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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。
To construct a recombinant baculovirus expressing both influenza virus M1 and HA antigen, the M1 gene of influenza A / PR / 8/34 strain was amplified by PCR and the HA gene was deleted from the signal peptide. The two genes were cloned into baculovirus The multiple cloning sites downstream of the two promoters of the pFastBac Dual cassette vector were screened out and the positive recombinant transposase pFastBac Dual-M1-HA was screened out. The recombinant baculovirus shuttle vector rBacmid-M1-HA was transformed into Bacillus subtilis DH10Bac competent cells. The recombinant baculovirus shuttle vector rBacmid-M1-HA was transfected into Sf9 by lipofectamine Insect cells to obtain recombinant baculovirus rBac-M1-HA. The recombinant virus genome was extracted and the exogenous gene was identified by PCR. Indirect immunofluorescence and Western-blot showed that the recombinant baculovirus successfully expressed M1 and HA in Sf9 insect cells. The successful co-expression of the influenza virus M1 and HA antigen using the baculovirus / insect cell system laid the foundation for the study of the formation mechanism of influenza virus VLP and the development of a novel influenza vaccine.