信号肽影响日本乙型脑炎假型病毒包装效果及感染能力的研究

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• 英文论文题名：The influence of packaging titer and infection capacity about introducing signal peptide to pseudotyped Japanese encephalitis virus
• 论文作者：刘瀚扬 ; 曹三杰 ; 伍锐 ; 袁磊 ; 刘洪明 ; 冯瑶 ; 曹玉琴 ; 田耕 ; 周玉鹏 ; 黄小波 ; 文翼平 ; 文心田
• 英文论文作者：Liu hanyang ; Cao sanjie ; Wu rui ; Liu hongming ; Feng yao ; Cao yuqin ; Tian geng ; Zhou yupeng ; Yuan lei ; Huang xiaobo ; Wen yiping ; Wen xintian ; Research Center of Swine Disease ; College of Veterinary Medicine ; Sichuan Agricultural University
• 年：2016
• 作者机构：四川农业大学预防兽医研究所猪病研究中心;
• 论文关键词：信号肽 ; 包装滴度 ; 日本乙型脑炎 ; 假病毒
• 英文论文关键词：signal peptide ; packaging titer ; Japanese encephalitis virus ; pseudotyped virus
• 会议召开时间：2016-05-25
• 会议录名称：第七届中国畜牧科技论坛论文集
• 英文会议录名称：Proceeding of the 7th China Animal Husbandry Sci-tech Forum
• 语种：中文
• 分类号：S852.65
• 学会代码：ZGXJ
• 会议名称：第七届中国畜牧科技论坛
• 会议地点：中国重庆
• 主办单位：中国畜牧兽医学会
• 学会名称：中国畜牧兽医学会
• 页数：9
• 文件大小：2166k
• 原文格式：O
• 会议级别：全国

摘要

猪乙型脑炎病毒(JEV)是一种人畜共患的虫媒病毒,具有传染性和危险性,研究这类病毒需要一种安全的研究代替工具,假型病毒技术为解决这些问题提供了一种有效的研究手段。为了构建1种高包装滴度的JEV假型病毒,我们设计并包装出了3种乙脑假型病毒(JEVpv),分别是融合表达了水泡口炎病毒(VSV)囊膜蛋白强信号肽和JEV囊膜蛋白(ME)的VSVMEpv,带有乙脑自身弱信号肽及ME蛋白的SPMEpv,以及不带有信号肽的MEpv。蛋白免疫印迹结果显示,3种JEVpv均能成功表达JEV的E蛋白及HIV的p24蛋白;透射电镜观察显示,3种JEVpv的粒子直径在1 20~180nm;Realtime RT-PCR结果显示,VSVpv的包装滴度是MEpv的17倍,而SPMEpv的包装滴度是MEpv的6倍,说明信号肽能够显著增强病毒的包装滴度,同时强信号肽的增强效果高于弱信号肽;对绿色荧光蛋白及荧光素酶的表达检测显示,3种JEVpv均能感染293T及BHK-21细胞,且感染滴度没有显著差异(P>0.05),说明信号肽不影响JEVpv的感染能力。因此,我们的研究提供了一种经济高效的日本乙型脑炎假型病毒的包装方法,为乙脑的入侵机制研究奠定了基础。
Japanese encephalitis virus(JEV) is a zoonotic arboviruses.A safe succedaneum model is needed when study such infectious and dangerous virus.The use of pseudotyped viruses can solve all of the problem above.In this study,we generated three pseudotyped JEVs that coexpressed a strong signal peptide of vesicular stomatitis virus(VSV)-G(VSVMEpv)or a weak signal peptide of JEV(SPMEpv),or without a signal peptide in front of JEV prM/E(MEpv).Western blotting demonstrated that JEV E protein and HIV p24 were present in the same particles of the three pseudotyped JEVs.Electron microscopy revealed that the three pseudotyped JEVs were 120-180 nm in diameter.Real-time quantitative reverse transcriptase polymerase chain reaction showed that the titer of VSVpv was 17-fold higher than that of MEpv,while the titer of SPMEpv was sixfold higher than that of MEpv.Signal peptide enhanced packaging titer of pseudotyped JEV,and a strong signal peptide helped them generate a higher number of virus particles.Green fluorescent protein and luciferase expression showed that the transduction titer or relative fluorescence units of VSVMEpv,SPMEpv and MEpv were not significantly different.We suggest that signal peptide does not influence the infectivity of pseudotyped JEVs.Therefore,our study provided a cost-effective method to generate pseudotyped JEV,which may represent a valid model to investigate some of the infectious properties of JEV.

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