【作者】 崔志刚；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 博士

【摘要】 目的:从噬菌体随机12肽库中筛选新的MUC1的模拟表位,观察其免疫学特性,以及诱导机体产生特异性免疫应答和对小鼠膀胱癌的治疗作用。构建MUC1模拟表位原核表达载体,获得特异、纯化的模拟表位融合蛋白。本研究为研制针对MUC1为靶点的肿瘤疫苗奠定基础,为膀胱癌治疗提供新方法。方法:1.选用抗MUC1多肽表位的单克隆抗体BC2为筛选配基,利用噬菌体展示技术,从噬菌体随机12肽库中,通过3轮生物淘洗、测定噬菌体滴度、扩增噬菌斑、挑选特异性阳性噬菌体克隆,通过ELISA实验鉴定阳性噬菌体克隆与单克隆抗体BC2结合活性;抽提阳性噬菌体克隆的插入肽ssDNA模板,测定其DNA序列,按遗传密码表推导出插入肽的氨基酸序列,同MUC1核心肽表位序列APDTR进行比较,从中选出与原表位序列不同的模拟表位。利用表位预测专用数据库SYFPEITHI database进行氨基酸序列的表位预测,根据抗原肽的基序特征计算其积分,判断其与MHCⅠ类分子结合能力,合成相应的模拟表位肽,通过竞争ELISA实验判断模拟表位肽能否特异性抑制单克隆抗体BC2与MUC1抗原结合,进一步鉴定筛选出的MUC1模拟表位活性。2.利用阳离子脂质体介导转染方法,将人MUC1全长cDNA转入T739小鼠膀胱癌细胞BST739中,经G418筛选和克隆化培养,基因组PCR、RT-PCR、免疫组化、流式细胞仪从不同水平检测MUC1的表达,建立稳定表达人MUC1T739小鼠膀胱癌细胞株,观察转入人MUC1肿瘤细胞体内外生长特性和体内致瘤性。3.无菌取T739小鼠股骨和胫骨,PBS冲出骨髓细胞,红细胞裂解液裂解红细胞,按2×106/ml的细胞密度加入含有树突状细胞(DC)专用培养基塑料培养皿,同时加入细胞因子rmGM-CSF,rmIL-4 1000U/ml,37℃,5% CO2体外培养,第6天加入LPS(1μg/ml)刺激DC成熟,第7天收集细胞,显微镜下观察成熟DC形态,流式细胞仪检测成熟DC标记33D1单抗的表达以判断DC纯度。4.体外用合成的模拟表位肽刺激成熟DC,PBS洗涤三次,然后分别用PBS,未刺激的DC,MUC1模拟表位肽刺激的DC,通过小鼠尾静脉注射,免疫T739小鼠,每只注射2×105细胞,每周免疫一次,共三次。更多还原

【Abstract】 Objective: To acquire new MUC1 mimic peptide epitopes by biospanning in phage random 12 peptide library and preliminarily investigate the functional immunological responses and effectiveness in treatment of MUC1-positive T739 mouse bladder cancer induced with the mimic peptide epitopes. To construct a recombinant plasmid expressing MUC1 antigen mimic epitope and acquire the specific purified fusion protein induced with IPTG. Based on our preliminary data, MUC1 mimic peptide epitopes will act as an important tumor-vaccine candidates for bladder cancer therapy.Methods: Monoclonal antibody BC2 which aims at MUC1 peptide epitopes was used as a ligand to screen MUC1 antigen mimic epitopes by phage display peptide library technology. Specific positive phage clones which can specifically recognize MUC1 monoclonal antibody Were screened after 3 rounds of biospanning, phage titer determination and plaque amplification in phage random 12 peptide library. Their banding capacity to BC2 antibody were tested by ELISA assay, then their DNA sequences were determined by rapid purification of sequencing templates and amino acid sequences were deducted by reduced genetic codes. Two MUC1 mimic epitopes were acquired comparing with MUC1 core sequence. Predictions of the two epitopes binding to MHC classⅠmolecules and its scores evaluated the peptide’s binding capacity based on the feature of motif were given from SYFPEITHI databases and competitive inhibition ELISA assay were taken to confirm the specificity of the synthetized epitope peptides. T739 mouse bladder cancer cell lines expressing human MUC1, named BST739-MUC1, were established by Lipofectamine-mediated transfection of human MUC1 full length cDNA into BST739 cells, followed by G418 selection and clonal culture. PCR, RT-PCR and immunohistochemistry were carried out to confirm the stable integration of MUC1 cDNA into the mouse genome and expression of MUC1 mRNA and proteins in the cells. The growth characteristics of更多还原