Recombinant adenovirus-mediated expression of tyrosine hydroxylase in 293 cells and activity assay of the expressed protein in vitro by capillary electrophoresis / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease.</p><p><b>METHODS</b>The TH gene was inserted into the shuttle plasmid, which was transformed into E.coli BJ-5183 for homologous recombination with the adenovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombinant virus, and the transcription and expression of TH were determined by RT-PCR and immunofluorescence assay, respectively. The production of L-DOPA in the in vitro reaction system was determined using capillary electrophoresis.</p><p><b>RESULTS</b>We have successfully constructed the recombinant adenovirus. The TH mRNA and the corresponding protein were detected by RT-PCR and immunofluoresence assay in 293 cells. L-DOPA was also detected in the reaction system.</p><p><b>CONCLUSION</b>The adenovirus constructed allows efficient expression of bioactive TH protein in vitro, which provides a basis for future study of gene therapy of Parkinson disease in animal models.</p>