Construction and Identification of adenovirus vector encoding mouse PGC1α gene

MA Shi-kun; et al

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Construction and Identification of adenovirus vector encoding mouse PGC1α gene

更新时间：2024-01-17

- Construction and Identification of adenovirus vector encoding mouse PGC1α gene
- Journal of Chongqing Medical University Vol. 34, Issue 1, Pages: 6-8(2009)
- 作者机构：
  
  重庆医科大学附属第一医院内分泌科
- 作者简介：
- 基金信息：
- DOI：
  CLC： R346
- Published：2009，
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[1]马仕坤,钟立,程庆丰,李卫平,青华,李启富.编码小鼠PGC1α基因的重组腺病毒载体构建及鉴定[J].重庆医科大学学报,2009,34(01):6-8.

MA Shi-kun, et al. Construction and Identification of adenovirus vector encoding mouse PGC1α gene. [J]. Journal of Chongqing Medical University 34(1):6-8(2009)
[1]马仕坤,钟立,程庆丰,李卫平,青华,李启富.编码小鼠PGC1α基因的重组腺病毒载体构建及鉴定[J].重庆医科大学学报,2009,34(01):6-8. DOI：

MA Shi-kun, et al. Construction and Identification of adenovirus vector encoding mouse PGC1α gene. [J]. Journal of Chongqing Medical University 34(1):6-8(2009) DOI：

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。

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.

关键词

PGC1α腺病毒载体同源重组

Keywords

PGC1αadenovirus vectorHomologous recombination

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