| 研究报告 |
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| The expression of gag-pol fusion gene in NIH3T3 and its ldentification |
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Abstract Construction of a fusion expression vector of MoMLV gag-pol and eukaryotic expression of GAG-POL protin. Methods The gag-pol gene of Moloney murine leukemin virus (MoMLV) were amplified by RT-PCR,they were subcloned into eukaryotic expression vector pcDNA4/HisMaxA.The recombinant plasmid pcDNA4/HisMaxA-gag-pol was thus constructed and identified by sequencing and digestion with restriction enzymes. The recombinant plasmid pcDNA4/HisMaxA-gag-pol was transfected into NIH3T3 cells by lipofecamine2000.At 72 hours after tranfection, passage the cells 1:10 into selective medium containing Zeocin for stable expression. The fusion protein gene was identified by RT-PCR and SDS-PAGE. A retroviral vector pMSCV/u6-HDVRZ, carrying HDV ribozyme gene, was transferred into the packaging cell line.The titer of retrovirus was assayed on NIH3T3 cells. The helper virus was tested by both PCR and marker rescue assay. Results A recombinant fusion expression plasmid was successfully constructed.The plasmid expressed GAG-POL protein. The fusion expression vector pcDNA4/HisMaxA-gag-pol can construct targeting retrovirus packaging cell lines.The efficiency and safety of this system in gene transfer might provide an optimal system for hepatocyte gene therpy.
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Received: 25 October 2005
Published: 25 June 2006
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