Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-alpha using computer modeling and deletion mutant technique.
The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-alpha were cloned, and according to the translated sequence of amino acids, the spatial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-alpha was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-alpha. hTNF-alpha molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92-157 of hTNF-alpha, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF-a 141-146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-alpha were specially recognized by Z12 antibody.
Zhang
Wei
W
Beijing Basic Medical Institute, Beijing 100850, China. great86@sohu.com
Feng
Jiannan
J
Shen
Beifen
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000276:immunology; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D006860:Hydrogen Bonding; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha