CAJ | 학술논문

应用免疫蛋白质组研究方法筛选、鉴定日本血吸虫尾蚴、童虫可溶性抗原蛋白质。方法日本血吸虫尾蚴可溶性粗抗原(SCAP)、童虫可溶性粗抗原(SLAP)分别用双向凝胶电泳(2-DE)分离蛋白质，每样本同时制3块胶，1块胶进行银染，2块胶通过电转印后再分别用感染兔和正常兔血清作Western印迹分析，确定特异性阳性反应点，再从相应银染胶图上找到匹配的抗原蛋白质点；用MALDI-TOF/TOF串联质谱鉴定抗原蛋白质。结果SCAP、SLAP与感染兔血清免疫反应分别获得94个和68个阳性点，在相应的银染胶图上分别获得33个和31个匹配蛋白质点；用MALDI-TOF/TOF串联质谱鉴定和NCBI数据库检索，鉴定成功率分别为100.0％(20/20)和68.2％(15/22)。已获鉴定的SCAP抗原中蛋白酶占62.5％(10/16)，SLAP抗原中蛋白酶占36.4％(4/11);2个抗原蛋白为SCAP和SLAP共有。结论2-DE能有效地分离日本血吸虫可溶性抗原蛋白质，2-D Western印迹法能较好地筛选特异性抗原；2-D Western印迹法阳性点与2-DE胶图蛋白质点匹配率低，低丰度的抗原蛋白质易被漏检；尾蚴和童虫的抗原蛋白质组差异较大。
응용면역단백질조연구방법사선、감정일본혈흡충미유、동충가용성항원단백질。방법일본혈흡충미유가용성조항원(SCAP)、동충가용성조항원(SLAP)분별용쌍향응효전영(2-DE)분리단백질，매양본동시제3괴효，1괴효진행은염，2괴효통과전전인후재분별용감염토화정상토혈청작Western인적분석，학정특이성양성반응점，재종상응은염효도상조도필배적항원단백질점；용MALDI-TOF/TOF천련질보감정항원단백질。결과SCAP、SLAP여감염토혈청면역반응분별획득94개화68개양성점，재상응적은염효도상분별획득33개화31개필배단백질점；용MALDI-TOF/TOF천련질보감정화NCBI수거고검색，감정성공솔분별위100.0％(20/20)화68.2％(15/22)。이획감정적SCAP항원중단백매점62.5％(10/16)，SLAP항원중단백매점36.4％(4/11);2개항원단백위SCAP화SLAP공유。결론2-DE능유효지분리일본혈흡충가용성항원단백질，2-D Western인적법능교호지사선특이성항원；2-D Western인적법양성점여2-DE효도단백질점필배솔저，저봉도적항원단백질역피루검；미유화동충적항원단백질조차이교대。
Objective To screen and identify specific antigenic proteins of Schistosoma japonicum cercariae and schistosomula using immunproteomics approaches. Methods Soluble antigenic proteins of Schistosoma japonicun cercarie (SCAP) and 15 days lung-stage schistosomulum (SLAP) were separated by two-dimensional electrophoresis (2-DE). For each sample, three gels were run in parallel with one gel for silver stain and the other two gels for Western blot using Schistosoma japonicum infected rabbit sera and normal rabbit sera separately. The specific antigenic protein spots were determined on the membrane of Western blot. The matched antigenic protein spots on the sliver stained gels were subsequently analysed by MALDI-TOF/TOF-MS/MS respectively. Results 94 and 68 positive spots were visualised respectively on the membranes of 2-D Western blot of SCAP and SLAP, incubated with Schistosoma japonicum infected rabbit sera. There were 33 and 31 precisely matched protein spots on the corresponding sliver stained gels of SCAP and SLAP, separately. All SCAP protein spots were identified successfully with MALDI-TOF/TOF-MS/MS and NCBI database retrieval while 68.2％ (15/22) of SLAP were confirmed. 62.5％ of antigenic proteins of SCAP were protease and that of SLAP was 36.4％. Two antigenic proteins existed both in SCLP and SLAP. Conclusions 2-DE could efficiently separate proteins of SCAP and SLAP and 2-D Western blot could screen specific antigens very well, but the matching rate between positive spots on 2-D Western blot and protein spots on 2-DE silver stained gels were low, and low-abundant antigenic proteins were easily missed to be detected. The antigenic proteomes were significantly different between SCAP and SLAP.