流感病毒核酸内切酶抑制剂高通量筛选模型的建立

Establishment of high throughput screening assay targeting the endonuclease activity of influenza virus PAN protein

目的:建立适于高通量筛选的流感病毒核酸内切酶(PAN)抑制剂筛选模型。方法:在大肠杆菌中诱导表达质粒,经蛋白纯化和浓缩,得到目的蛋白PAN。提取噬菌体M13K07的单链环状DNA为底物,应用荧光定量方法建立PAN抑制剂高通量筛选体系,并对应用虚拟筛选技术挑选的100个样品进行PAN抑制活性筛选。结果:反应体系在底物质量浓度0.2μg.mL-1,pH 9.0,Mn2+浓度1 mmol.L-1,37℃温孵60 min的条件下酶活性最佳。此时Z'因子为0.77,可用于高通量筛选。筛选结果发现7个样品对PAN具有抑制活性。结论:本研究建立的流感病毒核酸内切酶抑制剂高通量筛选体系可用于PAN抑制剂的筛选。

Objective ： To establish a high throughput screening （HTS） assay for the endonuclease activity of influenza virus PAN protein. Methods： The plasmid pET-28a/PAN was constructed and transformed into Esche- richia coli to express PAN which was purified and concentrated next. The single-stranded circular DNA of phage M13K07 was prepared and used as a substrate （ph-DNA） of PAN endonuclease activity. The fluorescence quantita- tive method was applied to establish the HTS assay for PAN inhibitors, and then 100 samples selected by virtual screening strategy were evaluated. Results： PAN endonuclease had high reaction activity under the condition of O. 2 μg· mL-1 ph-DNA substrate, 1 mmol. L-1 Mn2＋ , pH value 9.0, temperature 37 ℃ and reaction time 60 min. Z＇ factor was 0.77, indicating the assay could meet the HTS requirements, and the screening results showed that 7 samples displayed significant inhibitory activity on PAN. Conclusion： In this study, the HTS assay targeting the endonuclease activity of influenza virus PAN protein was established and optimized which was available for the screening of PAN inhibitors.