黄病毒检测工程细胞系的构建及功能鉴定  

Construction and Identification of the Cell Line for Detecting Flaviviruses

作　　者：尉研[1] 王焕琴[1] 吴萌[1] 张凤娟[1] 梁国栋[1] 朱武洋[1] 

机构地区：[1]中国疾病预防控制中心病毒病所,北京100052

出　　处：《中国生物工程杂志》2015年第9期35-41,共7页China Biotechnology

基　　金：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)

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pr M-E基因1 945bp,构建含有红色荧光蛋白m Cherry报告基因的登革病毒复制子载体P4-△pr ME-m Cherry。以P4-△pr ME-m Cherry为基础,通过融合PCR方法,以真核表达载体p CDNA3.1+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p CDNA3.1-P4-m Cherry。脂质体转染法将质粒p CDNA3.1-P4-m Cherry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BHKFlavivirus。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m Cherry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。Objective： To generate and identify the cell line for detecting the unknown flaviviruses. Methods：The defective replicon with a deletion of pr M-E gene and the insertion of the red fluorescent protein m Cherry reporter gene was constructed on the basis of the infectious clone of dengue virus type 4（ P4）,which was named P4-△pr ME-m Cherry. To select the packaging cell lines for detecting flaviviruses,The multi-fusion PCR method to construct the defective eukaryotic expression plasmid p CDNA3. 1-P4-m Cherry on the basis of P4-△pr MEm Cherry were used. BHK-21 cells were transfected with thep CDNA3. 1-P4-m Cherry,then the G418-resistant BHK-21 cellsexpressing the defective replicons were obtained,which were named BHK-Flavivirus. Results： The expression of m Cherry 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,m Cherry was silent when the BHK-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.

关 键 词：黄病毒 细胞系 红色荧光蛋白 

分 类 号：Q819[生物学—生物工程]