禽呼肠孤病毒σC基因在杆状病毒表达系统中的表达及其活性鉴定

本研究利用Bac-To-Bac杆状病毒表达系统构建重组禽呼肠孤病毒(Avian reovirus,ARV)σC基因的杆状病毒,感染sf9细胞获得表达重组蛋白。首先,将ARVσC基因克隆至pFastBacHTA载体,构建重组供体载体pFσC,将其转化大肠杆菌DHl0Bac感受态细胞,使σC基因整合到Bacmid穿梭载体中,获得重组穿梭载体BacmidσC。通过脂质体介导将其转染到sf9昆虫细胞中,获得重组杆状病毒rBacσC。通过Western Blot、间接免疫荧光试验(IFA)进行检测,结果显示:σC蛋白在重组杆状病毒rBacσC感染的sf9昆虫细胞中获得正确表达,分子质量约为37kD,表达的σC蛋白具有良好的反应活性。

Expression and identification of sigmaC gene of avian reovirus by baculovirus expression system

Recombinant baculovirus containing sigmaC gene of Avian reovirus was constructed using Bac-To-Bac Baculovirus expression system, and recombinant sigmaC protein was expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, sigmaC gene of Avian reovirus was cloned and inserted into donor plasmid pFastBacHTA to obtain recombinant donor plasmid pFsigmaC. Plasmid pFsigmaC was transformed into E. coli DH10Bac for integration into bacmid vector and the recombinant bacmid plasmid BacmidsigmaC was obtained. Recombinant baculovirus rBacsigmaC was obtained by transfection of the sf9 cells with BacmidsigmaC. Western blot and indirect immunofluorescence assay (IFA) were carried and the results showed that the recombinant sigmaC protein with 37 kDa molecular weight was expressed successfully.