The Preparation And Function Research Of Human Specific Anti-P-selectin Monoclonal Antibodies

Recent years, the effect of P-selectin on inflammatory reaction and neoplasm metastasis was more and more paid attention to. An attempt to block the binding between P-selectin and its ligands has been the hot spot for a long time. This study is to screen the human specific anti-P-selectin monoclonal antibody from the large fully synthetic human antibody libraries.And to identify the function on blocking the binding of P-selectin and its ligands.First of all, cell adhesive molecular P-selectin was cloned, expressed and archored on CHO cell membrane throught GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into an eukaryotic expression vector pMCEw2 containing an attenuated Neo gene together with a downsteam GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-P-selectin-GPI was then transfected to CHOdhfr- cells and secreened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided necessary basises for the following study of selection the antibodies targeting P-selectin.Secondly, 15 antibodies selected from the libraries showed P-selectin prokaryotic expression protein binding capacity and were expressed in soluble form in E. coli. At the same time, we did four rounds of subtractive selections on cell P6 which express P-selectin on the cell membrane, using normal CHO as a negative control. 1H11,2F8 selected from the libraries was proved to be with excellent specificity and binding capacity against P-selectin eukaryotic expression protein. At last, modificate 1H11,2F8 to be whole antibody form. Platelet which transport P-selectin to the membrance when stimulated by thrombin will combine with neutrophil which express P-selectin ligands. Primary biological result indicated 1H11,2F8 whole antibody was likely to inhibit the binding between them.