抗日本血吸虫噬菌体抗体库的筛选和阳性克隆的鉴定

Screening and Identification of Phage Antibodies from Phage Display Library Against Schistosoma japonicum

目的 研制抗日本血吸虫循环抗原Sj MAg的单链抗体。 方法 用日本血吸虫成虫代谢抗原Sj MAg包板 ,对已构建的抗日本血吸虫噬菌体抗体库进行 3轮富集 ,然后用ELISA法从富集的次级抗体库中筛选阳性克隆 ,最后借助ELISA、SDS PAGE和Western印迹等方法 ,根据阳性克隆表达产物与其它 4种吸虫抗原反应的情况 ,进行特异性鉴定。 结果 从随机挑取的 72个克隆中筛选到阳性克隆 6个 ,经交叉筛选和鉴定最终获得特异性抗日本血吸虫阳性克隆 2个。 结论 用固相富集和ELISA筛选法可以从抗体库中有效地筛选到抗日本血吸虫循环抗原的阳性克隆 ,获得的抗体克隆可用于抗日本血吸虫循环抗原单链抗体的制备。

Objective To obtain single chain variable fragment(ScFv) against the circulating antigen(CAg) from Schistosoma japonicum(Sj). Methods Metabolic antigen of adult worm of Sj (Sj-MAg) was used in the panning of phage library against Schistosoma japonicum. The activity of Sj-MAg-binding phage clones was assayed by ELISA. The specificity of expression products of the positive clones was analyzed by ELISA, SDS-PAGE and Western blotting. Results Seventy-two randomly selected clones were tested for the presence of anti-Sj-MAg ScFvs, 6 clones showed positive. The specificity of these 6 clones was confirmed by binding them to antigens of other four trematodes. Two clones (B04,C24) were found to bind to Sj-MAg but not to any of the antigens of other four trematodes and their expression products were about 31kDa in size. Conclusion ScFv antibodies against the circulating antigens from Schistosoma japonicum Sj-MAg can be selected and manufactured from the antibody library.