具有绿色荧光标记的表达T7 RNA聚合酶稳定细胞系的建立  

Establishment of BHK-21 Stable Cell Lines Expressing T7 RNAP and GFP

作　　者：宋玲玲[1] 李欣[1,2] 王洪梅[1] 高远东[1] 王立群[2] 仲跻峰[1] 何洪彬[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,山东济南250100 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《安徽农业科学》2011年第5期2550-2551,2553,共3页Journal of Anhui Agricultural Sciences

基　　金：国家转基因重大专项项目(2009ZX08007-006B);国家自然科学基金项目(31072160);山东省科技攻关项目(2009GG20002032);山东省自然基金项目(Y2008D20);兽医生物技术国家重点实验室开放课题基金项目(SKLVBF200806)

摘　　要：[目的]建立具有绿色荧光标记表达T7 RNA聚合酶的稳定细胞系。[方法]从BL21(DE3)大肠杆菌中克隆出T7RNAP基因,定向克隆质粒FG12后,构建了表达T7 RNA聚合酶基因(T7)的Lenti-virus重组质粒FG12-T7 RNAP。用此重组质粒转染293T细胞,WB可检测出瞬时表达的T7 RNAP蛋白;利用辅助质粒对表达T7的非复制型Lenti-virus病毒进行体外包装,所获得的非复制型Lenti-virus病毒感染BHK-21细胞,利用GFP通过流式细胞分选仪筛选表达T7的细胞系,并利用WB检测基因的表达。[结果]通过WB检测出不同代次的阳性细胞中均能稳定表达目的基因T7 RNA聚合酶。[结论]T7 RNA聚合酶能顺利在真核细胞内表达,并在此基础上建立的稳定细胞系为RNA病毒体内拯救技术平台的建立奠定了基础。[Objective] The aim of this study was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method] T7 RNAP gene was amplified from E.coli BL21（DE3） and inserted into FG12 vector.The Lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP Lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continually screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result] The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion] T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.

关 键 词：绿色荧光标记 T7 RNAP 真核表达 

分 类 号：S188[农业科学—农业基础科学]