兔出血症病毒主要结构多肽的纯化及免疫保护性试验  

Purification and Protective Test of Main Structural Polypeptide of Rabbit Haemorrhagic Disease Virus

作　　者：王恒安[1] 杜念兴[2] 徐为燕[2] 

机构地区：[1]上海交通大学农学院动科系,上海201101 [2]南京农业大学动物医学院,南京210095

出　　处：《江西农业大学学报》2000年第3期439-442,共4页Acta Agriculturae Universitatis Jiangxiensis

基　　金：农业部八五重点资助项目

摘　　要：人工感染RHDV -NJ株病死兔肝经 - 5 0℃反复冻融 ,氯仿处理 ,PEG沉淀 ,差速离心 ,Sepharose - 4B柱层析得纯化病毒 ,纯化RHDV经SDS -PAGE、CuCl2 负染色后 ,切取主要结构多肽VP1(6 4.5KD)凝胶带 ,除去CuCl2 和SDS获得VP1,高效液相色谱鉴定只有一个蛋白峰 ,Western -Blotting ,HA和HI结果显示纯化VP1具有良好的抗原表位稳定性。无母源抗体的 3月龄兔 1 2只 ,平均分为 3组 ,阳性对照组每只兔接种 1mL肝组织灭活苗 ,试验组每只兔接种 1mL ,0 .5mg/mL的VP1溶液 ,空白对照组每只兔接种 1mL正常兔肝悬液 ,接种后1 0d攻毒 ,每只兔攻击 1 .5mL1∶1 0稀释的RHDV -NJ株肝毒悬液 ,攻毒保护性试验结果显示阳性对照组和试验组的攻毒保护率均为 1 0 0 % ,空白对照组的 4只兔攻毒后 48h内全部死亡 ,证明VP1为RHDV的保护性抗原。Rabbit haemorrhagic disease virus (RHDV) was purified from the artificially infected liver emulsion by freezing and thawing, chloroform extraction, PEG6000 precipitation and Sepharose-4B chromatography. Negatively stained with 0.3M CuCl 2, the SDS-PAGE containing RHDV VP 1 were picked up, the Cu 2+ and SDS were removed, the VP 1 solution was summitted to High Performance Liquid Chromatography (HPLC) and showed only one peak. The purified VP 1 could combine with rabbit anti-RHDV sera by westem-blotting, and had hemagglutinating activity which could be inhibited by rabbit anti-RHDV sera. 12 3-month rabbits without maternal antibodies were divided into three groups, each group had four, each rabbit of the positive control group was vaccinated with 1mL formalinizedly infected liver vaccine, each of the experimental group with 1mL 0.5mg/mL purified VP 1 solution, and each of the negative control group with 1mL normal liver suspension, on the tenth day postinocunation, all rabbits were attacked with 1.5mL liver emulsion infected with RHDV-NJ strain, the protecting rate of the positive, experimental, and negative group arrived at 100%, 100% and 0, respectively. All of the above results came to a conclusion that VP 1 is the protective antigen of RHDV, and in that case VP 1 gene-engineering subunit vaccine may be developed.

关 键 词：兔 出血症病毒 结构多肽 纯化 免疫保护性 

分 类 号：S858.291[农业科学—临床兽医学]