Cloning and characterization of Plasmodium vivax thioredoxin peroxidase-1
Cloning and characterization of Plasmodium vivax thioredoxin peroxidase-1
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DOI：
10.1007/s00436-012-2864-3
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发表时间：
2012-08-01
影响因子：
2
通讯作者：
Kawazu, Shin-ichiro
中科院分区：
文献类型：
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作者：
Hakimi, Hassan;Asada, Masahito;Kawazu, Shin-ichiro
血红蛋白消化和宿主免疫系统产生的活性氧可能对疟疾寄生虫产生不利影响。为了保护自己，疟疾寄生虫高度依赖抗氧化酶，包括超氧化物歧化酶和依赖硫氧还蛋白的过氧化物酶。迄今为止，已经在恶性疟原虫中发现了几种硫氧还蛋白过氧化物酶（TPx），但间日疟原虫中尚未发现TPx。利用PCR扩增了间日疟原虫硫氧还蛋白过氧化物酶-1 （PvTPx-1）基因编码的完整序列，并进行了克隆。利用重组PvTPx-1 （rPvTPx-1）在小鼠体内产生多克隆抗体，将该酶免疫定位于疟原虫。采用混合功能氧化法评价rPvTPx-1的抗氧化活性。PvTPx-1在氨基酸序列的50和170位有两个保守的半胱氨酸残基，在非还原条件下形成二聚体。采用巯基混合功能氧化实验，揭示了rPvTPx-1的抗氧化活性。特异性抗体间接免疫荧光显微镜显示PvTPx-1在疟原虫红细胞期细胞质中呈点状表达。结果提示间日疟原虫利用TPx-1还原和解毒过氧化氢，以维持过氧化氢在宿主体内的氧化还原稳态和增殖。
Reactive oxygen species produced from hemoglobin digestion and the host immune system could have adverse effects on malaria parasites. To protect themselves, malaria parasites are highly dependent on the antioxidant enzymes, including superoxide dismutases and thioredoxin-dependent peroxidases. To date, several thioredoxin peroxidases (TPx) have been characterized in Plasmodium falciparum, but the TPx in Plasmodium vivax has not yet been characterized. The complete sequence of gene coding for thioredoxin peroxidase-1 of P. vivax (PvTPx-1) was amplified by PCR and cloned. Using the recombinant PvTPx-1 (rPvTPx-1), polyclonal antibody was produced in mice for immunolocalization of the enzyme in the parasite. The antioxidant activity of rPvTPx-1 was evaluated by mixed-function oxidation assay. PvTPx-1 has two conserved cysteine residues in the amino acid sequence at the positions 50 and 170 which formed a dimer under a non-reducing condition. Using a thiol mixed-function oxidation assay, the antioxidant activity of rPvTPx-1 was revealed. Indirect immunofluorescence microscopy with the specific antibody indicated that PvTPx-1 was expressed in the cytoplasm of the erythrocytic stage of the parasite in a dots-like pattern. The results suggest that P. vivax uses TPx-1 to reduce and detoxify hydrogen peroxides in order to maintain their redox homeostasis and proliferation in the host body.