草鱼呼肠孤病毒VP6蛋白与大肠杆菌LTB亚基植物融合表达载体的构建  

Construction of plant-based fusion expression vector of the GCRV VP6 antigenic protein and Escherichia coli LTB protein

作　　者：周勇[1] 曾令兵[1,2] 范玉顶[1] 罗晓松[1] 徐进[1] 肖艺[1,2] 

机构地区：[1]中国水产科学研究院长江水产研究所,湖北荆州434000 [2]华中农业大学水产学院,湖北武汉430070

出　　处：《中国水产科学》2011年第1期1-7,共7页Journal of Fishery Sciences of China

基　　金：农业部现代农业产业技术体系建设专项资金资助(nycytx-49-16);农业部公益性行业科研专项(200803013)

摘　　要：根据GenBank中草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)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)的草鱼(Ctenopharyngodon idellus)肾细胞(CIK)中提取病毒核酸作为模板,进行RT-PCR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PCR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(LTB)、绿色荧光蛋白(GFP)融合表达的植物载体pCAMBIA 1302-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。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 plant-de-rived 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 multi-advantages,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 anti-genic peptide coding gene（GCRV-VP6） and a green fluorescent protein gene（GFP） were included.In this study the specific primer pairs for ampli

关 键 词：草鱼出血病 呼肠孤病毒 VP6基因 LTB基因 植物表达载体 

分 类 号：Q786[生物学—分子生物学] S917[农业科学—水产科学]