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目的 利用噬菌体随机肽库技术筛选弓形虫抗原的有效表位 ,并探讨其免疫保护效果。 方法 用纯化的弓形虫免疫兔血清IgG对噬菌体 12肽库进行三轮筛选 ,对获得的日的噬菌体用间接ELISA和Dot -ELISA检测其特异性 ,并对其中的某些克隆进行Western -blot分析和序列测定 ;用混和噬菌体克隆及强阳性表位克隆免疫BABL/c小鼠 ,间接ELISA法测定其特异性抗体滴度 ,攻击感染后观察小鼠存活情况。 结果 经三轮筛选 ,特异性噬菌体得到富集 ,随机挑取 18个克隆经间接ELISA和Dot-ELISA鉴定 ,有 16个克隆能与弓形虫免疫兔血清lgG呈特异性反应。Western -blot分析显示P2克隆能被免抗弓形虫血清所识别 ,具有类似于弓形虫抗原的免疫原性 ,其序列为 5’ -CTTCAGTTGGATCGGGCTCCGTTTTGGAATCAGGGT -3’ ,与GenBank的弓形虫已知序列无一级结构的同源性 ;与原肽库对照组相比 ,P2实验组和P总实验组免疫小鼠均能诱导出较高滴度的IgG抗体 ,抗攻击感染后小鼠的存活率及存活时间也均高于对照组。 结论 利用噬菌体随机肽库技术获得了弓形虫抗原的有效模拟表位 ,这些表位能诱导对弓形虫的部分保护作用。
Objective To screen the effective epitopes of Toxoplasma gondii antigen using phage random peptide library technology and to explore its immunoprotective effect. Methods Rabbit serum IgG was purified from Toxoplasma gondii by three rounds of screening of phage 12 peptide library. The specificity of the obtained phage was assayed by indirect ELISA and Dot-ELISA, and some of the clones were analyzed by Western-blot Bcl - / c mice were immunized with mixed phage clones and strong positive epitope clones. The specific antibody titers were determined by indirect ELISA and the survival of mice was observed after challenge with the infection. Results After three rounds of screening, the specific phages were enriched. 18 clones were randomly selected and identified by indirect ELISA and Dot-ELISA. 16 clones were able to react specifically with lgG from rabbits immunized with Toxoplasma gondii. Western-blot analysis showed that the P2 clone could be recognized by anti-Toxoplasma gondii serum and had immunogenicity similar to that of Toxoplasma gondii antigens. The sequence of the clone was 5’-CTTCAGTTGGATCGGGCTCCGTTTTGGAATCAGGGT-3 ’ Compared with the original peptide library control group, the mice immunized with P2 and P groups showed higher titer of IgG antibody, and the survival rate and survival time Also higher than the control group. Conclusion The effective mimotopes of Toxoplasma gondii antigens were obtained by phage random peptide library technology. These epitopes can induce partial protection against Toxoplasma gondii.