中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
Chinese Journal of Experimental and Clinical Virology
2015年
5期
447-451
,共5页
张克佩%尉研%王焕琴%吴萌%张凤娟%梁国栋%王小平%朱武洋
張剋珮%尉研%王煥琴%吳萌%張鳳娟%樑國棟%王小平%硃武洋
장극패%위연%왕환금%오맹%장봉연%량국동%왕소평%주무양
甲病毒%细胞系%复制子载体
甲病毒%細胞繫%複製子載體
갑병독%세포계%복제자재체
Alphaviruses%Engineering cell lines%Replicon vector
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.
目的 構建和鑒定可用于甲病毒快速檢測的工程細胞株.方法 以辛德畢斯病毒缺陷型複製子載體繫統pVaXJ-EGFP-△NSP4為基礎,利用慢病毒錶達載體pCDH繫統,穫得含有綠色熒光蛋白(Enhanced green fluorescent protein,EGFP)報告基因的慢病毒錶達質粒pCDH-XJ-EGFP.使用脂質體轉染法將質粒pCDH-XJ-EGFP和包裝質粒pPACKHl Mix共轉染HEK293T細胞,48 h、72 h收集慢病毒顆粒用于感染BHK-21細胞,經嘌呤黴素篩選穫得用于蚊媒甲病毒檢測的細胞繫BHK-Alphavirus.結果 BHK-Alphavirus感染辛德畢斯病毒48 h後,可檢測到EGFP的錶達,錶明BHK-Alphavirus能夠用于外源甲病毒的檢測.BHK-Alphavirus細胞感染甲病毒屬中的辛德畢斯病毒(XJ-160和YN87448)、基孔肯雅病毒(SD08Pan)及蓋塔病毒(HB0234)均可以檢測到特異的綠色熒光,而被黃病毒屬的乙腦病毒(P3)、4型登革病毒(P4)和佈尼亞病毒屬的塔希納病毒(XJ0625)等其他正鏈RNA病毒感染後則檢測不到綠色熒光.結論 BHK-Alphavirus細胞可用于蚊媒甲病毒的快速檢測,且該檢測方法特異性良好.
목적 구건화감정가용우갑병독쾌속검측적공정세포주.방법 이신덕필사병독결함형복제자재체계통pVaXJ-EGFP-△NSP4위기출,이용만병독표체재체pCDH계통,획득함유록색형광단백(Enhanced green fluorescent protein,EGFP)보고기인적만병독표체질립pCDH-XJ-EGFP.사용지질체전염법장질립pCDH-XJ-EGFP화포장질립pPACKHl Mix공전염HEK293T세포,48 h、72 h수집만병독과립용우감염BHK-21세포,경표령매소사선획득용우문매갑병독검측적세포계BHK-Alphavirus.결과 BHK-Alphavirus감염신덕필사병독48 h후,가검측도EGFP적표체,표명BHK-Alphavirus능구용우외원갑병독적검측.BHK-Alphavirus세포감염갑병독속중적신덕필사병독(XJ-160화YN87448)、기공긍아병독(SD08Pan)급개탑병독(HB0234)균가이검측도특이적록색형광,이피황병독속적을뇌병독(P3)、4형등혁병독(P4)화포니아병독속적탑희납병독(XJ0625)등기타정련RNA병독감염후칙검측불도록색형광.결론 BHK-Alphavirus세포가용우문매갑병독적쾌속검측,차해검측방법특이성량호.
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.