We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constr...We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.展开更多
Through PCR amplification, 5’ flanking region and partial open reading frame（ORF） of gene of Nile tilapia（Oreochromis niloticus） was cloned by PCR amplification. Sequence analysis showed that no difference was fo...Through PCR amplification, 5’ flanking region and partial open reading frame（ORF） of gene of Nile tilapia（Oreochromis niloticus） was cloned by PCR amplification. Sequence analysis showed that no difference was found in known functional regions. This study was to construct and identify the mammalian expression vector of pEGFP-β-actin and to detect whether it could express in HEK 293T cell line. pEGFP-β-actin was transfected into HEK 293T cells with Lipofectamine 2000. The results showed that correct construction of recombinant pEGFP-β-actin has been shown by restriction enzyme digestion. The expression of gene in HEK 293T cells could be observed under microfluoroscope. pEGFP-β-actin could repress EGFP protein in HEK 293T cells. The results showed that β-actin gene promoter possessed effective transcription activities in eukaryotic cells. The work laid foundations for further study on the gene engineering and autotransgenic tilapia.展开更多
基金supported by the National Programs for High Technology Research and Development of China (2006AA10A204)the Gansu Key Technologies R&D Program(ZGS-052-A41-0006-03)the Programs for Director Fund of Lanzhou Veterinary Research Institute
文摘We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.
基金Supported by China Agriculture Research System(CARS-49)Fujian Seed Industry Innovation and Industrialization(2011FJZY)
文摘Through PCR amplification, 5’ flanking region and partial open reading frame（ORF） of gene of Nile tilapia（Oreochromis niloticus） was cloned by PCR amplification. Sequence analysis showed that no difference was found in known functional regions. This study was to construct and identify the mammalian expression vector of pEGFP-β-actin and to detect whether it could express in HEK 293T cell line. pEGFP-β-actin was transfected into HEK 293T cells with Lipofectamine 2000. The results showed that correct construction of recombinant pEGFP-β-actin has been shown by restriction enzyme digestion. The expression of gene in HEK 293T cells could be observed under microfluoroscope. pEGFP-β-actin could repress EGFP protein in HEK 293T cells. The results showed that β-actin gene promoter possessed effective transcription activities in eukaryotic cells. The work laid foundations for further study on the gene engineering and autotransgenic tilapia.
