高通量荧光底物HIV-1蛋白酶模型的建立

Adopting fluorescent substrate to establish the high throughput screening model for HIV-1 protease inhibitors

目的应用荧光方法建立、优化、评价艾滋病病毒1型(HIV-1)蛋白酶抑制剂高通量筛选模型,并用于大规模样品筛选。方法将p100w表达质粒转化E.coli细菌细胞,使其表达包涵体形式的HIV-1蛋白酶融合蛋白,制备纯化自剪切后成熟的HIV-1蛋白酶。通过测定Km值及已知HIV蛋白酶抑制药的IC50对酶进行鉴定。优化酶反应体系,评价模型的可靠性。建立、比较HPLC酶切分析法及荧光底物法两种测定酶活性方法。结果建立了高通量HIV-1蛋白酶筛选模型,经优化减少酶及底物用量,降低了成本。在模型可靠性评价中,Z因子达到0.786以上。在对9000个微生物发酵产物样品的筛选中,验证了方法的可应用性,并获得阳性菌株41个。结论所建立的荧光方法高通量筛选模型稳定可靠,效率比传统的HPLC酶切分析法提高,适用于大规模筛选HIV-1蛋白酶抑制剂。

Objective HIV protease inhibitors are demonstrated to be effective drugs against the HIV infection The purpose of the present study is to establish a high throughput screening model by adopting fluorescent substrate and optimize such a system so as to be able to screen a large number of samples. Methods Plasmid p100w was transferred into E. coil cells. HIV-1 protease fusion protein was expresaed and purified. The purified mature HIV-1 protease was identified by measuring Km value and IC50 of known inhibitor of HIV-1 protease. Two assays for estimating enzyme activity, HPLC and fluorescence methods, were established and compared. After identification of model reliability, the reaction system was optimized to reduce the cost. Result HIV-1 protease with high activity was obtained. The Z factor of the model was 0. 786 or above. 9 000 microorganism product samples were screened and 41 of them showed positive effect. Conclusions The fluot method of HIV-1 pmtease model is demonstrated reliable and stable. Efficiency of this model is much higher than HPLC assay. A vast number of samples can be screened conveniently, swiftly and sensitively.