分子信标方法研究HIV-1整合酶3'加工反应动力学  

Kinetic Study of The HIV-1 Integrase 3′-processing Reaction Using a Molecular Beacons Based Assay

作　　者：何红秋[1,2] 刘斌[1] 陈慰祖[1] 王存新[1] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]重庆市科学技术研究院重庆生物医药与器械研究中心,重庆401123

出　　处：《中国生物工程杂志》2012年第2期76-81,共6页China Biotechnology

基　　金：国家自然科学基金(30670497);北京市自然科学基金(5072002);重庆市科技攻关重点项目(CSTC2010AB5101)资助项目

摘　　要：HIV-1整合酶催化病毒cDNA与宿主细胞基因组DNA的整合,是病毒在宿主细胞中增殖的一个关键酶。3'加工是整合酶催化整合过程的第一步反应,3'加工反应动力学的研究对整合酶催化机理研究和以整合酶为靶点的药物研发都具有重要意义。构建了野生型HIV-1整合酶重组质粒,在大肠杆菌BL21中诱导表达,通过对包涵体变性、复性,纯化得到了整合酶蛋白。基于分子信标原理,设计了荧光和淬灭基团标记的DNA底物,通过荧光信号实时监测3'加工反应,对酶反应的动力学性质进行研究。结果表明,纯化的整合酶蛋白具有较高的活性,酶反应表现出显著的Mg2+偏好性。酶动力学研究(Km=131.79 nmol/L,Kcat=0.0042 min-1)表明,该分子信标方法和设计的DNA底物可用于整合酶3'加工反应动力学研究以及酶反应性质的研究。HIV-1 integrase （IN） mediates the integration of viral eDNA into the host genome, which is a key process for viral replication. The 3＇-processing of viral DNA extremities is the first step in the integration process, and kinetic studies on the 3＇-processing reaction will be helpful in mechanism studies of IN and anti-IN drug design. Wild type IN recombinant plasmid was constructed and IN was expressed in E. coli BL21 cell. Subsequently, IN was purified through denaturation and refolding procedure from inclusion bodies. Based on molecular beacons, fluorophore and quencher-labeled DNA substrate was designed and applied in real-time monitoring of 3＇-processing reaction as well as subsequent kinetic study of the reaction. As a result, the purified IN was active, and IN prefered Mg2＋ as the cationic cofactor in the reaction. The studies on its enzymatic kinetics （Km = 131.79 nmol/L and Kcat = 0. 0042 rain -1） elucidate that both the molecular beacons based real-time assay and the designed DNA substrate are suited for use in 3 ＇-processing kinetic study as well as other reaction characters studies.

关 键 词：HIV-1 整合酶分子信标3’加工动力学 

分 类 号：Q557[生物学—生物化学]