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人肠道病毒71型(human enterovirus 71,EV71)是近年来暴发危害较严重的手足口病的病原。利用家蚕杆状病毒表达系统在家蚕卵巢培养细胞(Bm N)中共表达EV71病毒衣壳蛋白VP1与VP2,分析二者组装成病毒样颗粒(VLPs)的效率,比较用不同启动子构建重组病毒的表达效果,并利用家蚕幼虫进行2种蛋白质的共表达及VLPs纯化条件的研究。结果表明,重组病毒v Bm Bac-vp1-vp2与v Bm Bac-vp1-IRES-vp2均可介导EV71的VP1、VP2蛋白在Bm N细胞中表达,并能够组装成病毒样颗粒,但采用Pph及Pp10启动子的表达和组装效率明显高于Pph及IRES启动子组合;将重组病毒v Bm Bac-vp1-vp2以1×10~4TCID_(50)/头的剂量注射5龄起蚕,Western blotting检测到VP1在幼虫血淋巴中特异性表达,并且随着感染时间的延长表达量增加;经蔗糖梯度密度离心能够有效地纯化蚕体血淋巴中的病毒样颗粒。研究结果为后续EV71疫苗研制奠定了一定的试验基础。
Human enterovirus 71 (EV71) is the pathogen of hand-foot-mouth disease which is more serious in recent years. The expression vectors VP1 and VP2 of EV71 virus capsid protein were coexpressed in silkworm ovary cultured cells (BmN) using the silkworm baculovirus expression system. The efficiency of assembling the two vectors into virus-like particles (VLPs) was analyzed. Expression of the effect, and the use of silkworm larvae two kinds of protein co-expression and VLPs purification conditions. The results showed that the VP1 and VP2 proteins of EV71 could both be expressed in BmN cells and could be assembled into virus-like particles by using recombinant baculovirus vBm Bac-vp1-vp2 and vBm Bac-vp1-IRES-vp2, respectively. However, And Pp10 promoter expression and assembly efficiency was significantly higher than the combination of Pph and IRES promoter; the recombinant virus v Bm Bac-vp1-vp2 1 × 10 ~ 4TCID_ (50) / head dose injection of fifth instar silkworm, Western blotting The expression of VP1 was detected in larval haemolymph and the expression of VP1 was increased with the extension of infection time. The virus-like particles in silkworm hemolymph could be effectively purified by sucrose gradient density centrifugation. The results laid a certain experimental basis for the subsequent development of EV71 vaccine.