Establishment of IBRS-2 cell line stably expressing T7 RNA polymerase and recovery of SVDV frorn IBRST7 cells

文献类型： 外文期刊

第一作者： Tian Hong

作者： Tian Hong;Jin Ye;Wu Jin-Yan;Shang You-Jun;Liu Xiang-Tao;Xie Qing-Ge;Zheng Hai-Xue

作者机构：

关键词： T7 RNA polymerase(T7 RNAP);IBRS-2 cell;pseudotype virus;retroviral vector;infectious clone

期刊名称：PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS （ 影响因子：0.351； 五年影响因子：0.272 ）

ISSN： 1000-3282

年卷期： 2008 年 35 卷 4 期

页码：

收录情况： SCI

摘要： The bacteriophage T7 RNAP gene was amplified via PCR from lambda-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 cotransilected 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 RNA-P 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 EBRST7 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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