黄病毒检测工程细胞系的构建及功能鉴定

doi:10.13523/j.cb.20150906

中国生物工程杂志

2015, Vol. 35

Issue (9): 35-41 DOI: 10.13523/j.cb.20150906

技术与方法

黄病毒检测工程细胞系的构建及功能鉴定

尉研, 王焕琴, 吴萌, 张凤娟, 梁国栋, 朱武洋

中国疾病预防控制中心病毒病所北京 100052

Construction and Identification of the Cell Line for Detecting Flaviviruses

WEI Yan, WANG Huan-qin, WU Meng, ZHANG Feng-juan, LIANG Guo-dong, ZHU Wu-yang

National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China

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摘要：

目的:构建黄病毒检测工程细胞系并对其功能进行鉴定.方法:以含有登革4型病毒814669株全长感染性克隆的质粒P4质粒为基础,缺失prM-E基因1 945bp,构建含有红色荧光蛋白mCherry报告基因的登革病毒复制子载体P4-△prME-mCherry.以P4-△prME-mCherry为基础,通过融合PCR方法,以真核表达载体pCDNA3.1+为骨架,构建用于黄病毒检测细胞筛选的缺陷型真核表达质粒pCDNA3.1-P4-mCherry.脂质体转染法将质粒pCDNA3.1-P4-mCherry转染BHK-21细胞,G418进行筛选,获得的阳性细胞克隆经96孔板系列稀释筛选、纯化克隆细胞BHK-Flavivirus.结果:BHK-Flavivirus细胞感染登革病毒60h后,能够在荧光显微镜下检测到红色荧光蛋白mCherry的表达,说明BHK-Flavivirus能够用于外源黄病毒的检测.BHK-Flavivirus细胞分别感染黄病毒属的乙脑病毒(P3)、4型登革病毒(P4),可以检测到红色荧光;而辛德毕斯病毒(XJ-160)、和基孔肯雅病毒(SD08Pan)、盖塔病毒(HB0234)等正链RNA病毒感染后则不出现红色荧光.结论:以上结果表明BHK-Alphavirus细胞可用于未知蚊媒黄病毒检测,该方法通过报告基因表达与否,能够高效、特异的甄别主要蚊媒甲病毒和黄病毒,同时该方法有利于稀缺病毒的分离、保存,操作方法简单、直观,有望应用于临床检测及病毒性生物战剂的早期甄别.

关键词： 黄病毒; 细胞系; 红色荧光蛋白

Abstract:

Objective:To generate and identify the cell line for detecting the unknown flaviviruses. Methods: The defective replicon with a deletion of prM-E gene and the insertion of the red fluorescent protein mCherry reporter gene was constructed on the basis of the infectious clone of dengue virus type 4 (P4), which was named P4-△prME-mCherry. To select the packaging cell lines for detecting flaviviruses,The multi-fusion PCR method to construct the defective eukaryotic expression plasmid pCDNA3.1-P4-mCherry on the basis of P4-△prME-mCherry were used. BHK-21cells were transfected with thepCDNA3.1-P4-mCherry, then the G418-resistant BHK-21cellsexpressing the defective replicons were obtained, which were named BHK-Flavivirus.Results: The expression of mCherry reporter gene were detected after the BHK-Flavivirus cells were infected by Flaviviruses,such as Japanese encephalitis virus (JEV,P3) and Dengue virus(DENV,P4). By contrast, mCherry was silent when theBHK-Flavivirus were infected by three related plus-strand RNA viruses, including Sindbis virus (SINV,XJ-160, YN87448), Chikungunya virus (CHIKV,SD08Pan) and Getah virus (GETAV,HB0234),and Tahyna virus (TAHV,XJ0625). Conclusion:These result indicated that the BHK-Flavivirus were specific and effective to detect Flavivirus in cell culture, which is a simple, color-visible detection method could detect corresponding mosquito-borne viruses quickly and specifically without affecting the viral multiplication capacity, making it possible to be used in the detection for clinical examinations and the screening of viral biological warfare agents.

Key words: Flaviviruses Engineering cell line Red fluorescent protein

收稿日期: 2015-05-12 出版日期: 2015-09-25

ZTFLH:

Q819

基金资助:

艾滋病和病毒性肝炎等重大传染病防治科技重大专项资助项目(2013ZX10004-101,2014ZX10004002-004-001,2013ZX10004-601-001)

通讯作者: 朱武洋 E-mail: zhuwuyang1971@sina.com

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引用本文:

尉研, 王焕琴, 吴萌, 张凤娟, 梁国栋, 朱武洋. 黄病毒检测工程细胞系的构建及功能鉴定[J]. 中国生物工程杂志, 2015, 35(9): 35-41.

WEI Yan, WANG Huan-qin, WU Meng, ZHANG Feng-juan, LIANG Guo-dong, ZHU Wu-yang. Construction and Identification of the Cell Line for Detecting Flaviviruses. China Biotechnology, 2015, 35(9): 35-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150906 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I9/35

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