人醛糖还原酶基因在酿酒酵母中的诱导表达及活性分析
DOI:
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

国家自然科学基金(30472172)


Induced expression and activity analysis of human aldose reductase gene in Saccharomyces cerevisiae
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    目的 建立人醛糖还原酶(AR)及其与绿色荧光蛋白(GFP)融合表达的酿酒酵母细胞表达株, 检测细胞中目的基因的表达及蛋白活性。方法 将含目的基因AR及融合基因AR∷GFP的重组质粒pYEX-BX转化酵母细胞INVSc1, 得到宿主菌株XAR, XAG及对照菌株YEX。观察酵母细胞生长曲线及GFP荧光信号; Northern blot检测细胞中AR mRNA转录; Western blot检测AR蛋白表达; 荧光法测定AR活性。结果 三菌株生长速率无明显差异; XAG菌株相对荧光强度与生长时间成线性关系; XAR, XAG菌株分别有持续、稳定的目的基因mRNA转录及目的蛋白表达, 并具有AR活性。结论 成功构建了AR基因的酵母表达株, 为进一步将此细胞株应用于研究AR的致病机制及AR抑制剂的初步筛选打下基础。

    Abstract:

    Objective To construct the Saccharomyces cerevisiae cell strains expressing aldose reductase(AR) and AR-green fluorescent protein(AR-GFP) fusion protein, and to detect the target gene expression and the AR enzyme activity. Methods The yeast expression plasmids pYEX-BX inserted with AR and AR::GFP fusion gene were transformed into the yeast strain INVSc1, which were named as XAR and XAG strains respectively. The blank pYEX-BX strain was used as the normal control. The growth curves and the fluorescence were determined in all strains. Northern blot, Western blot and the fluorescent method were used to detect the AR mRNA transcription, AR protein expression, and the AR activity respectively. Results There was no significant difference in the growth rates among three strains. There was a linear relationship between relative fluorescence of the XAG and the growth time. The mRNA transcription and protein expression of AR and AR::GFP were sustainable and stable in XAR and XAG strains. The AR activities in the two strains were both proved by the fluorescent method. Conclusion The yeast expression strain of AR was constructed successfully, which lays basis for its application in the researches on pathogenic mechanism of AR and preliminary screening of new AR inhibitors.

    参考文献
    相似文献
    引证文献
引用本文

翟冰, 叶玲, 刘静, 等.人醛糖还原酶基因在酿酒酵母中的诱导表达及活性分析[J].中华老年多器官疾病杂志,2011,10(4):340~343

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期:
  • 出版日期: