人源噬菌体抗体库的构建及抗人NH-LBP抗体的筛选与鉴定  

Construction of human phage antibody library and screening and characterization of phage antibodies against N terminal fragment of human lipopolysaccharide binding protein

作　　者：葛晓冬[1] 刘友生[1] 王晓东[1] 王长松[1] 邓军[1] 李红[1] 

机构地区：[1]第三军医大学西南医院病理学研究所,重庆400038

出　　处：《细胞与分子免疫学杂志》2005年第2期180-184,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(No. 30271342)

摘　　要：　目的: 构建人源噬菌体抗体库并制备抗人脂多糖结合蛋白氨基端片段 (NH- LBP)的单克隆抗体 (mAb)。方法:以噬菌体展示系统(pComb3H/VCSM13)建立人源噬菌体抗体库(Fab), 并以昆虫细胞sf21在无血清培养基 (SF 900Ⅱ )中,通过BAC- TO- BAC杆状病毒表达系统来表达NH -LBP。再以NH -LBP为抗原, 从人源噬菌体抗体库中筛选可产生抗NH- LBPmAb的菌株并进行鉴定。结果: 昆虫细胞sf21可表达人源NH- LBP, 经亲和纯化柱芯 (TALON)有效纯化后, 获得约8mg的NH- LBP。成功地建立人源噬菌体抗体库, 库容达5. 0×108 CFU。经 8轮筛选后, 抗体库被富集 1. 85×104 倍。经ELISA法鉴定, 获得 3株可产生抗NH -LBPmAb的菌株(核酸序列及氨基酸序列见GenBank中的: AY337713,AY337714 )。结论: 以昆虫细胞sf21表达NH LBP及以其为抗原制备噬菌体mAb是可行的。本研究为进一步建立抗NH- LBP的二硫键稳定的Fv抗体 ( disulfidestabilizedFvfrag ments, dsFv), 研究人体内LBP的变化规律和过度炎症反应的防治奠定了基础。AIM: To construct human phage antibody library and to prepare phage antibodies against N terminal fragment of human lipopolysaccharide binding protein (NH-LBP). METHODS: NH-LBP was expressed in insect cells sf21 cultured with serum-free culture medium(SF-900Ⅱ) by BAC-TO-BAC baculovirus expression system. Human antibody library (Fab) was constructed by phage display system (pComb3H/VCSM13) and screened with NH-LBP. RESULTS: NH-LBP was successfully expressed in insect cells sf21 cultured with serum-free culture and effectively purified via affinity chromatography column with TALON resin. About 8 mg NH-LBP was obtained. Human combinatorial phage display antibody library consisting of 5.0×108 CFU was constructed. After eight rounds of panning with NH-LBP we got 3 phage antibodies with high affinity for NH-LBP (DNA sequences and amino acid sequences of kappa chains of these antibodies were deposited in GenBank (AY337713 and AY337714). CONCLUSION: Phage antidodies against NH-LBP were obtained successfully, which lays the foundation for preparing disulfide stabilized Fv fragments antibody (dsFv) against NH-LBP, understanding the function of LBP in vivo, and prevention and therapy of excessive inflammatory reaction. N-terminal

关 键 词：人脂多糖结合蚩白氨基端片段 人源噬菌体抗体库 昆虫细胞 

分 类 号：R392.11[医药卫生—免疫学]