抗日本血吸虫噬菌体抗体库的筛选和阳性克隆的测序

Screening and Analysis of Phage Antibodies from Phage Display Library against Schistosoma Japonicum (Sj)

目的 :探索制备抗日本血吸虫循环抗原的单链抗体的新方法。方法 :用日本血吸虫成虫代谢抗原对已构建的抗日本血吸虫噬菌体抗体库进行富集 ,然后用ELISA法从富集的抗体库中筛选阳性克隆 ,然后借助ELISA、SDS PAGE和Westernblot等方法对阳性克隆表达产物进行了特异性鉴定 ,最后对阳性克隆插入片段进行了测序和序列分析。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。测序和序列分析结果也证实 :阳性克隆插入片段推导的氨基酸序列具有典型的抗体可变区特征。结论

Objective: To prepare single chain variable fragment(ScFv) against Schistosoma japonicum circulating antigen(CAg) in a new way. Methods: Metabolic antigen of adult worm of Sj(Sj MAg) was used in panning of phage clones against Schistosoma japonicum . The postive phage clones binding Sj MAg were selected from the panned library by ELISA. The specificity of expression products of the positive clones was identified with ELISA,SDS PAGE and Western blot. DNA sequences of the positive antibody clones were also determined and analyzed with some bioinformatics tools. Results: 72 random clones were tested for the presence of anti Sj MAg ScFvs,and 6 clones showed positive. Two specific clones (B04,C24) were finally obtained through further identification. It was shown that amino acid sequences deduced from DNA sequences of specific clones had typical features of Ig heavy and light chains variable regions. Conclusion: ScFv antibodies against Schistosoma japonicum CAg can be manufactured effectively by the phage antibody library technique.