抗肿瘤MUC1单链抗体的构建表达及其功能初步分析.docVIP

抗肿瘤相关Ⅰ型粘蛋白（MUC1）单链抗体的构建及其功能初步分析 陈庆峰1，马中良1，王笑峰2，李福年1，郑玲莉1 ( 山东 青岛，青岛大学医学院：附属医院乳腺外科1，微生物教研室2） [摘要] 目的 分离鼠源抗MUC1单克隆抗体可变区基因，构建有结合功能的单链抗体，并初步检测？其功能。方法 提取杂交瘤细胞总mRNA，合成cDNA后，PCR扩增鼠特异性抗体可变区编码基因，通过9个氨基酸的连接子构建表达单链抗体的原核重组表达载体，体外诱导表达纯化后，采用Western blot和流式细胞术等方法分析单链抗体与变性或天然状态下抗原的结合活性。结果 在杂交瘤细胞中成功分离到抗体轻重链可变区基因，重组链接后形成单链抗体能够在大肠杆菌中有效表达，Western blot和流式细胞术分析表明该单链抗体能够与变性或天然状态下的T47D肿瘤细胞表面MUC1特异性结合。结论 成功制备具有生物活性的抗肿瘤相关MUC1单链抗体，该单链抗体具有与MUC1特异性结合的生物学活性。 [关键词] 乳腺肿瘤；黏蛋白类Construction, expression and characterization of anti-tumour associated MUC1 single chain Fv antibody fragment Chen Qingfeng, Ma Zhongliang, Wang Xiaofeng, Li Funian (The Affiliated Hospital of Medical College Qingdao University，Department of breast surgery） [Abstract] Objective Construction of single chain variable fragment antibody(scFv) from a mouse monoclonal antiI-MUC1 antibody secreting cell lines. Methods Isolation of mRNA of hybridoma cells, synthesis of cDNA, and antibody genotyping was performed using mouse antibody specific primers, related variable genes were fused with a 9-amino-acid linker to obtain a scFv.,which was characterized on denatured and natural antigen by Western-blot and flow cytometry. Results Variable regions of antibodies heavy and light chain coding gene were isolated form a monoclonal anti-MUC1 hybridoma cell line. ScFv was obtained by the combination of the two variable genes, which could be produced in E.coli effectively. Western-blot and FACS analysis showed that the obtained scFv could bind nature and denatured MUC1 of T47D tumor cells specifically. Conclusion A functional anti tumour associated MUC1 scFv was obtained successfully, which could bind to tumour associated MUC1 specifically. [Key Words] breast neoplasms；mucins；single-chain antibody MUC1蛋白是上皮粘蛋白家族中的一员，大约在90%的乳腺癌患者存在MUC1高表达、糖基化不完全和糖基化异常等特征，导致MUC1在细胞表达中异常暴露或者某一肽段骨架或异常表位暴露，成为乳腺癌的诊断和治疗中的重要靶标，许多其他恶性肿瘤细胞也存在MUC1表达水平的增高，如胃癌、前列腺癌等[1-5]。 单克隆抗体类药物是生物制药产业中最大类别的产品，如今已被成功用于治疗肿瘤等多种疾病领域，在早期临床试验中所采用的单抗多为鼠源，易于诱导患者机体产生人抗鼠抗体（HAMA），限制了这类抗体新药的安全使