蓝舌病病毒血清Ⅰ型VP2与VP5、VP3与VP7两组蛋白在杆状病毒系统中的表达与鉴定  

Expression and Identification of VP2 and VP5,VP3 and VP7 Proteins of Bluetongue Virus Serotype 1 in Baculovirus System

作　　者：李占鸿 杨恒 宋子昂 高林 李华春 廖德芳 Li Zhanhong;Yang Heng;Song Ziang;Gao Lin;Li Huachun;Liao Defang(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Yunnan Animal Science and VeterinaryInstitute,Kunming,Yunnan 650224,China)

机构地区：[1]云南省畜牧兽医科学院热带亚热带动物病毒重点实验室,云南昆明650224

出　　处：《中国动物检疫》2019年第5期77-84,共8页China Animal Health Inspection

基　　金：国家自然科学基金项目(31760744);国家公益性行业(农业)科研专项(201303035);云南省中青年学术和技术带头人后备人才培养项目(2017HB055)

摘　　要：为表达具有天然构象的蓝舌病病毒血清1型(BTV1)主要结构蛋白VP2、VP3、VP5和VP7,研制针对流行于我国的BTV1型毒株的病毒样颗粒(virus-like particles,VLP)疫苗提供基础,将编码BTV1 VP2和VP5蛋白的基因分别克隆到双表达载体pFastBacDual的启动子pPH和pP10下游,构建重组质粒pFastBacDualBTV1-VP2-VP5,将重组质粒转化DH10Bac感受态细胞,获得重组穿梭质粒rBacmidBTV1-VP2-VP5,转染Sf9昆虫细胞,获得重组杆状病毒rBacBTV1-VP2-VP5;按照同样策略,制备重组杆状病毒rBacBTV1-VP3-VP7。使用兔抗BTV1-VP5蛋白多克隆抗体和鼠抗BTV-VP7蛋白单克隆抗体,分别对rBacBTV1-VP2-VP5和rBacBTV1-VP3-VP7感染的Sf9细胞进行间接免疫荧光试验(IFA)检测,结果病毒感染细胞可见特异性荧光出现;使用兔抗BTV1-VP2蛋白多克隆抗体和兔抗BTV1-VP3蛋白多克隆抗体,分别对rBacBTV1-VP2-VP5和r BacBTV1-VP3-VP7感染的Sf9细胞进行Western-blot检测,可见相对分子质量约100 kDa左右的条带,大小与预期相符。IFA检测和Western Blot检测结果显示,BTV1 VP2、VP3、VP5和VP7蛋白均得以表达,且具有良好的反应原性。In order to express the major structural proteins VP2,VP3,VP5 and VP7 of bluetongue virus serotype 1 (BTV1)with natural conformation,and to provide reference for the development of virus like particles(VLP) vaccine against BTV1 strain which was prevalent in China,the genes of BTV VP2 and VP5 were cloned into the downstream of pPH and pP10,the promoters of the double expression vector(pFastBacDual),to construct the recombinant plasmid pFastBacDual-BTV1-VP2-VP5 and then transformed it into DH10Bac competent cells to obtain the recombinant baculovirus shuttler plasmid,rBacmidBTV1-VP2-VP5,then the tecombinant baculovirus,rBacBTV1- VP2-VP5,was gained by transfection of the Sf9 cells. Also,recombinant baculovirus rbacBTV1-VP3-VP7 was prepared based on the same strategy. The Sf9 cells infected with rBacBTV1-VP2-VP5 and rbacBTV1-VP3-VP7 were detected through indirect immunofluorescence assay(IFA)respectively by rabbit anti-BTV1-VP5 protein polyclonal antibody and by mouse anti-BTV-VP7 protein monoclonal antibody. It was found that the specific fluorescence appeared in the infected cells;while the same Sf9 cells were detected through Western-blot respectively by rabbit anti-BTV1- VP2 protein poly-clonal antibodies and by rabbit anti-BTV1-VP3 protein monoclonal antibodies,the strip with relative molecular weight of about 100 kDa was shown,which was consistent with the expected value. These results showed that BTV1-VP2,VP3,VP5 and VP7 proteins were all expressed with good immunogenicity.

关 键 词：蓝舌病病毒血清1型 主要结构蛋白 杆状病毒表达 

分 类 号：S852.659.4[农业科学—基础兽医学]