应用生物素标记与蛋白质组学方法分离鉴定弓形虫表面蛋白和分泌蛋白

Separation and Identification of the Surface and Secreted Proteins in Toxoplasma gondii by Biotin Labeling and Proteomics Methods

目的分离和鉴定刚地弓形虫RH株速殖子表面蛋白和分泌蛋白。方法在绿猴肾Vero细胞株中体外培养弓形虫RH株速殖子,利用滤膜和Percoll细胞分离液分离纯化速殖子。将纯化的速殖子用生物素标记后,裂解虫体,采用亲和素凝胶珠分离生物素标记的弓形虫表面蛋白和分泌蛋白。对分离得到的蛋白进行浓缩和密度梯度凝胶电泳。将电泳条带分段切胶,回收蛋白后,酶切,应用液相色谱和两级质谱联用技术(LC-MS/MS)进行蛋白鉴定。结果共鉴定出785种弓形虫蛋白,其中81种为基因组注释的表面或分泌蛋白,占10.3%。785种蛋白中,检测到同一种蛋白多肽的次数(PSM值)>10的蛋白有65种,其中43种为基因组注释的表面蛋白或分泌蛋白,占66%,余22种为预测的未知蛋白。结论应用生物素标记和亲和素层析分离了弓形虫表面蛋白和分泌蛋白,并初步鉴定了一批潜在的弓形虫新表面蛋白和分泌蛋白。

Objective To separate and identify the surface proteins and secreted proteins of Toxoplasma gondii taehyzoties of RH strain. Methods T. gondii tachyzoties were cultured in Veto cells, and purified by filtration and Percoll cell separation solution. The biotin-labeled tachyzoites were ]ysed, and the surface and secreted proteins were separated by NeutrAvidin agarose beads. After condensation and SDS-PAGE, the protein were collected, digested and identified by LC/MS-MS. Results A total of 785 T. gondii proteins were identified, 81 （10.3%） of which were originally annotated as the surface or secreted proteins. Among the highly-expressed （PSM〉10） 65 proteins, 43 （66%） were originally annotated as surface or secreted proteins, while the others were predicted unknown proteins. Conclusion The surface and secreted proteins of T. gondii are separated by biotin labeling and avidin chromatography, among which some potential new surface or secreted proteins of T. gondii are identified.