H3N8亚型马流感病毒VLPs的构建及其生物学特性分析  

Construction and biological characterization of virus-like particles containing HA and M1proteins of equine influenza virus subtype H3N8

作　　者：刘春国[1] 王伟[1] 刘飞[2] 刘彦云[1] 王丹 吕让[1] 刘明[1] 相文华[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]中国农业科学院上海兽医研究所,上海200241 [3]黑龙江省人民医院,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2014年第3期240-244,共5页Chinese Veterinary Science

基　　金：公益性行业(农业)科研专项(201303046-08)

摘　　要：为了构建含H3N8亚型马流感病毒HA蛋白和M1蛋白的病毒样颗粒疫苗,把A/equine/Xinjiang/3/2007(H3N8)的HA基因和M1基因克隆到杆状病毒穿梭载体pFastBac Dual中,将得到的重组穿梭质粒pFBD-XJ3HA-M1转化至DH10Bac感受态细胞,与杆状病毒骨架质粒Bacmid进行重组从而获得重组杆状病毒转座子rBac-XJ3HA-M1,然后将其转染Sf9昆虫细胞,包装重组杆状病毒rBV-XJ3HA-M1。通过PCR鉴定、细胞免疫组织化学试验、Western-blot分析以及血凝试验证明,HA蛋白和M1蛋白在昆虫细胞中能够有效表达,且表达产物具有良好的反应活性,表达的HA蛋白具有血凝活性。电镜观察发现,HA蛋白和M1蛋白能够稳定形成病毒样颗粒。上述研究结果为研制马流感亚单位疫苗提供了技术储备,也为马流感病毒蛋白的功能研究奠定了基础。To construct the virus-like particles（VLPs） vaccine containing hemagglutinin（HA） and ma- trix 1（M1） proteins of H3N8 subtype equine influenza virus（EIV） ,the HA and M1 genes of H3N8 subtype EIV A/equine/Xinjiang/3/2007（H3N8） were cloned into the transfer plasmid pFastBac Dual and then the recombinant shuttle plasmid pFBD-XJ3 HA-M1 was transformed into DH10Bac competent cells to generate a recombinant baculovirus bacmid of rBac-XJ3 HA-M1. The rBac-XJ3 HA-M1 was then transfected into Sf9 insect cells for packing a recombinant baculovirus of rBV-XJ3 HA-Ml,which was confirmed by PCR, immunohistochemistry,Western-blot, and hemagglutination tests. In result, the HA and M1 proteins of EIV, which were expressed in insect cells, had the immunoreactivity and hemagglutinin activity. The VLPs were assembled in the insect cells confirmed by the electron microscope, which was similar to the native virus particles in size and shape. The assembly of VLPs containing the HA and M1 proteins provides the basis for the development of subunit vaccine against H3N8 subtype EIV.

关 键 词：马流感病毒 H3N8亚型 HA基因 M1基因 杆状病毒双表达 病毒样颗粒 

分 类 号：S852.659.2[农业科学—基础兽医学]