ABSTRACT
To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.
Subject(s)
Humans , Antibodies, Monoclonal , Genetics , Allergy and Immunology , Antibodies, Neutralizing , Genetics , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Methods , HEK293 Cells , HIV Antibodies , Genetics , Allergy and Immunology , HIV Envelope Protein gp120 , Genetics , Allergy and Immunology , HIV Infections , Allergy and Immunology , Virology , HIV-1 , Genetics , Allergy and Immunology , Immunoglobulin Fab Fragments , Genetics , Allergy and Immunology , Neutralization Tests , Peptide Library , TransfectionABSTRACT
With the fast development of biopharmacy,transgenic chicken oviduct bioreactor is going to be the focus of people’s attention.For its excellent advantages,transgenic chicken will be one of rising industries in the field of biopharmacy and hot spot of scientific study.Now,the most successful method for producing transgenic chicken is mediated by retrovirus transferring gene into it, and transgenic chicken have been produced by this method.The advantages and methods of producing transgenic chicken by retrovirus,and the progress in transgenic chicken research were summarized.The significance and existent problems of transgenic chicken are also discussed.