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
宋玲玲;李欣;王洪梅;高远东;王立群;仲跻峰;何洪彬;
摘要(Abstract)：
[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。
关键词(KeyWords)： 绿色荧光标记;T7 RNAP;真核表达
基金项目(Foundation): 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)
作者(Author): 宋玲玲;李欣;王洪梅;高远东;王立群;仲跻峰;何洪彬;
Email:
DOI: 10.13989/j.cnki.0517-6611.2011.05.060
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