Study for NTPase from the newly recognized parasitic protozoan Neospora caninum

我们已经在犬新孢子虫速殖子的细胞提取物中鉴定出I型核苷三磷酸水解酶（NTPase; EC 3.6.1.3）的活性，以前认为这种活性仅限于刚地弓形虫的毒力菌株。对Nc-1菌株的完整cDNA序列分析表明，NcNTPase基因与弓形虫NTPase的同源性约为69%，与NTPase- i同工酶最为相似。对Nc-1菌株的两个独立的NcNTPase克隆进行基因组DNA的Southern blot分析和序列分析，发现存在多个基因，其中至少有两个基因被转录。重组或部分纯化的天然NcNTPase对ATP和ADP水解的底物特异性和Km值与弓形虫I型毒力菌株中发现的I型同工酶（NT Pase-I）相同。NcNTPase以包涵体的形式在大肠杆菌中表达，并在重折叠后纯化。纯化的NcNTPase作为抗原在免疫功能正常的人血清诊断急性犬犬感染的有效性进行了测试。采用重组NcNTPase进行检测，并将酶联免疫吸附试验（ELISA）的吸光度与Sabin-Feldman染色试验滴度进行比较。重组NcNTPase在染料试验阳性血清中的总阳性率很低。重组NcNTPase有部分阳性血清，但这些血清对其他犬弓形虫抗原无反应，而对弓形虫抗原有反应，表明它们是弓形虫病血清。结果表明，重组NcNTPase是一种有用的血清诊断犬链球菌感染的抗原。

We have identified type I nucleoside triphosphate hydrolase (NTPase ; EC 3.6.1.3) activity, previously thought to be restricted to the virulent strains of Toxoplasma gondii, in the cell extracts of Neospora caninum tachyzoites. Sequence analysis of a complete cDNA from Nc-1 strain indicated that the N.caninum NTPase gene (NcNTPase) shared approximately 69% identity to the NTPases of T.gondii and is most similar to the NTPase-I isozyme. Southern blot analysis of genomic DNA and sequence analysis of two independent NcNTPase clones from Nc-1 strain revealed the presence of multiple genes, at least two of which are transcribed. Substrate specificity and Km values for ATP and ADP hydrolysis for recombinant or partially-purified native NcNTPase were the same as the type-I isozyme (NT Pase-I) found in type I virulent strains of T.gondii. NcNTPase was expressed in Escherichiacoli as inclusion bodies and purified after refolding. The purified NcNTPase was tested for their usefulness as antigen for the serodiagnosis of acute N.caninum infection in immunocompetent humans. The test was conducted by using the recombinant NcNTPase and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. The total positive rate in dye test positive sera was very low for refolded recombinant NcNTPase. There were some positive sera for this recombinant NcNTPase, however, these sera did not react to other N.canium antigens and reacted to T.gondii antigens, indicating that they were toxoplasmosis sera. The data suggested that the recombinant NcNTPase was an useful antigen for serodiagnosis of N.caninum infection.