Study for NTPase from the newly recognized parasitic protozoan Neospora caninum

我们已经鉴定了犬新孢子虫速殖子细胞提取物中的I型核苷三磷酸水解酶（NTH; EC 3.6.1.3）活性，以前认为其仅限于刚地弓形虫强毒株。对Nc-1株的全基因cDNA序列分析表明，NcNTR基因与弓形虫NTPases的同源性约为69%，与NTPase-I同工酶的同源性最高。基因组DNA的Southern印迹分析和来自Nc-1菌株的两个独立的NcNTR克隆的序列分析揭示了多个基因的存在，其中至少两个是转录的。底物特异性和Km值为ATP和ADP水解重组或部分纯化的天然NcNTARY是相同的I型同工酶（NT Pase-I）中发现的I型强毒株的刚地弓形虫。NcNTR在大肠杆菌中以包涵体形式表达，复性后纯化。测试纯化的NcNTR作为抗原用于免疫活性人中急性犬奈瑟氏菌感染的血清学诊断的有用性。通过使用重组NcNTR并比较酶联免疫吸附测定（ELISA）法和Sabin-Feldman染料试验滴度来进行测试。在染料试验阳性血清中，复性重组NcNTR的总阳性率很低。该重组NcNT抗体有一定的阳性血清，但这些血清不与其他犬型奈瑟氏菌抗原反应，而与弓形虫抗原反应，表明它们是弓形虫病血清。结果表明，重组NcNTR可作为犬奈瑟氏菌感染的血清学诊断抗原。

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.