BHK-T7 cell line稳定表达噬菌体T7 RNA聚合酶T7 RNAP细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心
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- 货 号：BHK-T7 cell line稳定表达噬菌体T7 RNA聚合酶T7 RNAP细胞株
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BHK-T7 cell line稳定表达噬菌体T7 RNA聚合酶T7 RNAP细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心 BHK-T7 cell line稳定表达噬菌体T7 RNA聚合酶T7 RNAP细胞株
Introduction •Reverse-genetics systems for the rescue of recombinant FMDV have proven to be of great value for foot-and-mouth disease virus (FMDV) research and vaccine development. The reverse-genetics techniques based on in vitro transcription system or similar techniques were successfully developed for recovery of FMDV O, A, SAT1and SAT2. However, these reverse genetics systems has low efficiency of virus recovery. To overcome this, using retroviral gene transfer technology, we developed a stable BHK-21 cell line (designated as BHKT7) constitutively expressing cytoplasmic bacteriophage T7 RNA polymerase (T7 RNAP) for efficient rescue of infectious FMDV from cloned cDNA. Materials and methods •T7 RNAP gene was inserted in the retroviral vector at first. Then, the retrovirus packaging cell line was co-infected with the recombinant retroviral plasmids and pVSV-G (the pseudotype virus enveloped with G glycoprotein of the vesicular stomatitis virus) was obtained. BHK-21 cell was infected by the pseudotype virus, and T7 RNAP gene was integrated into the chromosome of the BHK-21, resulting in a BHKT7. Results •By using retroviral genetransfer technology, the T7 RNAP gene was integrated into the chromosome of BHK-21. T7 RNAP was constitutively expressed in cytoplasm of BHKT7 (Fig1). The transcriptional activity in the different passage-time BHKT7 was confirmed by detection of expression level of EGFP reporter gene under T7 promoter (Fig2). The different passage-time BHKT7 cell line was then directly transfected with linearized full-length cDNA of FMDV and infectious FMDV was efficiently recovered from the cDNA. The recued FMDV recovered from transfected BHKT7 cells was designated as rFMDV.
•A cell line expressing T7 RNAP was developed using transient expressional vector or vaccinia virus. To our best knowledge, we firstly utilized retroviral gene transfer technology to integrate T7 RNAP into the cellular chromosome. As a result, T7 RNAP was constitutively expressed even cells was passaged a few times.
•Our data show that the BHKT7 cell line can be used as T7 polymerase donor for recovery of infectious RNA virus directly from full-length cDNAs.
BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心 电话：+86-010-53513060 网址：www.biovector.net [Supplier来源] http://www.biovector.net
Introduction •Reverse-genetics systems for the rescue of recombinant FMDV have proven to be of great value for foot-and-mouth disease virus (FMDV) research and vaccine development. The reverse-genetics techniques based on in vitro transcription system or similar techniques were successfully developed for recovery of FMDV O, A, SAT1and SAT2. However, these reverse genetics systems has low efficiency of virus recovery. To overcome this, using retroviral gene transfer technology, we developed a stable BHK-21 cell line (designated as BHKT7) constitutively expressing cytoplasmic bacteriophage T7 RNA polymerase (T7 RNAP) for efficient rescue of infectious FMDV from cloned cDNA. Materials and methods •T7 RNAP gene was inserted in the retroviral vector at first. Then, the retrovirus packaging cell line was co-infected with the recombinant retroviral plasmids and pVSV-G (the pseudotype virus enveloped with G glycoprotein of the vesicular stomatitis virus) was obtained. BHK-21 cell was infected by the pseudotype virus, and T7 RNAP gene was integrated into the chromosome of the BHK-21, resulting in a BHKT7. Results •By using retroviral genetransfer technology, the T7 RNAP gene was integrated into the chromosome of BHK-21. T7 RNAP was constitutively expressed in cytoplasm of BHKT7 (Fig1). The transcriptional activity in the different passage-time BHKT7 was confirmed by detection of expression level of EGFP reporter gene under T7 promoter (Fig2). The different passage-time BHKT7 cell line was then directly transfected with linearized full-length cDNA of FMDV and infectious FMDV was efficiently recovered from the cDNA. The recued FMDV recovered from transfected BHKT7 cells was designated as rFMDV.
•A cell line expressing T7 RNAP was developed using transient expressional vector or vaccinia virus. To our best knowledge, we firstly utilized retroviral gene transfer technology to integrate T7 RNAP into the cellular chromosome. As a result, T7 RNAP was constitutively expressed even cells was passaged a few times.
•Our data show that the BHKT7 cell line can be used as T7 polymerase donor for recovery of infectious RNA virus directly from full-length cDNAs.
BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心 电话：+86-010-53513060 网址：www.biovector.net [Supplier来源] http://www.biovector.net
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