质谱技术鉴定日本血吸虫可溶性虫卵抗原具有早期诊断价值的蛋白分子

Identification of early diagnostic molecules in soluble egg antigen of Schistosoma japonicum by MASS

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 the molecules of soluble egg antigen（SEA） for early diagnosis of Schistosoma japonicum infection by two-dimensional electrophoresis（2-DE）,immunoblotting and liquid chromatography with tandem mass spectrometry（LC-MS/MS）.Methods The 2-DE of SEA was executed through first direction isoelectric focusing（IEF） in immobilized pH gradient gel 3-10（IPG3-10） and second direction SDS-PAGE.The protein dots of SEA on the SDS-PAGE gel were transferred to nitrocellulose membrane.These nitrocellulose membranes were responded to the sera of healthy,sera of mice at 1 week,2 weeks and 6 weeks post-infection respectively,then the membrane color was developed with the second antibody of HRP labeled goat anti-mouse IgG conjugate and substrate DAB.The protein dots recognized by sera of mice in the early stage of schistosome infection were identified by LC-MS/MS.Results After matching and analyzing the Western blot patterns of SEA responding to acute infection sera（1 week and 2 weeks post-infection）,chronic infection sera（6 weeks post-infection） and healthy sera by PDQuest 1.0 software,two protein dots were found to be recognized by sera of mice at 1 week,2 weeks and 6 weeks post-infection,and three protein dots were only recognized by the sera of mice at 6 weeks post-infection,no protein dot was recognized by healthy mouse sera.The data of LC-MS/MS showed that the two protein molecules recognized by the sera of mice with schistosome infection in the early stage were heat shock protein 70（HSP70） and 78 kDa glucose-regulated protein（Grp78/Bip） respectively.Conclusion The results of this study preliminarily indicate that HSP70 and Grp78 in SEA have early diagnostic value for S.japonicum infection.