Experiment Study On Construction Of The Recombinant Plasmid PEGFP-BDNF And It's Eukaryotic Expression

Objevtive:Amply BDNF eDNA fragment from the total RNA of Hale cell to construct a recombinant pEGFP- BDNF for the gene therapy research of sensorineural deafness.Methods:(1)cloning human's BDNF eDNA:total RNA was extracted from Hale cell.Amplify BDNF eDNA fragment from the total RNA through RT-PCR method using Trizol Kit and cloned into the plasmid pMD18-T Vector.(2)construction of the recombinant plasmid pEGFP- BDNF:BDNF eDNA was inserted into the plasmid EGFP and constructed the plasmid pEGFP- BDNF.The recombinant plasmid was sequenced by Sanger-dideoxy-mediated chain termination.(3)transfection:The recombinant plasmid was transfeeted into Hale cell using Lipofectamine.(4) Expression of the recombinant plasmid pEGFP- BDNF:After 48 hours of transfection, the expression of pEGFP- BDNF in Hale cell was demonstrated though Western blot and fluorescent microscope.Results:The results showed that the hBDNF eDNA was cloned and the recombinant plasmid pEGFP- BDNF was constructed successfully,and the sequence of the transgene was identical to the published sequence.After 48 hours of transfection,the strong green fluorescence was observed in Hale cell.The Hale cells transfected with the pEGFP- BDNF plasmid had the band of the fusion protein: pEGFP- BDNF at 38KD by Western blot.Conclusion:These results indicated that the a recombinant pEGFP- BDNFwas constructed and expressed successfully in in eukaryotic cells.This date provided a basis of BDNF in geng therapy for disease of inner ear.