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将EV71P1和3CD基因片段克隆入同一杆状病毒穿梭质粒Bacmid中,构建出重组杆状病毒表达质粒Bac-mid-P1-3CD;脂质体介导其转染Sf9昆虫细胞获得共表达P1和3CD的重组杆状病毒(AcMNPV-P1-3CD)。用IFA和Western-blot法对表达产物进行鉴定和分析。电镜结果显示P1经3CD切割装配成了大小约为27nm的类球形颗粒(即EV71VLPs)。进一步分析影响杆状病毒表达系统的因素以对表达条件进行优化,结果显示MOI值和时间均可影响目的蛋白的表达,其中时间是主要因素。选择优化后条件利用无血清培养基对贴壁Sf9细胞在多层细胞培养器中进行VLPs的大量表达,密度梯度离心法纯化,SDS-PAGE结果可见三条大小约为39kD、34kD和26kD的VP1、VP0和VP3特异性条带。纯化后EV71VLPs颗粒结构完好,为下一步EV71蛋白结构的基础研究和基因工程疫苗的研究奠定了基础。
The EV71P1 and 3CD gene fragments were cloned into the same baculovirus shuttle plasmid Bacmid to construct a recombinant baculovirus expression plasmid Bac-mid-P1-3CD; liposome-mediated transfection of Sf9 insect cells to obtain P1 and 3CD Of recombinant baculovirus (AcMNPV-P1-3CD). The expression products were identified and analyzed by IFA and Western-blot. Electron microscopy results showed that P1 was assembled by 3CD into spherical particles of about 27 nm in size (ie EV71 VLPs). Further analysis of the factors influencing the baculovirus expression system to optimize the expression conditions showed that the MOI value and time all affected the expression of the target protein, with time being the major factor. After optimized conditions, large numbers of VLPs were expressed in multi-layer cell culture medium using adherent Sf9 cells in serum-free medium and purified by density gradient centrifugation. Three SDS-PAGE results showed that the three VPs were 39kD, 34kD and 26kD, VP0 and VP3 specific bands. After purification, the structure of EV71VLPs particles is intact, which lays the foundation for the further research on the basic structure of EV71 protein and the research of genetic engineering vaccine.