Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
Parasitology. 2012 Jul;139(8):998-1004. doi: 10.1017/S0031182012000285. Epub 2012 Mar 12.
Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42.9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.
免疫图谱蛋白 1（IMP1）是柔嫩艾美耳球虫中一种新发现的蛋白。它被认为是柔嫩艾美耳球虫的一种潜在疫苗候选物，也是顶复门寄生虫中高度保守的蛋白。虽然在刚地弓形虫基因组中预测了与柔嫩艾美耳球虫 IMP1 同源的 Neospora caninum IMP1（NcIMP1），但它仍未被鉴定和表征。在本研究中，通过从 Nc1 速殖子分离的 RNA 进行 RT-PCR 克隆了编码 NcIMP1 的 cDNA 序列。NcIMP1 由一个 1182bp 的开放阅读框编码，该框编码一个 393 个氨基酸的蛋白质，预测分子量为 42.9kDa。序列分析表明，NcIMP1 氨基酸序列中既没有信号肽也没有跨膜区。然而，预测了几种功能蛋白基序，包括一个 N-豆蔻酰化位点和一个棕榈酰化位点。在大肠杆菌中表达了重组 NcIMP1（rNcIMP1），然后用纯化的 rNcIMP1 在小鼠中制备特异性抗血清。针对 rNcIMP1 的小鼠多克隆抗体识别约 43kDa 的天然 IMP1 蛋白。免疫荧光分析表明，NcIMP1 定位于刚地弓形虫速殖子的膜上。N-豆蔻酰化位点和棕榈酰化位点被发现有助于 NcIMP1 的定位。此外，rNcIMP1 特异性抗体可抑制刚地弓形虫速殖子在体外的细胞入侵。所有结果表明，NcIMP1 可能是刚地弓形虫的一种膜蛋白，可能参与寄生虫入侵。
