Bezerra Marcos Alexandre, Pereira Luiz Miguel, Bononi Aline, Biella Carla Agostino, Baroni Luciana, Pollo-Oliveira Leticia, Yatsuda Ana Patrícia
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
Núcleo de Apoio à Pesquisa em Produtos Naturais e Sintéticos, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Parasitol Int. 2017 Apr;66(2):173-180. doi: 10.1016/j.parint.2017.01.010. Epub 2017 Jan 18.
Neospora caninum is a parasite of the Apicomplexa phylum responsible for abortion and losses of fertility in cattle. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed with the surface proteins known as the SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SAG related or SRS proteins have been a target of intense research due to its immunodominant pattern, exhibiting potential as diagnostic and/or vaccine candidates. The aim of this study was the cloning, expression and characterization of the gene NcSRS67 of N. caninum using a novel designed plasmid. The coding sequence of NcSRS67 (without the signal peptide and the GPI anchor) was cloned and expressed constitutively instead of the ccdB system of pCR-Blunt II-TOPO. The protein was purified in a nickel sepharose column and identified by mass spectrometry (MS/MS). The constitutive expression did not affect the final bacterial growth, with a similar OD 600nm compared to the non-transformed strains. The recombinant NcSRS67 was over expressed and the native form was detected by the anti-rNcSRS67 serum on 1D western blot as a single band of approximately 38kDa as predicted. On an in vitro assay, the inhibitory effect of the polyclonal antiserum anti-rNcSRS67 was nearly 20% on adhesion/invasion of host cells. The NcSRS67 native protein was localised on part of the surface of N. caninum tachyzoite when compared to the nucleus by confocal immunofluorescence.
犬新孢子虫是顶复门的一种寄生虫，可导致牛流产和生育能力丧失。作为其细胞内周期的一部分，该寄生虫与靶细胞的首次相互作用是通过称为SRS超家族（表面抗原糖蛋白相关序列）的表面蛋白进行的。由于其免疫显性模式，SAG相关或SRS蛋白一直是深入研究的目标，具有作为诊断和/或疫苗候选物的潜力。本研究的目的是使用新设计的质粒对犬新孢子虫的NcSRS67基因进行克隆、表达和表征。克隆并组成型表达了NcSRS67的编码序列（无信号肽和GPI锚），而不是pCR-Blunt II-TOPO的ccdB系统。该蛋白在镍琼脂糖柱上纯化，并通过质谱（MS/MS）进行鉴定。组成型表达不影响最终的细菌生长，与未转化菌株相比，OD 600nm相似。重组NcSRS67过量表达，在一维蛋白质免疫印迹上，抗rNcSRS67血清检测到天然形式为一条约38kDa的单带，与预测一致。在体外试验中，多克隆抗血清抗rNcSRS67对宿主细胞黏附/侵袭的抑制作用接近20%。通过共聚焦免疫荧光将NcSRS67天然蛋白与细胞核相比，定位在犬新孢子虫速殖子部分表面。
