【摘要】 目的:构建编码小鼠PGC1α基因的重组腺病毒载体,为进一步研究其蛋白功能提供工具。方法:穿梭质粒pAdTrack-CMV-PGC1α线性化后与骨架质粒pAdeasy-1在BJ5183菌中完成同源重组,筛选并提取重组正确的腺病毒质粒,PacⅠ线性化,脂质体转染293细胞包装出成熟的具有感染活力的腺病毒颗粒,TCID50法测定病毒滴度,Western bloting检测PGC1α蛋白在293细胞的表达。结果:测序及酶切鉴定表明目的基因正确插入穿梭质粒,读码框未发生移位。重组腺病毒扩增后滴度约3×1010TCID50/ml,能在293细胞中表达PGC1α蛋白。结论:利用细菌内同源重组的方法成功构建了编码小鼠PGC1α基因的重组腺病毒载体,并观察到其蛋白表达。更多还原

【Abstract】 Objective: To construct an adenovirus vector encoding mouse PGC1α gene for further study on its function. Methods: Recombinant shuttle plasmid pAdTrack-CMV-PGC1α was linearized by PmeⅠ and transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdeasy-1 to obtain adenovirus plasmid through homologous recombination. The recombinant adenovirus plasmid was digested by PacⅠ and transfected into HEK293 cells to form adenvirus particles. The virus titer was determined by TCID50 method. The expression of PGC1α in 293 cells was detected by Western blot. Results: The packaging cell showed typical CPE,and the virus titer was about 3×1010 TCID50/ml after amplification. The expression of PGC1α mediated by the recombinant adenovirus vector was verified by Western blot. Conclusion: The adenovirus vector encoding mouse PGC1α gene was successfully constructed,and the protein was observed in 293 cell.更多还原