好地研究对虾白斑综合征病毒(WSSV)蛋白VP19在WSSV感染过程中的作用,利用VP19的单克隆抗体直接对VP19进行了定位。从患白斑综合征的中国明对虾(Fenneropenaeus chinensis)鳃丝中提取WSSV,将提纯的WSSV经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,然后洗脱提纯其病毒蛋白VP19并免疫Balb/c小鼠,取免疫小鼠脾细胞和骨髓瘤细胞融合,用间接免疫荧光技术(IFAT)和Western-Blot技术筛选出1株阳性杂交瘤细胞,将检测出的阳性杂交瘤细胞经有限稀释法克隆,研制出抗VP19的单抗,再利用免疫胶体金技术对病毒蛋白VP19进行定位,结果显示,胶体金粒子位于WSSV病毒的囊膜上,说明病毒蛋白VP19位于WSSV囊膜上。
White spot syndrome virus （WSSV） which emerged in 1993 has been a severe threaten to shrimp culture industry up to now. Therefore, WSSV has been extensively studied in the aspects of epidemiologt, gene and protein characteristics, etc. And some exciting research progresses have been abtained. For example, it is proved that virus envelop proteins （VPs） play important roles in virus infection. In order to investigate the infection mechanism of one of the WSSV envelop proteins-VP 19, this study tried to locate VP 19 on WSSV using the Mab of VP19. WSSV was isolated from tissues of WSSV-infected Fenneropenaeus chinensis and electroeluted by SDS-PAGE. A prominent 19KD protein （VP19） was electroeluted from the SDS-PAGE gels, dialyzed in PBS and concentrated by a vacuum evaporator. Then monoclonal antibodys （McAb） were produced against VP 19. The 4-week-old Balb／c mice were immunized by WSSV and VP19. Spleen cells of the immunized mice were fused with P3-X63-Ag8U1 myeloma cell, and hybridoma were detected by Immunofluoresence assays （IFAs） and western blotting analysis, and then subcloned. One clone was eventually chosen that reacted in IFAs with WSSV and in western blotting analysis with the denatured viral protein VP19 of WSSV. The result suggests that this Mab against VP 19 target linear epitopes. The location of VP 19 was tested by Immuno-electron microscopy with goldlabelled antibody and Mabs against VP19, the result revealed that the gold particles were distributed in the outer envelope of WSSV virions and showed that VP 19 is a virus envelope protein.
出处
《中国水产科学》
CAS
CSCD
北大核心
2009年第1期69-74,共6页
Journal of Fishery Sciences of China
基金
国家重点基础研究发展规划(973)基金项目(2006CB101806)
上海高校选拔培养优秀青年教师科研专项基金项目(科07-05)
上海海洋大学博士启动基金(科06-315)
