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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 construct the flavivirus test cell line and to identify its function. Methods: Based on the plasmid P4 plasmid containing 814669 full-length infectious clones of dengue virus type 4, 1 945bp of pr ME gene was deleted to construct dengue virus replicon vector P4- △ pr containing red fluorescent protein m Cherry reporter gene ME-m Cherry. On the basis of P4- △ pr ME-m Cherry, the eukaryotic expression plasmid pCDNA3.1- P4-m Cherry. BHK-21 cells were transfected with plasmid pCDNA3.1-P4-m Cherry by Lipofectamine 2000. G418 cells were screened. Positive clones were screened by serial dilutions in 96-well plates to purify BHK-Fvivirus. Results: After being infected with Dengue virus for 60 hours, BHK-Flavivirus could detect the expression of red fluorescent protein m Cherry under the fluorescence microscope, indicating that BHK-Flavivirus can be used for the detection of foreign flavivirus. BHK-Flavivirus cells were infected with Flaviviridae JE virus (P3) and dengue virus type 4 (P4) respectively, and red fluorescence was detected. However, Sindbis virus (XJ-160) and Chikungunya virus ), Geeta virus (HB0234) and other positive-stranded RNA viruses do not appear after the red fluorescence. Conclusion: The above results indicate that BHK-Alphavirus cells can be used for detection of unknown mosquito-borne flaviviruses. By means of reporter gene expression or not, BHK-Alphavirus cells can efficiently and specifically screen the major mosquito vectors and flaviviruses. At the same time, Separation, preservation, operation method is simple and intuitive, is expected to be used in clinical testing and early detection of viral biological warfare agents.