CAJ | 학술논문

目的应用经典的免疫蛋白质组技术筛选、鉴定日本血吸虫雄虫抗原蛋白质,以期获得敏感性高、特异性强的诊断抗原.方法从人工感染日本血吸虫的家兔门静脉中收集血吸虫雄性成虫,提取可溶蛋白；采用双向凝胶电泳与Western印迹相结合(2-D Western印迹)筛选特异性抗原蛋白质点；MALDI-TOF/TOF串联质谱鉴定各抗原蛋白质；将筛选出的体被抗原、ER Calcistorin基因用PCR扩增后插入到pIVEX 1.4WG和pET-28a载体上,分别用麦胚无细胞表达体系和原核表达系统获得重组抗原,Western印迹鉴定其抗原性.结果用2-D Western印迹共筛选到36个阳性蛋白质点,其中12个点同时也能与健康兔血清反应；24个特异性抗原蛋白质点经MALDI-TOF/TOF串联质谱鉴定,其中19个(79.2％)在NCBI数据库找到匹配蛋白质数据资料.体被抗原均能在麦胚无细胞体系和原核表达体系中表达；免疫印迹显示麦胚无细胞体系表达的重组蛋白有较好抗原性,检测敏感性90％,特异性100％.结论基于凝胶电泳的免疫蛋白质组学技术用于诊断抗原筛选、鉴定切实可行；与E.coli原核表达体系相比,麦胚无细胞表达体系表达的重组蛋白具有较好的抗原性.
목적 응용경전적면역단백질조기술사선、감정일본혈흡충웅충항원단백질,이기획득민감성고、특이성강적진단항원.방법 종인공감염일본혈흡충적가토문정맥중수집혈흡충웅성성충,제취가용단백；채용쌍향응효전영여Western인적상결합(2-D Western인적)사선특이성항원단백질점；MALDI-TOF/TOF천련질보감정각항원단백질；장사선출적체피항원、ER Calcistorin기인용PCR확증후삽입도pIVEX 1.4WG화pET-28a재체상,분별용맥배무세포표체체계화원핵표체계통획득중조항원,Western인적감정기항원성.결과 용2-D Western인적공사선도36개양성단백질점,기중12개점동시야능여건강토혈청반응；24개특이성항원단백질점경MALDI-TOF/TOF천련질보감정,기중19개(79.2％)재NCBI수거고조도필배단백질수거자료.체피항원균능재맥배무세포체계화원핵표체체계중표체；면역인적현시맥배무세포체계표체적중조단백유교호항원성,검측민감성90％,특이성100％.결론 기우응효전영적면역단백질조학기술용우진단항원사선、감정절실가행；여E.coli원핵표체체계상비,맥배무세포표체체계표체적중조단백구유교호적항원성.
Objective To screen and identify antigens from Schistosoma japoricum male adult worms with immunoproteomics techniques,so as to obtain high sensitivity and specificity diagnostic antigen.Methods Soluble proteins were extracted from the Schistosoma japonicum male adult worms which were collected from infected rabbits.Antigenic proteins were screened and identified with 2-D Western blot and MALDI-TOF/TOF.Genes of tegumental antigen and ER calcistorin were PCR-amplified and inserted into plVEX 1.4 WG and pET-28a vectors and the recombinant antigens were expressed by cell-free and prokaryotic systems respectively.Antigenicities of recombinant proteins were identified with Western blot.Results A total of 36 positive reactive protein spots were detected using 2-D Western blot,among 36 spots 12 spots could also react with serum of healthy rabbit.Tweenty four spots were specifically identified by infected rabbit sera,among these spots,19 spots (79.2％) were successfully identified by MALDI-TOF/TOF and matched with NCBI protein data.The recombinant tegument antigen (reTA) could be expressed in both cell-free and E.coli systems.The sensitivity and specificity of reTA expressed in cell-free system were 90％ and 100％,which were better than those in E.coli system.Conclusions Gel-based immunoproteomics technology is a practical approach for antigen discovery and the antigenicity of the recombinant antigens expressed in cell-free system is higher than that in E.coli system.