Biochemical and molecular biological studies on DNA polymerase of Toxoplasma gondii

在弓形虫RH株速殖子粗提物中检测到DNA聚合酶活性，并对其进行了鉴定。该酶的沉降系数为6.4S，对应的分子量约为15万。与哺乳动物的阿尔法型DNA聚合酶一样，弓形虫的酶对N-乙基马来酰亚胺敏感，但受到高离子强度的抑制。然而，哺乳动物DNA聚合酶α、β和epsilon的抑制剂aphidiclin不抑制该酶的活性，而哺乳动物DNA聚合酶β和γ的抑制剂ddTTP可抑制该酶的活性。酶活性需要镁离子，较高浓度的K~(2+)对酶有明显的抑制作用。抗人DNA聚合酶α的单抗不与弓形虫酶结合。因此，弓形虫的酶不同于人类的酶，可能是设计弓形虫药物的有用靶点。强毒菌株RH的酶活力显著高于无毒菌株ME49，说明强毒菌株酶活性的提高有助于提高微生物的增殖速度。为了克隆弓形虫DNA聚合酶α基因，合成了含有保守碱基的寡核苷酸探针，并将其应用于弓形虫基因组文库的筛选，获得了1个阳性克隆。对插入的DNA进行分析，发现其大小为15kb，然后将其亚克隆到pUC18中进行测序。为了克隆弓形虫DNA聚合酶Delta基因，用RT-PCR方法扩增出一条cDNA片段，测序证实为真基因。利用该片段构建了弓形虫GT-10基因文库，鉴定出两个克隆，并将其插入pBluescrip中进行测序。

A DNA polymerase activity has been detected and characterized in crude extracts from tachyzoites of virulent RH strain of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6.4 S,corresponding to an approximate molecular weight of 150,000. Like mammalian alpha-type DNA polymerases, the T.gondii enzyme was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerase alpha, delta and epsilon, ant the activity was inhibited by ddTTP which is an inhibitor of mammalian DNA polymerase beta and gamma. Mg^<2+> was required for activity and a higher concentration of K^+ markedly inhibited the activity. Monoclonal antibodies against human DNA polymerase alpha did not bind to the T.gondii enzyme. Thus the T.gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs. The activity of virulent RH strain was siginificantly higher than that of avirulent ME49 strain, suggesting that this increased activity of the enzyme of virulent strain contributes to a faster rate of multiplication of the organisms as aompared with that of avirulent strain. For cloning of DNA polymerase alpha gene of T.gondii, an oligonucleotide probe which included the conserved bases was synthesized and used for screening a T.gondii genomic DNA library in lambda GEM.One positive clone was obtained. The insert DNA was analyzed, found to be 15 kb in size, and then subcloned into pUC18 for sequencing. For cloning of DNA polymerase delta gene of T.gondii, a cDNA fragment was produced by RT-PCR and then sequenced and confirmed as the real gene. A T.gondii cDNA library in lambda gt 10 was screened using this cDNA fragment and two slones were identified and their insert DNAs were subcloned into pBluescript for sequencing.