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

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rSj38的表位。方法：用纯化的rSj338/26GST免疫家兔获得抗rSj338/26GST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Western blot免疫识别，核苷酸序列测定分析其获得的表位并与rSj338/26GST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Western blot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rSj338/26GST,26GST，日本血吸虫成虫及虫卵抗原进行Western blot识别。结果：经5轮免疫学筛选后挑取的32个克隆，用Western blot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rSj338/26GST，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，这将为日本血吸虫病的疫苗研究开辟新的途径。

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

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.