人源抗人免疫缺陷病毒1型噬菌体抗体库的构建及筛选

Construction and screening of type 1 human immunodeficiency virus specific phage antibodies combinatorial library

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－１ｇｐ１２０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－１ｇｐ１２０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－１ｇｐ１２０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－１ｇｐ１２０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。

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95×107. 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.