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采用Bac-to-Bac系统,分别将Fd、轻链连同其信号肽基因克隆于pFastBac Dual不同启动子下,转化DH10Bac后,快捷方便地获得重组病毒,并且在sf9细胞中表达出具有HAV抗原结合活性的Fab。 1.材料和方法:(1)质粒、细胞株:pBSFdS:含重链及其信号序列基因的质粒,pBSLS:含轻链及其信号序列基因的质粒,由本室构建;pFastBac Dual质粒购自美国Gibco公司。草地贪夜蛾细胞(sf9),购自美国Invitrogen公司。(2)重组Bacmid的筛选:重组转座载体pFBFab转化DH10Bac,涂布于X-gal和IPTG的平板,挑选较大的白色菌落,提取质粒,即为重组的Bacmid。(3)转染sf9细胞:用Lipofectamine Plus转染,27℃培养过夜,次日换液,继续培养72h。收集上清液,500×g离心10min,将上清液分成小份作病毒原种。(4)重组蛋白的表达及检测:每瓶加入含适量重组杆状病毒毒种的完全培养基2ml,加入
Using Bac-to-Bac system, the Fd, light chain and their signal peptide genes were cloned into different promoters of pFastBac Dual respectively. After transformed into DH10Bac, the recombinant virus was quickly and conveniently obtained and expressed in sf9 cells with HAV antigen binding Active Fab. 1. Plasmid and cell line: pBSFdS: plasmid containing heavy chain and its signal sequence gene, pBSLS: plasmid containing light chain and its signal sequence gene, constructed by our laboratory; pFastBac Dual plasmid was purchased from USA Gibco Corporation. Spodoptera frugiperda cells (sf9), purchased from the United States Invitrogen Corporation. (2) Screening of Recombinant Bacmid: Recombinant transposase pFBFab was transformed into DH10Bac and coated on the plates of X-gal and IPTG. Larger white colonies were picked and plasmids were extracted to obtain recombinant Bacmid. (3) Transfection of sf9 cells: Transfection with Lipofectamine Plus, overnight at 27 ℃, liquid replacement the next day, continue to culture 72h. The supernatant was collected and centrifuged at 500 × g for 10 minutes. The supernatant was divided into small parts for virus stock. (4) Expression and detection of recombinant protein: Add 2ml of complete medium containing proper amount of recombinant baculovirus to each bottle, add