犬新孢子虫巨噬细胞转移抑制因子（McMIF）克隆表达与其免疫调节作用鉴定

Functional Identification of Neospora Caninum Macrophage Migration Inhibitory Factor Enzyme

关键词:  犬新孢子虫  巨噬细胞转移抑制因子  互变异构酶  氧化还原酶。

Key words:  Neospora caninum  Macrophage migration inhibitory factor  Tautomerase  Oxidoreductase

摘要:

对犬新孢子虫巨噬细胞转移抑制因子(NcMIF)生物学特性进行鉴定,将NcMIF在大肠杆菌中以3种不同的形式进行表达,三种蛋白分别为NcMIF (成熟的蛋白质),NcMIFm (脯氨酸突变为甘氨酸)和NcMIFhis (在N端添加多组氨酸标记),对三种蛋白的多聚体状态、互变异构酶、氧化还原酶及是否与MIF受体(CD74)结合等进行分析。实验结果显示这三种重组的NcMIFs (rNcMIF)均不具备互变异构酶和氧化还原酶活性；甘氨酸替代脯氨酸的重组NcMIF减少了二聚体和三聚物的形成；N端额外添加的HIS标签增加了三聚物的形成；rNcMIF无法与重组人MIF竞争与MIF受体(CD74)结合,这表明CD74不是NcMIF受体结合；免疫荧光染色结果证明NcMIF定位于犬新孢子虫速殖子的顶端。免疫电镜结果进一步显示NcMIF存在于微线体、棒状体、致密颗粒及细胞核中。为进一步分析NcMIF在寄生虫免疫逃逸过程中发挥的作用提供参考。

Abstract:

NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase and oxidoreductase activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of neospora caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. This study provides a reference for further analysis of the role of NcMIF in the immune escape of parasites.