酪氨酸羟化酶重组腺病毒在293细胞中的表达及体外活性测定

Recombinant adenovirus-mediated expression of tyrosine hydroxylase in 293 cells and activity assay of the expressed protein in vitro by capillary electrophoresis

目的构建一种能够携带酪氨酸羟化酶(TH)进入帕金森氏病动物模型体内的重组腺病毒,该病毒能够使TH基因转录并且能够翻译成具有活性的功能蛋白。方法将TH基因插入穿梭质粒,重组穿梭质粒与腺病毒基因组在BJ-5183中进行同源重组,将重组成功的腺病毒基因组转染293细胞,使其在293细胞中包装出重组腺病毒,RT-PCR和免疫荧光检测目的基因的转录和表达。利用毛细管电泳法检测L-DOPA的生成。结果成功构建能够表达TH基因的重组腺病毒,RT-PCR能够扩增目的插入片段,毛细管电泳能够检测到L-DOPA的生成。结论该重组腺病毒能够在体外很好地表达具有活性的酪氨酸羟化酶,为利用该基因对帕金森氏病动物模型进行基因治疗打下基础。

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.Conclusion 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.