摘要
应用杆状病毒表达系统,将猪圆环病毒2型ORF2基因插入供体质粒pFastBacTMDual pH启动子控制下的多克隆位点,引入EGT信号肽取代ORF2原有核定位信号肽以实现在昆虫细胞中分泌型表达,并在C端融合6个组氨酸标签以便于后期的纯化.将构建质粒转化DH10Bac感受态细胞获得重组穿梭载体,提取重组Bacmid质粒.将阳性重组Bacmid质粒转染Sf9昆虫细胞,72 h收集培养上清液获得重组杆状病毒.间接免疫荧光试验和Westernblot表明,该重组杆状病毒可以在昆虫细胞实现猪圆环病毒2型Cap蛋白的分泌表达.
The objective of this study was to utilize baculovirus expression vector system to express capsid （Cap）protein （in secreted form）of porcine circovirus 2.The gene sequences encoding Cap protein fused with a C-terminal 6 Histidine tag were cloned into the baculovirus pFastBac TM Dual vector under the control of pH promoter .The authentic signal peptide of porcine circovirus type 2 ORF2 was substituted with the ecdysteroid UDP-glucosyltransferase （EGT） signal peptide.Plasmid was transformed into Escherichia coli DH10Bac competent cells to obtain the recombinant shuttle vector Bacmid .The Bacmid was transfected in-to Sf9 cells to produce the recombinant baculovirus .Indirect immunofluorescent assay and Western-blot in-dicated that the recombinant baculovirus could express Cap protein in infected Sf 9 cells.
出处
《华南农业大学学报》
CAS
CSCD
北大核心
2013年第4期564-568,共5页
Journal of South China Agricultural University
基金
863计划项目(2011AA10A209)
973计划项目(2011CB504701)
广州市"珠江科技新星专项"项目(2012J2200086)
公益性行业(农业)科研专项(201303034)
