全人源胃癌噬菌体单链抗体库的构建及其初步筛选

发布时间：2018-06-24 22:56

本文选题：抗体库 + 胃癌　；参考：《浙江大学》2010年硕士论文

【摘要】： 研究目的:构建全人源胃癌噬菌体单链抗体库,并从中筛选出与人胃癌细胞特异结合的单链抗体,并对其进行鉴定分析。研究方法和研究结果:首先分离胃癌病人外周血淋巴细胞,提取总RNA,RT-PCR合成cDNA第一链,采用兼并引物PCR扩增抗体重链(V_H)和轻链可变区(V_L)基因。经重叠PCR(SOE-PCR),将V_H和V_L连接成单链抗体(scFv)基因,并连接到噬菌粒载体pCANTAB-5E中,转化至E.coli TG1,经辅助噬菌体超感染,构建了全人源胃癌噬菌体单链抗体库,库容为2.6×10~6,质粒片段插入率95%。单链抗体库经与正常人胃黏膜上皮细胞GES-1孵育后,去除与正常细胞结合的噬菌体单链抗体,再以人胃癌细胞MGC-803为靶标对抗体库进行4轮富集筛选,结果显示富集了50倍。选取第四轮筛选后的菌落制备单克隆抗体,并进行ELISA特异性鉴定,获得五株与人胃癌细胞MGC-803有较强特异性结合的噬菌体抗体。送5株抗体进行测序,选取阳性值最高的单克隆对其序列进行分析并预测其空间结构。结果表明:可变区基因与人胚系基因有很高的同源性(大于90%)。结论:成功构建了库容为2.6×10~6的全人源胃癌噬菌体单链抗体库,抗体库多样性良好。获得的噬菌体单克隆抗体具有较强的特异性,为胃癌的诊断和治疗提供了新载体。
[Abstract]:Objective : To construct a single - chain antibody library of human gastric cancer phage and to identify the single - chain antibody specific to human gastric cancer cells .

A single - chain antibody library of human gastric cancer cells was amplified by PCR ( SOE - PCR ) , and then transformed into E . coli TG1 . After incubation with human gastric cancer cell MGC - 803 , a single - chain antibody library was constructed .

Conclusion : The phage single - chain antibody library of human gastric cancer with a volume of 2.6 脳 10 ~ 6 has been successfully constructed . The antibody library has a good diversity . The obtained phage monoclonal antibody has strong specificity and provides a new carrier for the diagnosis and treatment of gastric cancer .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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