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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。
Research evidence suggests that beta-amyloid peptide, Aβ, is an important causative agent of Alzheimer’s disease (AD). Therefore, A [beta] aggregation inhibitors are considered as potential AD therapeutics. In this study, we established a screening system for Aβ aggregation inhibitors based on the yeast two-hybrid technique. The cDNA of human Aβ42 was cloned by insert PCR and inserted into two cDNAs containing the GAL4_ (AD) transcriptional activator (GAL4_ (AD)) and the DNA binding domain (GAL4_ (BD) Expression vector. Co-transformation of the two fusion proteins GAL4_ (AD) -Aβ42 and GAL4_ (BD) -Aβ42 in the AH109 yeast strain was achieved by co-transformation of the above two vectors. Due to the self-interaction of Aβ42 in yeast, the activity of GAL4 transcription factor was reconstructed in yeast and the transcription and expression of four reporter genes HIS3, ADE2, lacZ and MEL1 which depend on GAL4 activity was activated. The normal expression of the above reporter genes gave AH109 yeast with multiple auxotrophic phenotypes the ability to grow normally on synthetic selection medium lacking histidine and adenine. By using growth inhibition as a screening marker, this system was used to analyze the Aβ aggregation inhibitory activity of Rhodiola rosea plants, and then it was found that this plant is likely to be an important resource for Aβ aggregation inhibitor discovery.