禽呼肠孤病毒σB和σC蛋白在昆虫细胞中的共表达

Expression ofσB andσC Proteins of Avian Reovirus in Insect Cells

本研究旨在获得具有天然构象和活性良好的禽呼肠孤病毒(ARV)σB和σC蛋白,采用杆状病毒表达系统在昆虫细胞中共表达σB和σC蛋白,并对其活性进行鉴定。根据NCBI数据库中ARVσB和σC基因序列设计引物,将两个基因克隆至pFast-Dual载体,转化大肠杆菌DH10Bac感受态细胞,筛选获得重组穿梭杆粒。通过转染sf9细胞,筛选到含有σB和σC基因的重组病毒。将重组病毒感染sf9细胞,通过优化接毒量和收获时间等参数,确定最佳表达条件,在sf9细胞中高效共表达σB和σC蛋白。Western blotting和间接免疫荧光(IFA)检测结果表明,成功在sf9细胞中共表达了ARVσB和σC蛋白,并具有很好的生物学活性。本试验结果为开发鉴别诊断试剂以及ARV颗粒疫苗的研究奠定了基础。

To obtain the native conformation and antigenicity ofσB andσC proteins of avian reovirus,theσB andσC proteins was expressed by Bac-to-Bac baculovirus expression system,and its reactivity was identified.TheσB andσC genes of avian reovirus were amplified by designing two pairs of primers according to the sequence published on NCBI,inserted into donor plasmid pFast-dual to obtain recombinant donor plasmid,then transformed into E.coli DH10Bac,and the recombinant bacmid plasmid was obtained.Recombinant baculovirus was transfected into sf9 insect cells using cellfection reagent to obtain the production of recombinant viral contained the sequence ofσB andσC genes.sf9 insect cells were inoculated by recombinant viral to explore the MOI and harvest timing expression condition,and the expression condition was optimized.The results of Western blotting and indirect immunofluorescence assay(IFA)showed that the recombinantσB andσC proteins were expressed successfully in sf9 insect cells and had good biological activity.This study provided a further research direction of ARV vaccine of virus-like particles and ARV subtype antibody ELISA detection kit.