人源噬菌体抗体库的构建及抗VEGF抗体的初步筛选分析

Construction of Human Antibody Library and Analysis of its Diversity

应用噬菌体表面呈递技术构建人抗体组合文库 .筛选获得了结合血管内皮细胞生长因子( VEGF)的人噬菌体 Fab抗体 ,并对所获抗体的多样性进行了进一步分析 .从不同人群外周血淋巴细胞提取总 RNA,经反转录后采用家族特异性免疫球蛋白可变区基因引物与免疫球蛋白信肽序列引物 ,通过改变 PCR条件或半套式扩增分别获得全部亚型的轻、重链抗体 Fab段 ,并重组到噬粒载体 p Comb3H中 ,经电转化大肠杆菌 XL- 1 Blue,构建了 1 .5× 1 0 8完整组合抗体库 .利用 VEGF12 1对该库经过 4轮固相筛选后 ,获得 1 2个可与 VEGF特异结合的阳性克隆 .酶谱分析表明了所获抗体克隆的多样性 .为通过基因工程改造 ,进一步获得可用于临床的人源 VEGF抗体奠定了基础 .

Human antibody library was constructed by using phage display technology and phage Fab antibodies against vascular endothelial growth factor(VEGF)were screened from this library.Enzyme analysis of these Fab antibodies was accomplished for diversity.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 as half nested PCR.The heavy and light chain PCR products were then cloned into phagemid vector pComb3H and a repertoire of 1.5×10 8 clones of Fab fragments displayed on the surface of phage was constructed.After 4 rounds panning with VEGF 121 ,280 clones were checked for their binding activities with ELISA and 12 clones could bind to VEGF 121 specifically.Restriction enzyme analysis showed that these clones contained vast diversity.These data provided a new way for preparation of high affinity human anti VEGF monoclonal antibodies through antibody engineering.