Songsivilai S, Dharakul T
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Asian Pac J Allergy Immunol. 1998 Mar;16(1):31-41.
Specific single-chain Fvs (scFvs) of human immunoglobulin that specifically recognized the recombinant hepatitis C virus (HCV) nucleocapsid protein were isolated from a large phage display antibody library. This universal library of genetically engineered filamentous phagemids displayed random pairings of the variable regions of both human heavy and light chain immunoglobulin in the scFv format. Specific clones were isolated by affinity selection with purified recombinant HCV protein fused to glutathione-S-transferase (GST). The GST-specific clones were excluded by blocking the phagemid library with GST prior to the selection. After 4 rounds of selection, the HCV-reactive clones were enriched by a factor of 100,000. About 4% and 9% of the clones from rounds 4 and 5, respectively, specifically reacted to the HCV portion of the fusion protein in an enzyme immunoassay. The specificity was confirmed by specific binding inhibition with plasma from an HCV-infected individual. Nucleotide sequence analysis of 3 HCV-specific clones indicated that all 3 clones contained an almost identical VH gene sequence which was derived from the VH3 germline gene family. These clones had different VL gene sequences of the lambda type. There were some differences between nucleotide and amino acid sequences of the HCV-specific scFv genes and those of the closest matched germline genes, indicating the presence of somatic mutation. This study illustrated the feasibility of using antibody engineering technology with the universal phage display library to isolate human antibodies with predefined specificity to important microbial pathogen which may be useful for future therapeutic purpose.
从一个大型噬菌体展示抗体文库中分离出了特异性识别重组丙型肝炎病毒（HCV）核衣壳蛋白的人免疫球蛋白特异性单链抗体片段（scFvs）。这个基因工程丝状噬菌粒通用文库以scFv形式展示了人重链和轻链免疫球蛋白可变区的随机配对。通过用与谷胱甘肽 - S - 转移酶（GST）融合的纯化重组HCV蛋白进行亲和选择来分离特异性克隆。在选择之前，用GST封闭噬菌粒文库以排除GST特异性克隆。经过4轮选择，HCV反应性克隆富集了100,000倍。在酶免疫测定中，分别来自第4轮和第5轮的约4%和9%的克隆与融合蛋白的HCV部分发生特异性反应。通过用HCV感染个体的血浆进行特异性结合抑制来确认特异性。对3个HCV特异性克隆的核苷酸序列分析表明，所有3个克隆都包含一个几乎相同的VH基因序列，该序列源自VH3种系基因家族。这些克隆具有不同的λ型VL基因序列。HCV特异性scFv基因的核苷酸和氨基酸序列与最匹配的种系基因的序列之间存在一些差异，表明存在体细胞突变。本研究说明了使用抗体工程技术与通用噬菌体展示文库来分离对重要微生物病原体具有预定义特异性的人抗体的可行性，这可能对未来的治疗目的有用。
