人源抗呼吸道合胞病毒基因工程单克隆抗体Fab段基因的筛选和表达  

Generation of human monoclonal antibody Fab fragments against respiratory syncytial virus (RSV) using phage display technology

作　　者：肖玮[1] 梁米芳[2] 钱渊[1] 邓洁[1] 李川[2] 

机构地区：[1]首都儿科研究所病毒研究室北京感染与免疫中心实验室 [2]中国疾病预防控制中心病毒病预防控制所

出　　处：《中华微生物学和免疫学杂志》2007年第7期587-592,共6页Chinese Journal of Microbiology and Immunology

基　　金：北京市自然科学基金重点项目(7001005);北京市科委项目(H010910060113)

摘　　要：目的采用噬菌体表面呈现和重组抗体技术构建人噬菌体抗体基因库，筛选获得人源抗呼吸道合胞病毒（RSV）Fab段基因并在原核细胞中表达。为研制安全、有效的预防和治疗RSV感染的制剂奠定基础。方法从RSV感染患儿恢复期外周血淋巴细胞中提取细胞总RNA，用一组人IgGF出特异性引物，通过RT—PCR扩增得到一组轻链（κ和λ）和重链Fab段基因，并将此轻链和重链基因片段克隆于pComb3噬菌体载体，电转化XL1-Blu菌构建成抗RSV噬菌体抗体基因库。用纯化病毒颗粒作抗原对此抗体库进行了富集筛选，得到了特异性针对RSV的人源单克隆抗体Fab段基因，并在大肠杆菌中获得有效表达。用ELISA方法检测了此Fab抗体的抗原特异性，并对阳性克隆进行了基因序列分析。结果所构建的抗体库库容为108，从此抗体库中筛选得到的阳性克隆所表达的Fab抗体能与RSV纯化抗原特异性结合，保留了对RSV的抗原特异性。核苷酸序列分析证实，所获得的阳性克隆基因为人源IgG基因。结论本实验获得了特异性抗RSVFab抗体基因并在大肠杆菌中获得表达，表达产物能与RSV抗原特异性结合。Objective To develop a safe and effective medicine for RSV infection. Methods The buman IgG Fab genes of heavy and light chains were amplified from peripheral lymphocytes of four infants in convalescent phase of asthma-like pneumonia caused by RSV infection in an outbreak in Henan province, and cloned into phage display system （pComb3/VCSM13）. The combinatorial phage Fab antibody library was constructed and followed by screening with purified RSV virions. The specific human antibody Fab fragnents against RSV were selected and expressed in XL1-Blu, and the specificity of the expressed RSV Fabs were characterized by ELISA. Finally the sequence of Fab genes from the positive clones were analyzed by alignment with GenBank. Results The human antibody library consists of 108 independent clones. Six rounds of selection against McAb captured RSV partides showed specific enrichment of phage antibodies. Direct ELISA verified that the positive phage antibodies were specific against RSV, Analysis of the DNA sequence indicates that the human immunoglobulin Fab sequence was identified and confirmed. Conclusion Human IgG Fab fragnents anti-RSV were obtained with phage display technique.

关 键 词：呼吸道合胞病毒 噬菌体表面表达 人源基因工程单克隆抗体 

分 类 号：R392[医药卫生—免疫学]