弓形虫有效抗原表位的筛选及其对小鼠免疫保护性的研究

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IgC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IgG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’-CTTCAGTTGGATCGGGCTCGGTTTTGGAAT CAGGGT-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睥位，这些表位能诱导对弓形虫的部分保护作用。

Studies on the identification of antigenic epitopes of Toxplasmoa gondii and its protective effect in mice

Objective To screen the mimic antigen epitopes of Toxoplasma gondii from phage display random peptide library and explore their immunoprotection against T. gondii. Methods Phage random peptide library of 12 amino acids was immunoscreened with purified lgG from rabbit sera immunized with T. gondii. Positive clones collected after three rounds of biopanning were detected by ELISA and Dot-ELISA, and some of them were analyzed by western-blot and DNA sequencing. BABL/c mice were immunized with mixed positive phage clones or the selected positive clone. Detected the titer of IgG antibody by ELISA and observed the date of death after challenged with T.gondii. Results The specific phages binding to IgG were enriched after three rounds of biopanning. Among 18 clones randomly selected from the third round biopanning, 16 positive clones were obtained. The results of Western-blot indicated that P2 clone could be recognized by immune rabbit sera and its immune antigenicity resembled to the antigen of T. gondii. The sequence of P2 has been found, to be 5' -CTI' CAG TTG GAT CGG GCT CCG TI'T TGG AAT CAG GGT-3', which showed no homology with the genomic sequences of T. gondii in the Genbank. Antibody lgG titer of the immunized mice with mixed positive phage clones or P2 were much higher than that of the control group, and the survival time of them were also longer than that of the control group. Conclusion The specific antigen peptides of T. gondii were obtained, which could induce partial protection against T. gondii.