稳定表达T7 RNA聚合酶IBRS-2细胞系的建立以及利用该细胞系对SVDV的体内拯救
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国家重点基础研究发展计划(973)(2005CB523201)和国家科技支撑计划(2006BAD06A03)资助项目.


Establishment of IBRS-2 Cell Line Stably Expressing T7 RNA Polymerase and Recovery of SVDV From IBRST7 Cells
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This work was supported by a grant from National Basic Research Program of China (2005CB523201) and National Key Technology R&D Program of China (2006BAD06A03).

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    摘要:

    利用PCR从?姿溶原性细菌DE3中扩增出T7 RNA聚合酶基因,定向克隆进逆转录病毒载体pBABEpuro,得到阳性重组质粒pT7BABEpuro.把该重组质粒与pVSV-G共感染GP2-293包装细胞系,形成假型病毒.通过polybrene介导假型病毒感染IBRS-2细胞,利用嘌呤霉素进行传代筛选,形成细胞系IBRST7.对不同代次的IBRST7细胞基因组进行PCR和RT-PCR鉴定,结果表明,T7 RNA聚合酶基因在细胞传代过程中能稳定存在,并能表达目的蛋白的mRNA.为了鉴定T7 RNA聚合酶在IBRST7细胞内是否具有转录活性,扩增口蹄疫病毒(FMDV)的内部核糖体进入位点(IRES)片段和EGFP基因,定向克隆于原核表达载体pET-43.1a-c(+)中,构建了T7启动子控制下转录的具有非帽依赖性表达的重组质粒pIERS-EGFP-ET,把该质粒转染IBRST7细胞,能够在紫外显微镜下观察到绿色荧光,说明EGFP得到了表达,表明IBRST7细胞系内的T7 RNA聚合酶具有转录活性.然后,利用该细胞系成功拯救出具有感染性的猪水泡病病毒(SVDV),并对其生物学功能进行了鉴定.该细胞系的建立为利用T7 RNA聚合酶转录系统体内高效拯救病毒提供了基础.该拯救病毒的策略使RNA拯救简化为一步快速的拯救方法,为进一步探索SVDV病毒致病的分子机制及研制新型SVD疫苗奠定了良好的基础.

    Abstract:

    The bacteriophage T7 RNAP gene was amplified via PCR from λ-lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.

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郑海学,田 宏,靳 野,吴锦艳,尚佑军,刘湘涛,谢庆阁.稳定表达T7 RNA聚合酶IBRS-2细胞系的建立以及利用该细胞系对SVDV的体内拯救[J].生物化学与生物物理进展,2008,35(4):449-456

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  • 收稿日期:2007-08-29
  • 最后修改日期:2008-02-20
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  • 在线发布日期: 2008-03-07
  • 出版日期: 2008-04-20