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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。
OBJECTIVE: To construct a human phage antibody library and prepare monoclonal antibodies (mAbs) for the NHLP fragment of anti-human lipopolysaccharide binding protein. METHODS: Human phage antibody library (Fab) was created with a phage display system (pComb3H / VCSM13) and expressed by the insect cell sf21 in a serum-free medium (SF 900 II) by the BAC-TO-BAC baculovirus expression system NH -LBP. NH-LBP was used as antigen to screen and identify anti-NH-LBP mAb from human phage antibody library. Results: Insect cell sf21 could express human NH-LBP. After purified efficiently by TALON, about 8 mg NH-LBP was obtained. Successfully established human phage antibody library, storage capacity of 5. 0 × 108 CFU. After 8 rounds of screening, the antibody library was enriched 1.85 × 104 times. Three strains producing anti-NH-LBP mAb were obtained by ELISA assay (for the nucleotide and amino acid sequences, see: AY337713, AY337714 in GenBank). Conclusion: The expression of NH LBP in insect cells sf21 and the preparation of phage mAb using it as an antigen are feasible. This study laid the foundation for the further study of the changes of LBP and the prevention and treatment of excessive inflammatory reaction in order to further establish disulfide-stabilized Fvfragments (dsFv) against NH-LBP.