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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 ,这将为日本血吸虫病的疫苗研究开辟新的途径
Objective To screen and identify the epitope of mitochondria-associated protein rSj338 of the recombinant Schistosoma japonicum (Chinese strain). Methods Rabbit was immunized with purified rSj338 2 6GST to obtain anti-rSj338 2 6GST polyclonal IgG. The antibody was further purified to obtain anti-rSj338 monospecific polyclonal antibody IgG. The purified anti-rSj338 antibody was used to immunoprecipitate the phage 12 peptide library and the clones were picked out. The protein was identified by Western blot and the nucleotide sequence was used to analyze the epitopes. The homology was compared with rSj338 2 6GST. The positive clones obtained from different epitopes were respectively immunized with the mice and the positive clones that could stimulate the mouse to produce high titer anti-rSj338 antibody were screened by Western blot and dot ELISA. The positive phage-immunized mouse sera were identified by western blot with purified rSj338 2 6GST, 2 6GST, adult worms of Schistosoma japonicum and egg antigen. Results Thirty-two clones picked after 5 rounds of immunological screening were identified by anti-rSj338 antibody using Westernblot method. A total of 11 epitopes were found by nucleotide sequence analysis, showing no primary structure homology with rSj338. A total of 4 positive clones with strong immunogenicity were screened from the preliminary experiments of animal immunization. All the mice immunized with rSj338 2 6GST and adult schistosomiasis japonica could be identified. CONCLUSIONS: Four epitopes of mitochondria-associated protein rSj338 from Chinese mainland Schistosoma japonicum were obtained and all of them were mimotopes. This will open up new avenues for vaccine research on schistosomiasis japonica