杆状病毒表达EV71病毒样颗粒的装配、制备纯化与鉴定

The Assemblage,Purification and Characterization of EV71 VLPs Expressed in Baculovirus

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。

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71（EV71）, the P1 and 3CD genes were cloned into the same baculovirus shuttle vector（Bac- mid）. Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-Pl-aCD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid Pl was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles（VLPs） about 27nm diameter. Different values of MOI and time points of expression were com- pared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Cham- bers to produce EV？I VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1（39kD）, VP0（34kD） and VP3（26kD） as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.