人肠道病毒71型类病毒颗粒在家蚕BmN细胞及幼虫体内的表达

发布时间：2018-06-02 19:42

本文选题：人肠道病毒型 + 病毒样颗粒　；参考：《蚕业科学》2016年03期

【摘要】：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。
[Abstract]:Human enterovirus 71 (human enterovirus 71 / EV71) is the pathogen of hand, foot and mouth disease (HFMD) which is a serious outbreak in recent years. The EV71 capsid protein VP1 and VP2were co-expressed in silkworm ovary culture cells (BmNs) by using the baculovirus expression system of Bombyx mori. The efficiency of assembling them into virus-like granulocytes was analyzed, and the expression effects of recombinant viruses constructed by different promoters were compared. The co-expression of two proteins and the purification conditions of VLPs were studied by using silkworm larvae. The results showed that both vBm Bac-vp1-vp2 and vBm Bac-vp1-IRES-vp2 could mediate the expression of VP1pVP2 protein of EV71 in BMN cells, and could assemble virus-like particles. However, the expression and assembly efficiency of Pph and Pp10 promoters were significantly higher than those of Pph and IRES promoter combinations. The recombinant virus vBm Bac-vp1-vp2 was injected into the head of 5 instar silkworm at the dose of 1 脳 10~4TCID_(50)/. Western blotting was used to detect the specific expression of VP1 in haemolymph of larva, and the expression increased with the prolongation of infection time. The virus-like particles in hemolymph of silkworm body can be effectively purified by sucrose gradient density centrifugation. The results of the study laid a certain experimental foundation for the further development of EV71 vaccine.
【作者单位】：江苏科技大学;中国农业科学院蚕业研究所;
【基金】：江苏省自然科学基金项目(No.BK20151321) 江苏省教育厅科技项目(No.11KJB180002)
【分类号】：R373.2;Q78

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