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《Chongqing Medicine》 2008-10
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Construct and identification of the pIRES2-enchanced green flurescent protein expression vector carrying hBDNF gene

XU Zhong-ye1,CHENG Yuan1,ZHU Xiao-feng2,et al.(1.Department of Neurosurgery,Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China;2.Neuro Science Research Institul Jiamusi University,Jianmusi 154003,China;3.Department of Neuro Surgery,First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)  
Objective To construct the pIRES2-enchanced green flurescent protein expression vector carrying hBDNF gene and detect the expression of recombinant plasmid in 293-T cells.Method To amplify hBDNF gene from normal human genomics by PCR,insert the products of hBDNF PCR products restriction digested with SalⅠ and XhoⅠinto eucaryotic expression vector pIRES2-EGFP with EGFP gene and internal ribosomes entry site(IRES).To indetify recombinant plasmid by enzyme digestion and sequence analysis.To transfect recombinant plasmid into 293-T cells with Lipofectamine 2000 and detect the expression of recombinant plasmid by RT-PCR and fluorescence microscope.Results Results of enzyme digestion and sequence analysis of recombinant plasmid demonstrated that hBNDF gene was correctly inserted into eucaryotic expression vector pIRES2-EGFP.After tranfected into 293-T cells,recombinant plasmid could be expressed successfully in cells.Conclusion EGFP/hBDNF plasmid has been constructed successfully,thus providing an important and convenient tool to treat central nervous system diseases with hBDNF.
【Fund】: 国家自然科学基金资助项目(No.30170963及No.30540062)
【CateGory Index】: Q78
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