[Recombinant adenovirus-mediated expression of tyrosine hydroxylase in 293 cells and activity assay of the expressed protein in vitro by capillary electrophoresis].
OBJECTIVE
To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Wen-ju
WJ
Institute of Medical Biology, Chinese Academy of Medical Science, Peking Union Medical College, Kunming 650118, China. wangwenju225@sina.com
Sun
Mao-sheng
MS
Yan
Min
M
Xie
Tian-hong
TH
Zhang
Guang-ming
GM
Li
Hong-jun
HJ
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007980:Levodopa / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D010300:Parkinson Disease / Q000628:therapy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D007980:Levodopa; D014446:Tyrosine 3-Monooxygenase