Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
Pathog Glob Health. 2013 Apr;107(3):116-21. doi: 10.1179/2047773213Y.0000000082.
Neospora caninum is considerd a major cause of abortion in cattle worldwide. The antigenic domain of NcSRS2 in N. caninum is an important surface antigen present in the membrane of this parasite. In the present study, the Pichia pastoris expression system proved to be a useful tool for the production of recombinant protein. The truncated NcSRS2 gene (by removal of the N-terminal hydrophobic sequence), was cloned in the vector pPICZalphaB, and integrated on the genome of the methylotrophic yeast P. pastoris. Subsequently, the NcSRS2 protein was expressed, purified, and characterized using naturally infected cattle sera and Mab 6xhistag. The recombinant protein NcSRS2 was present in the supernatant of the culture, where later it was concentrated and purified using ammonium sulfate (∼100 mg/ml). An indirect immunoenzymatic assay (ELISA) was performed using cattle sera from endemic N. caninum area.
刚地弓形虫被认为是全世界牛流产的主要原因。刚地弓形虫 NcSRS2 的抗原结构域是这种寄生虫膜上的一种重要表面抗原。在本研究中，毕赤酵母表达系统被证明是生产重组蛋白的有用工具。截短的 NcSRS2 基因（去除 N 端疏水性序列）被克隆到载体 pPICZalphaB 中，并整合到甲醇营养型酵母 P. pastoris 的基因组中。随后，使用天然感染牛血清和 Mab 6xhistag 表达、纯化和表征 NcSRS2 蛋白。重组蛋白 NcSRS2 存在于培养物的上清液中，随后使用硫酸铵（约 100mg/ml）进行浓缩和纯化。使用来自刚地弓形虫流行地区的牛血清进行间接酶联免疫吸附试验（ELISA）。
