用噬菌体肽库筛选重组日本血吸虫线粒体相关蛋白的表位  

Identification of mitochondria epitope-related protein of Schistosoma japonicum with phage-displayed random peptide library

作　　者：胡雪梅[1] 张兆松[2] 吴海玮[2] 李春林[2] 苏川[2] 季旻珺[2] 王诗宁 王勇[2] 吴观陵[2] 

机构地区：[1]山东滨州医学院免疫学教研室 [2]南京医科大学分子生物学研究所,分子免疫寄生虫学研究室

出　　处：《中华微生物学和免疫学杂志》2002年第2期180-184,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 3980 0 316 )

摘　　要：目的　筛选和鉴定重组的日本血吸虫 (中国大陆株 )线粒体相关蛋白rSj338的表位。方法　用纯化的rSj338 2 6GST免疫家兔获得抗rSj338 2 6GST的多克隆抗体IgG ,将抗体进一步纯化 ,获得抗rSj338单特异多克隆抗体IgG。用纯化抗rSj338抗体对噬菌体 12肽库进行 5轮免疫学筛选 ,挑取克隆 ;采用Westernblot免疫识别 ,核苷酸序列测定分析其获得的表位并与rSj338 2 6GST进行同源性比较。将获得的不同表位的阳性克隆分别免疫小鼠 ,并采用Westernblot及dot ELISA方法筛选能刺激小鼠产生较高滴度抗rSj338抗体的阳性克隆。并将阳性噬菌体免疫的小鼠血清对纯化的rSj338 2 6GST、2 6GST、日本血吸虫成虫及虫卵抗原进行Westernblot识别。结果　经 5轮免疫学筛选后挑取的 32个克隆 ,用Westernblot方法 30个克隆能被抗rSj338抗体识别。核苷酸序列分析发现共有 11种表位 ,与rSj338无一级结构的同源性。经动物免疫初步实验筛选 ,共获得 4个免疫原性较强的阳性克隆 ,其免疫鼠血清均可识别rSj338 2 6GST、日本血吸虫成虫及虫卵抗原。结论　获得了 4种日本血吸虫中国大陆株线粒体相关蛋白rSj338的表位 ,均为模拟表位 。Objective To identify the epitopes of rSj338 of Schistosoma japonicum. Methods Polyclonal IgG antibody against rSj338/26GST was obtained by immunizing rabbits with purified recombinant protein(rSj338/26GST). The antiserum was further purified to obtain the polyclonal IgG antibody against rSj338 and then used to screen the epitopes of rSj338 with a 12-mer phage display library. Five rounds of biopanning were carried out and thirty-two clones from the fifth round biopanning were randomly selected and identified by Western blot. The positive clones were sequenced and compared their deduced amino acid sequence to that of rSj338. BALB/c mice were immunized with the obtained positive clones and the antibodies were titered. The rSj338/26GST, 26GST, SWAP and SEA were identified with the sera of the mice immunized respectively with the four positive phages. Results Thirty of the thirty-two selected clones could be recognized by anti-rSj338 antibody with Westeren blot. The positive clones include eleven different epitopes and their amino acid sequences showed no homology with those of rSj338. Four epitopes stimulated the mice to produce anti-rSj338 antibodies as analysed by Westeren blot. The rSj338/26GST, SWAP and SEA were recognized by the sera of the mice immunized respectively with the four positive phages. Conclusion Four epitopes of rSj338 were obtained and all are mimic epitopes as potential vaccine candidates.

关 键 词：重组日本血吸虫 噬菌体肽库 筛选 线粒体 相关蛋白 表位 

分 类 号：R346[医药卫生—基础医学]