关键词：抗体库；人源单克隆抗体；人免疫缺陷病毒
【摘要】 目的 构建人免疫缺陷病毒1型(HIV-1)特异性噬菌体抗体库，制备人源抗HIV-1 gp120单克隆抗体。方法 以半巢式聚合酶链反应(PCR)从HIV-1感染者外周血单个核淋巴细胞中扩增抗体重链Fd和轻链(k)基因，与噬菌体载体pComb3连接，构建噬菌体抗体Fab组合文库。对抗体库进行3轮吸附-洗脱-扩增的亲和选择后，以ELISA法筛选抗HIV-1 gp120噬菌体抗体，并进行DNA序列分析和Fab的可溶性表达。结果 半巢式PCR有效地扩增出Fd和k基因，以此构建成容量为1.95×107 的噬菌体抗体库。3轮亲和选择使特异性抗体得到高度富集，抗HIV-1 gp120噬菌体抗体阳性克隆占32%。对一阳性克隆抗体基因CH1和CL部分DNA序列进行了测定，并在大肠杆菌表达出可溶性Fab。结论 抗HIV-1特异性噬菌体抗体库的构建和人源抗HIV-1 gp120单克隆抗体的制备为今后筛选抗HIV中和抗体奠定了基础，具有重要的应用价值。
Construction and screening of type 1 human immunodeficiency virus specific phage antibodies combinatorial library
He Yuxian, Wang Xue, Liu Shunai, et al. Research Center of Virology, Beijing Ditan Hospital, Beijing 100011
Abstract Human monoclonal antibodies to type 1 immunodeficiency virus (HIV-1) gp120 were generated from phage antibody combinatorial library. Methods: The human immunoglobulin heavy chain Fd and light chain k genes were amplified by half-nested PCR from PBMC of patient infected with HIV. Phage antibody combnatorial library was constructed with the Fd and k chain genes using Pcomb3 as vector. The affinity selection and ELISA were adopted for generating specific phage antibodies. Partial DNA of a positive clone was sequenced and its soluble Fab was expressed in E coli. HIV-1 specific phage antibodies combinatorial library were constructed using the Fd and k genes and Pcomb3 vector. The library capacity was about 1.95×107 . The specific phage antibodies were highly enriched after three rounds of biopanning selection against HIV-1 gp120 and 32% positive clones were detected by ELISA screening. DNA fragment coding for CH1 and CL derived from a positive clone was sequenced and its product was successfully expressed as soluble Fab which was specific for HIV-1 gp120. The HIV-1 specific phage antibody combinatorial library and human monoclonal antibodies to HIV-1gp120 have been used as tools for screening of neutralizing antibodg to HIV-1, and the methods seem to be very crucial and applicable.
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