Application of a yeast two-hybrid based screening system in the identification of amyloid-beta aggregation inhibitors in pharmaceutical plants

应用酵母双杂交筛选系统从药用植物中发现Aβ聚集抑制剂(英文)

The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD therapy. In the present study, we developed a novel screening tool based on the yeast two-hybrid system to screen Aβ aggregation inhibitors. The human Aβ42 peptide cDNA was cloned using assembly PCR and inserted into each of the yeast expression plasmids containing either the GAL4 activation domain （GAL4AD） or the DNA-binding domain （GAL4BD）. Co-transformation of the above plasmids led to the expression of the fusion proteins GAL4AD-Aβ42 and GAL4BD-Aβ42 in the AH 109 yeast strain. The self interaction of Aβ42 fragments reconstructed the GAL4 transcriptor and thus activated the GAL4 responsive transcription of four reporter genes including HIS3, ADE2, lacZ and MEL1. The expression of the reporter genes rendered the multiple auxotrophic yeast cells capable of growing on the synthetic SD media lacking adenine and histidine. Growth arrest was used as a marker for screening Aβ aggregation inhibitors in this system, and the evaluation of Rhodiola species revealed potential resources for the development of Aβ aggregation inhibitors.

研究证据表明,β淀粉样肽即Aβ的自我聚集是阿尔兹海默病(AD)重要的发病因素。因此,Aβ聚集抑制剂被认为是潜在的AD治疗候选药物。在本研究中,我们建立了一个基于酵母双杂交技术的Aβ聚集抑制剂筛选系统。通过采用拼接PCR技术(assembly PCR),克隆了人源Aβ42的cDNA并将其插入到分别含有酵母转录因子GAL4转录激活区(GAL4_(AD))与DNA结合区(GAL4_(BD))的两个表达载体中。通过以上两个载体的共转化实现了两个融合蛋白GAL4_(AD)-Aβ42与GAL4_(BD)-Aβ42在AH109酵母菌株中的共表达。由于Aβ42片段在酵母中的自我相互作用使GAL4转录因子的活性在酵母中得到重建,从而激活了依赖于GAL4活性的四个报告基因HIS3,ADE2,lacZ与MEL1的转录与表达。以上报告基因的正常表达使具有多种营养缺陷表型的AH109酵母获得了在缺乏组氨酸与腺嘌呤的合成选择培养基上正常生长的能力。通过采用生长抑制作为筛选标记,应用本系统对红景天属植物的Aβ聚集抑制活性进行了分析,进而发现本属植物很可能成为Aβ聚集抑制剂发现的重要资源。