Kobayashi T, Narabu S, Yanai Y, Hatano Y, Ito A, Imai S, Ike K
Laboratory of Veterinary Parasitology, Department of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan, Musashino, Tokyo 180-8602, Japan.
J Parasitol. 2013 Jun;99(3):453-8. doi: 10.1645/12-65.1. Epub 2012 Dec 17.
Neospora caninum is an Apicomplexan parasite that causes repeated abortion and stillbirth in cattle. The aim of this study was to clone the gene encoding the N. caninum orthologue (NcBAG1) of the Toxoplasma gondii bradyzoite-specific protein TgBAG1 and characterize its expression pattern in the parasite. Isolation of the full-length 684-bp gene revealed that it shared 78.3% sequence similarity with TgBAG1. NcBAG1 encodes a predicted protein of 227 amino acids with 80.3% similarity to TgBAG1. A putative signal peptide sequence and an invariant GVL motif characteristic of small heat-shock proteins were identified in the predicted N. caninum amino acid sequence. We expressed the NcBAG1 gene as a recombinant glutathione S-transferase fusion protein (rNcBAG1) in Escherichia coli and used the purified 60 kDa protein to obtain a monoclonal antibody (Mab). rNcBAG1 reacted to Mabs specific for NcBAG1 and TgBAG1. No reaction between the NcBAG1 Mab and N. caninum tachyzoites was observed. Although the predicted molecular mass of NcBAG1 is 25 kDa, Western blot analysis of parasite lysates using the NcBAG1 Mab revealed a cross-reactive protein of approximately 30 kDa. Additionally, immunofluorescence assays using the tachyzoite-specific Mab for NcSAG1 and the bradyzoite-specific Mab for TgBAG1 or NcSAG4 revealed NcBAG1-specific expression in bradyzoites in cultures exposed to sodium nitroprusside, a reagent that increases the frequency of bradyzoites. Interestingly, the NcBAG1 protein was identified in the cytoplasm of the bradyzoite-stage parasites. This preliminary analysis of the NcBAG1 gene will assist investigations into the role of this protein in N. caninum .
犬新孢子虫是一种顶复门寄生虫，可导致牛反复流产和死产。本研究的目的是克隆编码犬新孢子虫中与刚地弓形虫缓殖子特异性蛋白TgBAG1直系同源的蛋白（NcBAG1）的基因，并对其在该寄生虫中的表达模式进行表征。全长684 bp基因的分离显示，它与TgBAG1的序列相似性为78.3%。NcBAG1编码一个预测的227个氨基酸的蛋白质，与TgBAG1的相似性为80.3%。在预测的犬新孢子虫氨基酸序列中鉴定出一个推定的信号肽序列和一个小热休克蛋白特有的不变GVL基序。我们在大肠杆菌中表达NcBAG1基因作为重组谷胱甘肽S-转移酶融合蛋白（rNcBAG1），并使用纯化的60 kDa蛋白获得单克隆抗体（Mab）。rNcBAG1与针对NcBAG1和TgBAG1的单克隆抗体发生反应。未观察到NcBAG1单克隆抗体与犬新孢子虫速殖子之间的反应。尽管NcBAG1的预测分子量为25 kDa，但使用NcBAG1单克隆抗体对寄生虫裂解物进行的蛋白质印迹分析显示，存在一种约30 kDa的交叉反应蛋白。此外，使用针对NcSAG1的速殖子特异性单克隆抗体和针对TgBAG1或NcSAG4的缓殖子特异性单克隆抗体进行的免疫荧光测定显示，在暴露于硝普钠（一种增加缓殖子频率的试剂）的培养物中，缓殖子中存在NcBAG1特异性表达。有趣的是，在缓殖子阶段寄生虫的细胞质中鉴定出了NcBAG1蛋白。对NcBAG1基因的这一初步分析将有助于研究该蛋白在犬新孢子虫中的作用。
