甲病毒检测细胞株的构建及其功能鉴定

Establishment and identification of the cell lines for the detection of mosquito-borne alphaviruses

目的 构建和鉴定可用于甲病毒快速检测的工程细胞株.方法 以辛德毕斯病毒缺陷型复制子载体系统pVaXJ-EGFP-△NSP4为基础,利用慢病毒表达载体pCDH系统,获得含有绿色荧光蛋白（Enhanced green fluorescent protein,EGFP）报告基因的慢病毒表达质粒pCDH-XJ-EGFP.使用脂质体转染法将质粒pCDH-XJ-EGFP和包装质粒pPACKHl Mix共转染HEK293T细胞,48 h、72 h收集慢病毒颗粒用于感染BHK-21细胞,经嘌呤霉素筛选获得用于蚊媒甲病毒检测的细胞系BHK-Alphavirus.结果 BHK-Alphavirus感染辛德毕斯病毒48 h后,可检测到EGFP的表达,表明BHK-Alphavirus能够用于外源甲病毒的检测.BHK-Alphavirus细胞感染甲病毒属中的辛德毕斯病毒（XJ-160和YN87448）、基孔肯雅病毒（SD08Pan）及盖塔病毒（HB0234）均可以检测到特异的绿色荧光,而被黄病毒属的乙脑病毒（P3）、4型登革病毒（P4）和布尼亚病毒属的塔希纳病毒（XJ0625）等其他正链RNA病毒感染后则检测不到绿色荧光.结论 BHK-Alphavirus细胞可用于蚊媒甲病毒的快速检测,且该检测方法特异性良好.

Objective To construct and identify the cell line for detecting Alphaviruses.Methods We used the lentiviral vector pCDH to construct the expressing plasmid pCDH-XJ-EGFP containing enhanced green fluscent protein （EGFP） reporter gene,which was flanked into the defective eukaryotic cassette from a defective replicon system pVaXJ-EGFP-△NSP4.To obtain lentivirus,we transfected the lentiviral expression plasmid pCDH-XJ-EGFP together with the pPACKF1 packaging plasmids into HEK293T cells by liposomes.Then,the target cells BHK-21 were infected with the lentivirus particles.Puromycin-resistant cell colonies were detached from the 6 well plate and sub-cloned by use of 96 well plate.Finally,we selected the packaging cell lines that could express the defective replicons stably,named BHK-Alphavirus cells.Results The BHK-Alphavirus cells infected with SINV XJ-160 could express EGFP at 48 hours postinfection,demonstrating that the selected cells based on the defective replicon expression systems could be used to detect alphaviruses infection.Several alphaviruses and other single-stranded positive-sense RNA virus were used to analyze the specificity of the BHK-Alphavirus cells.EGFP expression assays indicated that BHK-Alphavirus cells expressing the defective replicon stably produced green fluorescence,when were infected with alphaviruses,including two Sindbis virus （SINV,XJ-160,YN87448）,Chikungunya virus （CHIKV,SD08Pan） and Getah virus （GETAV,HB0234）.While no green fluorescence was observed after the BHK-Alphavirus cells were infection with the Japanese encephalitis virus （JEV,P3）,Dengue virus （DENV,P4） or Tahyna virus （TAHV,XJ0625）.Conclusion Theses results indicated that the BHK-Alphavirus cells based on the defective replicon expression systems were specific and effective to detect alphaviruses from tissue culture.