草鱼出血病病毒vp6核酸疫苗的免疫效果评估  

Evaluation of immune efficacy of GCRV vp6 DNA vaccine

作　　者：刘林[1] 徐诗英[2,3] 李婧慧 邹勇 倪金俤 杨鸢劼 曹广力[1] 薛仁宇[1] 陈辉 贡成良[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,苏州215123 [2]江苏省水生动物疫病预防控制中心,南京210036 [3]南京师范大学生命科学学院,南京210036

出　　处：《中国水产科学》2012年第5期841-847,共7页Journal of Fishery Sciences of China

基　　金：江苏省科技支撑计划项目(BE2009387);苏州市自然科学基金(SYN201106);江苏省农业科技自主创新资金项目(SCX(11)2165)

摘　　要：为了评估草鱼出血病病毒(GCRV)vp6基因核酸疫苗的免疫效果,将vp6基因克隆进pFastBacTMDual载体杆状病毒的多角体蛋白(Ph)启动子下游,同时将团头鲂(Megalobrama amblycephala)β-actin启动子控制的vp6基因克隆进杆状病毒的P10启动子下游,获核酸疫苗载体pFastBac-FA-VP6-ph-VP6。按每尾分别注射疫苗载体10、30、60μg的剂量免疫草鱼(Ctenopharyngodon idellus)(体长14～20 cm,体质量60～120 g),同设pFastBacTMDual载体(30μg/尾)阴性对照组及空白对照组(0.4 mL/尾无菌水)。于免疫后不同时间通过RT-PCR检测免疫鱼体中vp6基因的表达,并于免疫后第14、21、28、49、70天分别通过间接凝集反应检测血液中的抗体水平,并在免疫第21天感染GCRV评估免疫保护效果。结果显示,核酸疫苗免疫草鱼后,各免疫组均有抗体产生,抗体效价在免疫后第28天达到最高;攻毒后每尾注射疫苗载体10、30、60μg组的死亡率分别为0%、0%、5%,pFastBacTMDual载体对照组和空白对照组分别为30%和100%。表明构建的核酸疫苗对草鱼病毒性出血病有较好的免疫保护效果。To evaluate the immune efficacy of a grass carp reovirus （GCRV） vp6 DNA vaccine, We constructed a baculovirus transfer vector pFastBacTMDual-derived DNA vaccine vector pFastBac-FA-VP6-ph-VP6 in which one vp6 gene was controlled by the baculovirus polyhedron （Ph） promoter, and another vp6 expression cassette was driven by the β-actin promoter of grass carp, Megalobrama amblycephala at the downstream of the baculovirus pl0 promoter. Grass carp （weight 60-120 g, length 14-20 cm） were injected with 10, 30, or 60 pg of the vaccine vector, the negative control group was inject with 30 ~tg of pFastBacTMDual, and the unvaccinated control group was injected with 0.4 mL of sterile water. We then analyzed the pattern of vp6 expression in vaccinated fish using RT-PCR, measured antibody titers in the blood by indirect agglutination at 14, 21, 28, 49, and 70 d post-vaccination and evaluated immune efficacy by challenging GCRV at 21 d post-vaccination. The specific an- tibodies were detected in all vaccinated groups, and its levels peaked at 28th day after immunization. The mortali- ties were 0, 0, and 5% in the vaccinated groups injected with 10, 30, and 60 μg of DNA vaccine, and 30 % and 100% in the negative control group and the unvaccinated control group, respectively. Our results suggest that the DNA vaccine had strongly immunoprotective efficacy against GCRV.

关 键 词：草鱼出血病病毒 VP6基因 核酸疫苗 免疫 相对存活率 

分 类 号：S942[农业科学—水产养殖]