Li Jun, Zhang Wen-Bao, Loukas Alex, Lin Ren-Yong, Ito Akira, Zhang Li-Hua, Jones Malcolm, McManus Donald P
Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research and The University of Queensland, Brisbane, Queensland 4029, Australia.
Gene. 2004 Feb 4;326:157-65. doi: 10.1016/j.gene.2003.10.027.
A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible.
通过使用感染六钩蚴的小鼠血清池进行免疫筛选，从绵羊株原头蚴cDNA文库中分离出编码细粒棘球绦虫硫氧还蛋白过氧化物酶（EgTPx）的全长cDNA序列。以与谷胱甘肽S-转移酶（GST）融合蛋白形式表达的EgTPx表现出显著的硫醇依赖性过氧化物酶活性，在体外可保护质粒DNA免受金属催化氧化（MCO）的损伤。此外，在体内试验中进一步证实了EgTPx的抗氧化作用，与对照相比，其在BL21细菌细胞中的表达显著提高了细胞对高浓度H2O2的耐受性和存活率。免疫定位研究表明，EgTPx在原头蚴和育囊的所有组织中均有特异性表达。在原头蚴的许多（但不是全部）石灰小体细胞中检测到更高强度的标记。纯化的重组EgTPx蛋白用于筛选重度感染小鼠和确诊包虫感染患者的血清。在蛋白质印迹中，只有一部分血清与EgTPx-GST融合蛋白呈阳性反应，这表明EgTPx可能形成抗体-抗原复合物，或者对EgTPx抗原的反应可能受到免疫调节。重组EgTPx可能有助于筛选特异性抑制剂，这些抑制剂可作为治疗包虫病的新药。此外，鉴于来自不同寄生门类的TPx在系统发育上与宿主TPx分子相距甚远，开发不与宿主TPx反应的抗寄生虫TPx抑制剂可能是可行的。
