鸭乙型肝炎病毒复制复合体中内源性DNA聚合酶测定方法的建立及其应用  

ENDOGENOUS DNA POLYMERASE IN DUCK HEPATITIS VIRUS REPLICATIVE COMPLEXES AND ITS INHIBITION

作　　者：郭巨涛[1] 陈鸿珊[1] 

机构地区：[1]中国医学科学院医药生物技术研究所

出　　处：《病毒学报》1990年第3期210-215,共6页Chinese Journal of Virology

基　　金：<七.五>攻关课题

摘　　要：应用~3H-TTP作为放射性掺入底物,建立了鸭乙型肝炎病毒复制复合体(DHBVRCs)内源性DNA聚合酶的测定方法,研究了该酶的生化特性,试验了12种药物对该酶的抑制作用。结果该方法测定的内源性DNA聚合酶为DHBV特异的,其活性依赖于4种dNTP和Mg~ ++的存在。最适Mg^++浓度为20mM。NP-40可刺激其活性。放线菌素D仅能抑制其30％的活性,膦羧基甲酸钠(PFA)、膦羧基乙酸钠(PAA)和苏拉明可抑制此酶,它们的半数抑制浓度分别为8.5μM、860μM和9.25μM。而Ara-AMP等几种药物对该酶无抑制作用。应用放射性掺入电泳自显影法证明,PFA对DHBV RCs中依赖DNA和RNA的DNA聚合酶均有显著的抑制作用。Hepadna viruses have an unique reverse transcription mechanism in their genomic DNA replication which is catalyzed by a DNA-dependent DNA polymerase ( DNAp ) and RNA-dependent DNA polymerase ( reverse transcriptase, RT ) This latter enzyme is an ideal target for anti-hepadna viruses chemotherapy. We have established a method for the endogenous DNA polymerase assay in DHBV replicative complexes ( RCs ) and tested the inhibitory effects of some drugs on it. The results showed that PFA, PAA and suramin are potent inhibitors, their IC50 are8.5, 860 and 9.25μM respectively. Ara-A, Ara-AMP, ACV, AZT, ddT, FMAU, RBV,Gan-ling and refampicin did not inhibit it.This assay can be used to screen anti-HBV drugs. Its advantages are that. ( 1 )it is simple; suitable for the screening of anti-HBV drugs on a large scale.(2) substrate labelled by the isotope of long half life, 3H-TTP, was used, so that the experiment can be done at any time. The results also showed that actinomycin D inhibited only 30% of this endogenous DNA polymerase activity, indicating that the DNA polymerase included both DNAp and RT. In order to differentiate the inhibitory effects of drugs on DNAp and RT, the products of the DNA polymerase reaction on α-32P-dCTP labelled substrate were analysed by agarose gel electropioresis and autoradiography. The result showed that PFA inhibited both DNAp and RT.

关 键 词：乙型肝炎病毒 DNA聚合酶 

分 类 号：R373.21[医药卫生—病原生物学]