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为构建同时表达流感病毒M1和HA抗原的重组杆状病毒,采用PCR扩增流感病毒A/PR/8/34株的M1基因和去除信号肽的HA基因,将两基因克隆到杆状病毒转座载体pFastBac Dual的两个启动子下游的多克隆位点,筛选出阳性重组转座载体pFastBac Dual-M1-HA。将其转化含有杆状病毒穿梭载体(Bacmid)的DH10Bac感受态细胞,通过抗生素、蓝白斑筛选和PCR鉴定获得重组杆状病毒穿梭载体rBacmid-M1-HA,在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒rBac-M1-HA。提取重组病毒基因组,通过PCR鉴定外源基因插入成功。间接免疫荧光和Western-blot检测表明,该重组杆状病毒在Sf9昆虫细胞中成功地表达了M1和HA。应用杆状病毒/昆虫细胞系统成功共表达流感病毒M1和HA抗原,为研究流感病毒VLP的形成机制和开发新型流感疫苗奠定了基础。
Abstract：The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid.The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion.After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid(bacmid) in DH10Bac competent cells,the colonies were identified by antibiotics and blue-white selection.The rBacmid-M1-HA was verified by PCR and transfected into Sf9 cells to produce recombinant baculovirus(rBac-M1-HA).Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot,demonstrating M1 and HA were co-expressed successfully.This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002271
中图分类号:R373.1
引用信息:
[1]徐鹏卫,郭建强,姚立红,等.共表达甲型流感病毒M1和HA抗原的重组杆状病毒的构建[J].病毒学报,2012,28(03):231-236.DOI:10.13242/j.cnki.bingduxuebao.002271.
基金信息:
“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项(NO.2009ZX10004-710)
2012-05-15
2012-05-15