| 稳定表达T7RNA聚合酶PK-15细胞系的建立 |
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| 引用本文： | 黄俊华,贺番,孙元,张鑫,常天明,李宏宇,仇华吉.稳定表达T7RNA聚合酶PK-15细胞系的建立[J].中国预防兽医学报,2011,33(5). |
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| 作者姓名： | 黄俊华 贺番 孙元 张鑫 常天明 李宏宇 仇华吉 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室,黑龙江,哈尔滨,150001 |
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| 摘 要： | 为构建稳定表达T7 RNA聚合酶(RNAP)的猪肾细胞系(PK-15),本研究采用PCR方法,以E.coli BL21(DE3)细菌基因组为模板扩增T7 RNAP基因,将其克隆到逆转录病毒载体pLXSN中,构建重组质粒pLXSN-T7.采用脂质体将pLXSN-T7转染至PT67细胞中,经G418筛选培养获得重组逆转录病毒rMLV-T7,将其接种于PK-15细胞进行G418加压筛选和纯化;应用PCR、间接免疫荧光试验及T7启动子控制下的红色荧光蛋白的重组表达质粒(pET-RED)进行瞬时表达,检测T7 RNAP的表达及活性,结果表明筛选所得的细胞系PK/T7的不同代次均具有转录活性.本研究建立稳定表达T7 RNAP的细胞系PK/T7,为实现细胞内T7启动的转录和猪源RNA病毒等的反向遗传操作提供了有效的方法.
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| 关 键 词： | 逆转录病毒载体 T7RNA聚合酶 稳定细胞系 |
Establishment of a PK-15 cell line stably expressing T7 RNA polymerase |
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| HUANG Jun-hua,HE Fan,SUN Yuan,ZHANG Xin,CHANG Tian-ming,LI Hong-yu,QIU Hua-ji.Establishment of a PK-15 cell line stably expressing T7 RNA polymerase[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(5). |
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| Authors: | HUANG Jun-hua HE Fan SUN Yuan ZHANG Xin CHANG Tian-ming LI Hong-yu QIU Hua-ji |
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| Institution: | HUANG Jun-hua,HE Fan,SUN Yuan,ZHANG Xin,CHANG Tian-ming,LI Hong-yu,QIU Hua-ji (Division of Swine Infectious Diseases,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China) |
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| Abstract: | To establish a PK-15 cell line stably expressing T7 RNA polymerase (T7 RNAP), the T7 RNAP gene was amplified by PCR from E. coli BL21 (DE3) cells and inserted into the retroviral plasmid vector pLXSN to construct the recombinant plasmid pLXSN-T7. The pLXSN-T7 was transfected into PT67 cells to prepare the recombinant retrovirus (rMLV-T7) in the presence of G418. The PK-15 cells stably expressing the T7 RNAP. designated PK/T7, was established by rMLV-T7 infection, G418 selection and immunofluorescence assay.... |
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| Keywords: | retroviral vector T7 RNA polymerase stable cell line |
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