CAJ | 학술논문

目的：建立与优化HIV-1整合酶3′端加工抑制剂筛选方法。方法利用荧光共振能量转移原理建立筛选方法,用DNaseⅠ切割底物 DNA确定底物检测波长,在该检测波长下,对缓冲液成分、底物浓度、酶浓度、金属离子浓度等影响整合酶活性的条件进行优化,并用阳性药物雷特格韦和杨梅黄素进行方法验证。结果检测波长为495 nm/525 nm,使用缓冲液1、底物浓度500 nmol·L-1,整合酶浓度1μmol· L-1,镁离子浓度20 mmol · L-1时整合酶3′端加工活性最强。在此反应条件下雷特格韦和杨梅黄素能有效地抑制整合酶3′端加工活性。用所建立的方法筛选到2个有较强抑制整合酶3′端加工活性的抑制剂。结论该文成功建立并优化了HIV-1整合酶3′端加工抑制剂筛选方法。
목적：건립여우화HIV-1정합매3′단가공억제제사선방법。방법이용형광공진능량전이원리건립사선방법,용DNaseⅠ절할저물 DNA학정저물검측파장,재해검측파장하,대완충액성분、저물농도、매농도、금속리자농도등영향정합매활성적조건진행우화,병용양성약물뢰특격위화양매황소진행방법험증。결과검측파장위495 nm/525 nm,사용완충액1、저물농도500 nmol·L-1,정합매농도1μmol· L-1,미리자농도20 mmol · L-1시정합매3′단가공활성최강。재차반응조건하뢰특격위화양매황소능유효지억제정합매3′단가공활성。용소건립적방법사선도2개유교강억제정합매3′단가공활성적억제제。결론해문성공건립병우화료HIV-1정합매3′단가공억제제사선방법。
Aim To establish and optimize a method for screening HIV-1 integrase 3′-processing inhibitor. Methods Fluorescence resonance energy transfer ( FRET) was used to create an assay for screening in-tegrase 3′-processing inhibitors; wavelength was de-fined by DNaseⅠ; factors affecting IN activity were optimized, including buffer composition, substrate con-centration, enzyme concentration, metal ion concentra-tion. Results Integrase 3′-processing optimizing reac-tion conditions were buffer 1 , 500 nmol · L-1 sub-strate, 1 μmol·L-1 integrase, 20mmol·L-1 magne-sium ion. Positive drug raltegravir and myricetin could effectively inhibit integrase 3′-processing activity using this assay. Two integrase 3′-processing inhibitors were screened by this method. Conclusion The method for screening HIV-1 integrase 3′-processing inhibitor is successfully established and optimized.