【摘要】 采用BAC TO BAC 杆状病毒表达载体系统构建了表达鸡传染性支气管炎病毒 (IBV)呼吸型毒株SD/ 97/ 0 1S1蛋白的重组杆状病毒 .含SD/ 97/ 0 1株S1基因的重组质粒pMDSD970 1S1用BamHI和SalI双酶切后 ,回收目的片段并克隆到杆状病毒转座载体pFASTBAC HTa中多角体基因启动子的下游 ,筛选出重组转座质粒pFASTSD970 1S1并转化大肠杆菌DH10 BAC 后 ,获得重组穿梭质粒rBacmidSD970 1S1.用重组穿梭质粒DNA转染昆虫Sf9细胞 ,获得了含SD/ 97/ 0 1S1基因的重组杆状病毒rAcSD970 1S1.重组病毒感染Sf9细胞后 ,用SDS PAGE、Westernblot和IFA对细胞表达产物进行检测和分析 .结果表明 :构建的重组杆状病毒能够在昆虫细胞中表达SD/ 97/ 0 1的S1蛋白 ,该蛋白具有天然蛋白的抗原性 .更多还原

【Abstract】 The recombinant baculovirus expressing S1 glycoprotein of respiratory strain SD/97/01 of infectious bronchitis virus was generated by using BAC TO BAC baculovirus expression systems. The Bam H I Sal I fragment containing S1 gene from the recombinant plasmid pMDSD9701S1 was purified and cloned in frame into the baculovirus transposing vector pFASTBAC HTa under the polyhedrin gene promoter. The recombinant transposing plasmid pFASTSD9701S1 was screened and then transformed into Escherichia coli DH10BAC. The resulting recombinant bacmid rBacmidSD9701S1 was transfected into cells of the insect Spodoptera frugiperda (Sf9) and recombinant baculoviruse rAcSD9701S1 was obtained. The lysate of cells infected with rAcSD9701S1 was analyzed by SDS PAGE and expression product of S1 gene was detected by western blot and immunofluorescence assay (IFA). The results showed that the recombinant baculovirus was fully capable of expressing S1 glycoprotein of SD/97/01. Maybe owing to the incomplete glycosylation in insect cells, the S1 gene product had an Mr of only 61 kD. In immunofluorescence test and western blotting, expression product could react with polycolonal antibody against IBV M41 strain, indicating that it possessed the antigenic properties specific for native S1 glycoprotein.更多还原