表达鸡传染性支气管炎病毒SD/97/01株S1蛋白的重组杆状病毒的构建

采用BAC－TO－BAC杆状病毒表达载体体系构建了表达鸡传染性支管炎病毒（IBV）呼吸型毒株SD／97／01S1蛋白的重组杆病病毒，含SD／97／01株S1基因原重组质粒p MDSD9701S1用BamHI和SalI双酶切后，回收的片段并克琶杆病病毒转座载体pFASTBACHTa中多角体基因启动子的下游，筛选出重组转座质粒pFASTSD9701S1U并转化大肠杆菌DH10BAC后,获得重组穿梭质粒rBacmidSD9701S1，用重组穿俊质粒DNA转染昆虫Sf9细胞，获得了含SD／97／01S1基因的重组杆状病毒rAcSD9701S1，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／97／01的S1蛋白，该蛋白具有天然蛋白的抗原性。

Construction of Recombinant Baculovirus Expressing S1 Glycoprotein of Infectious Bronchitis Virus Isolate SD/97/01

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.