人泌尿系上皮肿瘤疫苗初步探究.docVIP

人泌尿系上皮肿瘤疫苗初步探究 　【摘要】 目的通过筛选噬菌体随机12肽库获得MUC1糖链抗原模拟表位。方法利用纯化获得的Ma695抗体筛选噬菌体随机12肽库，通过夹心ELISA分析噬菌体克隆，测定阳性克隆DNA序列并进行同源性及氨基酸分析，竞争性抑制实验鉴定噬菌体克隆。结果经3轮筛选，获得了14个阳性克隆，DNA序列分析并推导出氨基酸序列：KHYDPFHHRMPQ，QADTARSVALAG，VPSKPDLHVRSI，MTPIHYWNHNRV。鉴定结果表明4个噬菌体展示肽克隆抑制率均在50%以上。结论所得序列KHYDPFHHRMPQ，QADTARSVALAG，VPSKPDLHVRSI，及MTPIHYWNHNRV模拟了MUC1抗原表位。 【关键词】 MUC1抗原 模拟表位 噬菌体随机12肽库 A study on epithelium tumor vaccine in urologic syste Abstract ObjectiveTo identify and characterize the mimic epitope of MUC1 from phage display random 12 peptide library. Methods The purified Ma695 antibody was used to screen in phage display random 12 peptide library. The positive clones were identified by sandwich ELISA， competitive inhibition assay. The homologous comparison of amino acid sequences of positive clones was conducted through searching the protein sequence databank. The digestion analyses of amino acid sequences were also conducted by software. Results 14 positive clones were acquired after 3 rounds of screening. Amino acid sequences deduced from DNA sequences showed four different sequences as KHYDPFHHRMPQ， QADTARSVALAG， VPSKPDLHVRSI， MTPIHYWNHNRV. The inhibitory assay showed that the 4 mimic epitope peptides displaying on the phage surface could effectively inhibit the combination of antibody with antigen and the inhibitory rates of each mimic epitope were 50% higher than that in the control. Conclusion The results shows that KHYDPFHHRMPQ，QADTARSVALAG，VPSKPDLHVRSI，MTPIHYWNHNRV are the mimotopes which could mimic the epitope of MUC1. Keywords: Mucin 1; mimic epitope; phage display random 12 peptide library 泌尿系肿瘤是泌尿外科疾病中最常见的疾病,深入研究其治疗方法具有重要的临床意义。正常情况下泌尿生殖道腺上皮细胞的管腔面顶部都有MUC1的表达。但在膀胱癌、前列腺癌、精囊癌等腺癌中MUC1呈异常表达：①表达量增高；②极性分布消失；③糖链的糖基化不全，糖链变短，分枝少，结构简单，导致新糖链表位形成和正常情况下隐蔽的肽链表位暴露，使MUC1成为可被免疫系统识别的肿瘤抗原［1］。由于MUC1具备以上特点，使其成为多种上皮性肿瘤免疫治疗的新靶点，通过机体的体液和细胞免疫机制能有效免除MUC1高表达肿瘤细胞。为研发人泌尿系上皮肿瘤疫苗，本文应用MUC1糖链抗体筛选噬菌体随机12肽库获得了MUC1模拟表位，报告如下。 1材料与方法 1.1材料 Ph.D.12TM肽库(NEW ENGLAND Biolabs)为展示在噬菌体pIII蛋白N端的12肽库，库滴度为4×1012 pfu/ml， 随机多样性（c