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目的采用二维电泳、免疫印迹法和质谱技术鉴定日本血吸虫可溶性虫卵抗原(SEA)成分中具有早期诊断价值的抗原分子。方法通过等电聚焦以及十二烷基磺酸钠聚丙烯酰胺凝胶电泳对SEA进行二维电泳,将胶上蛋白质斑点转移到硝酸纤维素膜上,再分别与日本血吸虫感染前、后不同时间点的小鼠血清反应,以辣根过氧化物酶标记的羊抗鼠IgG作为二抗,进行免疫印迹试验,寻找SEA中能与血吸虫感染早期血清反应的蛋白斑点。通过高效液相色谱串联质谱(LC MS/MS)分析鉴定其蛋白成分。结果对血吸虫感染1、2、6周以及未感染健康小鼠血清识别SEA的二维电泳免疫印迹图谱进行匹配分析发现,SEA中有2个蛋白斑点能被血吸虫感染后1、2、6周小鼠血清识别,另有3个蛋白斑点仅被血吸虫感染后6周小鼠血清识别。经LC MS/MS鉴定,2个被血吸虫感染早期血清识别的SEA蛋白分子分别为热休克蛋白(HSP70)和葡萄糖调节蛋白(Grp78/Bip)。结论 SEA中HSP70和Grp78/Bip可能具有血吸虫感染早期诊断价值。
Objective To identify antigen molecules with early diagnostic value in soluble antigen-of-swine antigen (SEA) of Schistosoma japonicum by two-dimensional electrophoresis, Western blot and mass spectrometry. Methods Two-dimensional electrophoresis of SEA was carried out by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the protein spots on the gel were transferred to nitrocellulose membrane and then incubated with SEA at different time before and after infection Point mouse serum reaction with horseradish peroxidase-labeled goat anti-mouse IgG as a secondary antibody, Western blotting test to find SEA can react with early schistosome serum protein spots. The protein components were identified by high performance liquid chromatography tandem mass spectrometry (LC MS / MS) analysis. Results Two-dimensional electrophoresis Western blot analysis of SEA in sera of uninfected healthy mice at 1, 2, 6 weeks after infection of schistosomiasis showed that there were 2 spots in SEA that could be spotted 1, 2, 6 weeks after schistosome infection Mouse serum identification, and another 3 protein spots were only recognized by schistosome infection 6 weeks after the serum of mice. By LC MS / MS, two SEA proteins recognized by schistosome-infected early serum were heat shock protein (HSP70) and glucose regulatory protein (Grp78 / Bip). Conclusion HSP70 and Grp78 / Bip in SEA may have early diagnostic value of schistosomiasis infection.