【作者】 韩者艺；

【导师】 吴开春； 樊代明；

【作者基本信息】 第四军医大学 ， 内科学（消化系病）， 2001， 硕士

【摘要】 肿瘤抗原和表位在诱导机体产生抗肿瘤免疫应答方面起着十分重要的作用。理想的表位疫苗应包含一组优化的CTL表位、Th表位和B细胞表位。因此，筛选并鉴定肿瘤B细胞表位对肿瘤疫苗设计具有重要意义。利用噬菌体表面呈现技术可构建含有大量的具有不同结构和组成信息的噬菌体呈现表位文库；这一技术以其高效的可筛性、有效的放大性和基因型与表型的相连性等几大特色在蛋白质的相互作用研究中占据了重要地位。由于可以用抗体作为筛选的配基通过噬菌体呈现的随机肽库来寻找相应的抗原或抗原表位，而且不需要预先知道抗原分子本身的结构及组成，因此近年来此项技术在肿瘤诊断试剂和疫苗研制方面的应用潜力日益受到重视。 本研究以MGb1-Ab为筛选配基，应用噬菌体随机肽库筛选技术，对胃癌相关抗原MGb1-Ag的模拟表位进行筛选，并检测筛选所得的噬菌体克隆与MGb1-Ab的结合活性，以及阻断MGb1-Ab与胃癌细胞系KATO-Ⅲ结合的能力，一 进而将所得的噬菌体免疫小鼠，检测噬菌体呈现多肽的免疫原性，旨在获得 MGb IAg的多肽模拟表位，为胃癌表位疫苗的设计提供依据。 本研究共进行三轮生物淘洗，滴定每轮产出噬菌体的滴度，计算噬菌体 的产率。结果显示，噬菌体的产率富集了约200倍。 经ELISA初筛，从第三轮淘洗的噬菌体中挑取10个克隆，经DNA测序 后推导出呈现随机肽的氨基酸序列。依据氨基酸序列同源程度，得到保守的 表位信息，结果提示：H（X）Q、LXS是出现频率较高的表位基序。 采用ELISA法检测阳性噬菌体呈现多肽与MGbl个 的结合活性，结果显 示阳性噬菌体呈现多肽均能与 MGb IAb发生特异性结合反应，而与无关单抗 的结合较弱。阻断实验的结果表明阳性噬菌体呈现多肽均能部分抑制 MGb IAb与 KATO—Ill细胞的结合，抑制率为 20％－40％。 用阳性噬菌体呈现多肽兔疫Babl八小鼠，研究了噬菌体呈现模拟表位 的免疫原性。经ELISA和 FACS检测，得到一株噬菌体呈现多肽，其诱导的 免疫血清可与胃癌细胞KATO｛ 发生特异性结合反应，为胃癌多肽疫苗的设 计奠定了初步基础。更多还原

【Abstract】 Tumor antigens and epitopes play important roles in inducing immune responses against tumor. The ideal epitope vaccine includes a group of optimized CTL epitopes, Th epitopes and B-cell epitopes. Selection and identification of B-cell epitopes are important for tumor vaccine designing. Filamentous bacteriophages have been used extensively in recent years for the display on their surface of large repertoires of peptides. Phage display system has many advantages: efficacy of biopanning, the ability for amplification and the linkage of the displayed peptide with its encoding DNA sequence within the phage particle, enabling quick and simple elucidation of binding sequences. Many papers have shown that it is possible to select phage display peptides that mimic the original characteristic of antigen binding to specific antibodies, without previous knowledge of the antigen structure. In this respect, phage display technology is well established as an important experimental approach in the development of -5- novel vaccines and drugs. In the present study, to select the possible mimic epitope of gastric cancer, a random phage display peptide library constructed on pill was biopanned using MGb 1 -Ab as selective molecule. After three biopanning procedures , there was remarkable enrichment for the titer of bound phages in each biopanning procedure. Then, 10 phage clones were selected from the third biopannirtg with ELISA. Single chains of DNA were sequenced and amino acid sequences were deduced. According to the homology of amino acid sequences of displayed peptides, preserved epitope information was obtained. Most of the positive phage dons had motifs of H(x)Q or L(x)S. ELISA showed that the positive phage clones had activity of specific binding to MGbI-Ab and could inhibit the binding of MGbl-Ab to gastric cancer cell KATO桰Il. After immunization of Babl/c mice with different purified positive phage display peptides, ISA and FACS were conducted. The results showed that one phage display peptide could induce specific serum which could bind to gastric cancer cell KIATO桰II.更多还原