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

Human monoclonal antibodies to type 1 immunodeficiency virus (HIV-1) gp120 were generated from phage antibody combinatorial library.

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 combinatorial 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 x 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-1 gp120 have been used as tools for screening of neutralizing antibody to HIV-1, and the methods seem to be very crucial and applicable.