ABSTRACT
Objective Human immunoglobulin combinatorial library was generated by using phage surface display expression system, and phage antibodies (Fabs) to platelet were screened.Methods:PBMC were separated from a patient who Were transfusion refractory to platelet. mRNA was isolated and cDNA was synthesized by reverse transcriptase.The immunoglobulin heavy chain Fd and the light chain ? genes were amplified by half nested PCR and the PCR products were cloned into the expression vector pCome3 respectively.The immunoglobulin combinatorial library was constructed and screened by 3 rounds of affinity selection.Results Human Immunoglobulin Combinatorial Library was successfully constructed.The specific phage antibodies were highly enriched after 3 rounds of biopanning selection against platelet membrane proteins.Conclusion The antibody library and human monoclonal antibodies to platelet may be useful as molecular tools to study the anti platelet drugs, platelet related diseases, epitope of human platelet antigens.
ABSTRACT
The emergence of repertoire cloning using phage antibody libraries has revolutionized the antibody engineering technology. We prepared phage antibody libraries from both human breast cancer cell immunized murine spleen cells and human peripheral lymphocytes from volunteer received HBsAg vaccination. Mouse anti-human breast cancer cell antibodies and human anti-HBsAg antibodies were successfully cloned by panning of the phage antibody libraries against breast cancer cells and immobilized HBsAg. The resulting Fab fragments were expressed in E.coli cells and the specificity, affinity and DNA sequences were analyzed. The results showed the potential of the phage display system in antibody preparation.