Grass carp reovirus (GCRV) is the first aquatic animal virus isolated and well characterized in China,which causes severe hemorrhage in fingerlings and yearlings of grass carp (Ctenopharyngodon idellus) with above 90％ morbidity and leads to huge economic losses. GCRV has been classified to the genus of aquareoviruses and shares the typical physico-chemical properties and morphological characteristics with other aquareovirueses,which consists of a double-layered capsid in spherical structure, containing a genome composed of 11 segments of double-stranded RNA. GCRV has been considered to be the most pathogenic aquareovirus in fishes, therefore, it represents a model system for the studies of replication and pathogenesis of aquareoviruses both in vitro and in vivo. Currently, there are limited vaccines available for the prevention and control of the disease, moreover, none of them is commercialized and able to be delivered through oral administration. The application of plants as a production system for vaccines facilitates a novel and safe approach of vaccination. Besides their application in human beings, they can be used as oral veterinary vaccines in animals. It should be noted that several plantderived vaccines for different animals, utilizing various plant production systems, are under investigation.Although up to date not yet readily available, the development of these plant-based vaccines is in an advanced stage and shows great potential for use in future. Especifically in aquaculture, oral vaccination has multiadvantages, since it is a relatively effortless, cost effective and stress-free immunization method, which can be used at almost any age of fish.For the purpose to generate a plant-based vaccine against grass carp hemorrhage, here we presented the result of constructing a plant-based fusion expression vector, in which a gut adhesion molecule gene (LTB), a GCRV antigenic peptide coding gene (GCRV-VP6) and a green fluorescent protein gene (GFP) were included. In this study the specific primer pairs for amplifying the GCRV-VP6 gene and the Escherichia coli LTB gene were designed and synthesized based on the sequences of GCRV-VP6 gene and Escherichia coli LTB gene posted in GenBank. GCRV genome was extracted from the virus infected grass carp kidney cell line (CIK) and the GCRV VP6 coding region was amplified by RT-PCR. The LTB coding region was amplified from Escherichia coli H44815 genome. The PCR products were cloned into pCR2.1 vector respectively and then sequenced and subcloned into vector pCAMBIA 1302, which contained a green fluorescence protein gene marker in order to track the route of antigen uptake. This study has made a solid base for the innovation of edible transgenic plant vaccine against grass carp hemorrhage through oral immunization.%根据GenBank中草鱼呼肠孤病毒(Grass Carp Reovirus,,CCRV)VP6蛋白的全基因序列(AF403394)和大肠杆菌不耐热肠毒素B亚单位(LTB)的基因序列(M17874),设计并合成特异性引物,从感染草鱼呼肠孤病毒(GCRV)09草鱼(Ctenopharyngodon idellus),肾细胞(CIK)中提取病毒核酸作为模板,进行RT-FCR扩增,得到约1.3 kb的GCRV VP6基因读码框片段,同时提取大肠杆菌(Escherichia coli)H44815全基因组作为模板,进行FCR扩增得到约370 bp的LTB基因读码框片段.将获得的2个目的片段分别克隆到pCR2.1载体上,经酶切、PCR扩增检测和序列测定确认后,将其克隆到携带绿色荧光蛋白标记的植物表达载体pCAMBIA1302上,成功构建了可将草鱼呼肠孤病毒VP6蛋白、大肠杆菌不耐热肠毒素B亚单位(ETB)、绿色荧光蛋白(GFP)融合表达的植物载体pCAMBIA1302-LTB-VP6.本研究旨为草鱼出血病可饲化转基因植物疫苗研制奠定基础.
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