【作者】 张扬；

【导师】 刘秀梵； 张如宽； 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2001， 硕士

【摘要】 沙门氏菌是一种十分重要的人畜共患病病原体，在全世界范围内广泛存在。脂多糖是沙门氏菌的血清学分类抗原之一，也是重要的毒力因子。由于多糖为非胸腺依赖性抗原，免疫原性较弱，通常需合成多糖并与蛋白载体相连接才能诱发产生再次免疫应答，操作十分烦琐。利用噬菌体展示技术来模拟多糖的抗原表位则可以解决这个问题。 用生物素标记沙门氏菌O9单克隆抗体（3-47-O）作为分子探针，用亲和筛选法从两个九肽噬菌体展示文库（9-aa pⅧ线性库，9-aa pⅧ Cys限制性库）中筛选该单抗所针对的抗原模拟表位。经过三轮的亲和筛选、洗脱、扩增之后，测定每一轮洗脱物及其扩增物的空斑形成单位（pfu）及转导单位（TU），其产出率明显增加，对三轮筛选的洗脱物的扩增物进行DOT-ELISA检测，其阳性值也不断升高。 从第三轮洗脱物中挑取单克隆进行扩增，用DOT-ELISA来选取阳性单克隆，提取DNA进行测序，得到的两个序列：YQKWYLPKS走SHHVRGGGG分别来自于噬菌体gm68和噬菌体gm62。竞争ELISA结果表明，10¹⁰TU的噬菌体gm68对肠炎沙门氏菌与3-47-O的抑制率可以达到65％，而噬菌体gm62的抑制率仅为20％，这表明短肽YQKWYLPKS可以模拟O9抗原的模拟表位，而且优于SHHVRGGGG。 扬州大学硕士学位论文Z 为探究其免疫生物学特性，将噬菌体n 62、gm68和M13KO7免疫ICR ’J’鼠， 通过间接荧光试验、竞争ELISA试验来检测免疫血清 结果表明，噬菌体驯68可 以诱导产生针对Og的抗体，免疫血清与肠炎沙门氏菌、鸡沙门氏菌等D群沙门 氏菌反应，不与鼠伤寒沙门氏菌等其它群内沙门氏菌反应，而噬菌体gm62和 M13KO7却不能诱导出针对Og特异性抗体。测定免疫血清的竟争ELISA结果， 噬菌体n 68的免疫血清可以抑制3叶70与肠炎沙门氏菌结合，而噬菌体n 62 的免疫血清则不能有效抑制，这充分说明九肽YQKWYLPKS是具有免疫原性的 Og抗原模拟表位。 我们首次报道了沙门氏菌多糖抗原的模拟表位：YQKWYLPKS。这为研究沙 门氏菌Og抗原致病作用与免疫保护机理研究提供新方法，且为模拟多糖的多肽疫 苗或编码该模拟表位的DNA疫苗研制奠定基础。更多还原

【Abstract】 Salmonella is a major zoonotic pathogens causing of morbidity and mortality worldwide in both animals and human beings. The lipopolysaccharide (LPS) is one of the major virulent determinants and serotypic antigens of Salmonella. Since polysaccharide is poorly immunogenic due to its T-independent nature, it is necessary to take the conventional and tiresome approach is to synthesize polysaccharide-carrier protein conjugates for inducing T-dependent immune response. A novel alternative way to elicit a T-dependent immune response is by selecting peptides that mimic polysaccharide antigens from a phage display library. We used two phage display libraries consisting of 9-random amino acids inserted in the N-terminal region of the major coat protein pVIII with or without two cysteine flanking the insert of phagemid PC89, named as pVllI-9aa and pVIII-9aa Cys, to screen minotopes by monoclonal antibody (MAb) 3-47-0 which is specific for antigen 09 of Salmonella. After each round of biopanning, the plaque forming units (pfu) and transducing units (TU) of the phage eluates and their amplified samples were titrated. The results of the yields and dot-ELJSA showed that the enrichment of positive signals increased gradually following three rounds of biopanning. 4 The single phage clones were amplified from the third round eluate, and then the specific clones were selected by using dot-ELISA. Two positive clones designated as gm68 and gm62 were identified and amino acid sequence YQKWYLPKS and SIIIIVRGGGG of two oligopeptides, respectively, were deduced after DNA sequencing. The phage clone gm68 blocked the binding of MAb 3-47-0 to antigen 09 of Salmonella enteritidis, and the inhibition rate was higher than 65% by 1010 TU phages in cELISA, while the inhibition rate of gm62 was only 20%. This result showed that peptide YQKWYLPKS displayed by gm68 mimicked polysaccharide of antigen 09, and was better than that of gm62. In order to detennine immunogencity of the selected mimicking peptides, ICR mice were immunized subcutaneously with phage gm68, gm62 and M13K07, respectively, and their sera were tested by indirect immunofluorescent assay (IFA) and competitive ELISA. The results demonstrated showed that gm68 can elicit a substantially high level of antibodies against antigen 09, which can bind to Salmonella spp. of serogroup D including S. enteritidis, S. gallinarum, but cannot react with Salmonella strains of other serogroups, including S. kiel, S. typhimurium, S. bonn, S. give, S. illinois, S. aberdeen, S. urbana. In contrast, the phage clone gm62 and Ml 3K07 did not induce specific antibodies against antigen 09. These results revealed that peptide YQKWYLPKS was an immunogenic minotope of antigen 09 with high specificity. We reported here for the first time an immunogenic peptide YQKWYLPKS mimicking of Salmonella polysaccharides. It would be very useful for the study pathogenesis and immune protection of Salmonella antigen 09 and for developing anti-carbohydrate peptide vaccines or DNA vaccines.更多还原