人源噬菌体抗体库中血管内皮细胞生长因子抗体的筛选及活性鉴定  

Selection and characterization of human antibodies against vascular endothelial growth factor from human phage display antibody library

作　　者：田学军[1] 寿成超[1] 董志伟[2] 

机构地区：[1]北京医科大学临床肿瘤学院,北京市肿瘤研究所,100034 [2]中国医学科学院肿瘤研究所

出　　处：《中华医学杂志》2000年第8期567-570,共4页National Medical Journal of China

基　　金：国家"八六三"高技术计划资助!项目 (8631 0 2 0 9);国家杰出青年科学基金!资助项目 (3952 50 2 1 )

摘　　要：目的　从人噬菌体Fab抗体库中筛选抗人血管内皮细胞生长因子 (VEGF)抗体克隆 ,并对其特异性及活性进行分析 ,为后续工作奠定基础。方法　从不同人群外周血淋巴细胞提取总RNA ,经逆转录聚合酶链式反应 (RT PCR)分别扩增轻、重链抗体Fab段 ,用噬菌体表面呈递技术构建抗体库 ,经过 4轮固相筛选及单克隆差异筛选 ,获得能结合VEGF的抗体克隆。在此基础上通过ELISA、Westernblot和3 H TdR参入分别对所获抗体克隆的特异性及中和活性进行分析 ,并对阳性克隆DNA进行测序分析。结果　构建了 1.5× 10 8人组合噬菌体抗体库 ,并筛选获得了结合VEGF的人噬菌体抗体Fab段。Westernblot表明所获抗体能较好结合VEGF ,3 H TdR参入实验显示其中一株抗体可中和VEGF引起的内皮细胞增殖作用。DNA序列分析表明所获抗体片段为人源VH6亚群抗体重链基因和VJC重排后的VL4亚群抗体轻链基因。结论　利用噬菌体抗体库技术可直接获得特异人源VEGF抗体 ,为进一步通过基因工程改造 ,获得有临床应用价值的VEGF抗体提供了新的可能性。Objective To select anti VEGF (vascular endothelial growth factor) antibodies (Fab fragments) from human phage display antibody library and to identify their specification and activities. Methods Human immunoglobulin heavy chain and light chain genes were separately amplified by RT PCR from human peripheral lymphocytes using family specific primers and signal sequences of immunoglobulin. Human antibody library was constructed by phage display technology, and phage Fab antibodies to VEGF were screened from this library. ELISA, Western blot and 3H thymidine incorporation assays were used for the specification and neutralization activities of these Fab antibodies. Sequencing analysis was carried out for further identification of the antibodies. Results The repertoire of human phage display Fab library was 1.5×10 8. After 4 round panning with VEGF 121 , 280 clones were checked for their binding activities with ELISA and 12 clones could bind to VEGF 121 specifically. Western blot demonstrated that bacterially expressed soluble Fab could specifically recognize VEGF. Results of 3H thymidine incorporation showed that one clone of soluble Fab could neutralize the mitogenic activity of VEGF 165 on HUVEC. Sequencing analysis showed that the obtained V H gene belonged to human VH6 subgroup and the light chain was VJC rearranged human VL4 gene. Conclusions Human anti VEGF antibodies can be obtained from human phage display library, which provides a basis for preparation of high affinity human anti VEGF monoclonal antibodies through antibody engineer technique.

关 键 词：DNA噬菌体 内皮生长因子 Fab抗体库 VEGF 抗体 

分 类 号：R730[医药卫生—肿瘤]