草鱼出血病病毒vp6核酸疫苗的免疫效果评估

摘要

To evaluate the immune efficacy of a grass carp reovirus (GCRV) vp6 DNA vaccine, We constructed a baculovirus transfer vector pFastBac?Dual-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 p10 promoter. Grass carp (weight 60-120 g, length 14-20 cm) were injected with 10, 30, or 60 μg of the vaccine vector, the negative control group was inject with 30 μg of pFastBac?Dual, 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 antibodies were detected in all vaccinated groups, and its levels peaked at 28th day after immunization. The mortalities 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.%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、60 μg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(30 μg/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、60 μg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.