表面展示鸡传染性支气管炎病毒M41株S1蛋白的重组假型杆状病毒的构建

Construction of a Recombinant Baculovirus Surface Displaying S1 Protein of M41 Strain of Infectious Bronchitis Virus

摘要: 以鸡传染性支气管炎病毒M41毒株基因序列为模板，利用PCR方法扩增膜定位信号缺失的S1基因（dS1），将其亚克隆入杆状病毒表面展示质粒pBACsurf-1中，再次将S1及gp64的基因片段克隆到杆状病毒转移质粒pFastBac^TMDual，得到重组质粒pFastBac-gp64-dS1.将该重组质粒转化到DH10Bac感受态细胞中，获得重组穿梭载体Bacmid-gp64-dS1，提取重组Bacmid质粒, 以PCR验证其正确性.将阳性重组Bacmid质粒利用脂质体转染Sf9昆虫细胞，获得重组杆状病毒BV-dS1.间接免疫荧光试验表明，该重组杆状病毒可以Sf9昆虫细胞膜表达鸡传染性支气管炎病毒S1蛋白.

Abstract: The S1（Spike）protein gene without membrane localization signal of infectious bronchitis virus (dS1）was amplified from infectious bronchitis virus(M41) by PCR and sub-cloned into pBACsurf-1. The fusion gene containing S1 and gp64 was then inserted into baculovirus transfer vector pFastBac^TM Dual to construct the recombinant plasmid pFastBac-gp64-dS1.Then,the plasmid was transformed into Escherichia coli DH10Bac competent cells to obtain the recombinant shuttle vector Bacmid-gp64-dS1. Extracted Bacmid-gp64-dS1 was transfected into Sf9 cells to produce the recombinant baculovirus BV-dS1 by using Lipofectamine^TM 2000. Indirect immunofluorescent assay indicated that the recombinant baculovirus BV-dS1 could express S1 protein on infected Sf9 cell membrane.