[关键词]
[摘要]
目的 建立以正向转录延伸因子(p-TEFb)为靶点的抗HIV药物快速高通量筛选模型,并运用此模型筛选抗艾滋病中药。方法 构建 BD-Tat和AD-CyclinT1融合的酵母双杂交质粒,分别转入AH109和Y187,经接合实验获得二倍体菌株并对其进行毒性检测、自激活实验和报告基因表达检测,建立基于酵母双杂交的筛选模型。结果 运用此系统筛选具有提高机体免疫功效的20种中药,获阳性中药2种,其抗HIV活性已被体外抑制HIV实验证实。结论 该筛选模型可成功用于抗艾滋病化合物和中药的高效筛选,得到的2种阳性中药狗脊和黄连,值得进一步进行干扰HIV Tat和CyclinT1互作的有效成分分离研究。
[Key word]
[Abstract]
Objective To establish a rapid high-throughput screening model with positvive transcription elongation factor (p-TEFb) as target for screening the inhibitors of HIV. Methods Double cross plasmid of BD-Tat and AD-CyclinT1 was established and transformed into yeast AH109 and Y187, respectively. AH109/pGBKT7-Tat was mated with Y187/pGADT7-CyclinT1 and the diploids were subjected to autoactivation, toxicity test, and reporter gene assay. The screening model based on double hybrid system was established. Results Two out of 20 kinds of Chinese materia medica possessing immunity-enhancing effects were identified as primary hits, the anti-HIV activity of these two positive drugs was already demonstrated by other researchers through detecting the suppression effects on HIV duplication in vitro. Conclusion The system established in this paper can be used for rapid high-throughput screening anti-HIV chemicals or herbs. Further research for the obtained two positive Chinese materia medica, Cibotii Rhizoma and Coptidis Rhizom should be carried out to isolate the effective component for disrupting the interaction between HIV Tat and CyclinT1.
[中图分类号]
[基金项目]
河南省重点科技攻关项目(092102310026);河南省高校科技创新团队支持计划资助项目(13IRTSTHN009)