Screening Of Human CD55 Antigen Mimic Epitope And Its Antitumor Effect On Breast Cancer Cells

Objective: Ph.D.12 peptide library was screened for specific binding to human CD55 monoclonal antibody(CD55 Mc Ab)by using phage peptide library display technology.Further,the antigenic mimic epitope of CD55 was found and the short peptides of the homology of human tumor immune escape protein CD55 were designed to lay the theoretical foundation for tumor therapy.Methods: The specifical binding phage clones were identified after four cycles of screening with human CD55 monoclonal antibody as a target,and the recovery rate per round was calculated.After 4 rounds of screening,we competed the random selection of11 phage monoclones and wild-type phage to calculate its binding power.ELISA and competition binding were used to identify the phage which had strong binding ability.Phages were expressed and purified by E.coli ER2378 host bacteria.The positive phage clones were sequenced by company and the short peptide sequences with high frequency were found out.Short peptides were designed,which had high affinity and specificity for human CD55 Mc Ab and its effect on anti-breast cancer in vitro was studied.The effects of short peptide on proliferation of breast cancer cells(MDA-MB-231 and MCF-7 cells)were detected by CCK8.Fluorescence microscopy was used to verify the englobement and distribution of short peptides in cells.The influences of short peptide on apoptosis of MDA-MB-231 and MCF-7 cells were determined by flow cytometry and transmission electron microscope(TEM).Result: After four rounds of screening,the phage recovery rate reached a high order of magnitude.The results of competition experiment showed that 11 phage monoclones and the phages after round 3,4 screening were very significantly combined with CD55 monoclonal antibody.ELISA results showed that there were 8 phage monoclines with high affinity.According to sequencing results,two short peptide sequences with high expression were found: HAHTPTRGVMHA(H peptide)and QVNGLGERSQQM(Q peptide).CCK8 proved that the Q peptide sequences had significant toxic effects on breast cancer MDA-MB-231 cells and MCF-7 cells and the toxic effects were dose-dependent.However,the effect of H peptide was poor compared with Q peptide and need higher concentration to reach the same tumor suppression.Q peptide was observed on the surface of cell membrane by fluorescence microscope.Flow cytometry and TEM results showed that Q short peptide could induce the apoptosis of MDA-MB-231 and MCF-7 cells.Conclusion: We were succeeded in obtaining two peptide(HAHTPTRGVMHA and QVNGLGERSQQM)which is specificly binding with CD55 monoclonal antibody.The antitumoral effect of Q short peptide was more significant than H peptide,and it could inhibit the proliferation and induce the apoptosis of breast cancer cells,which provided new idea and new target for the treatment of breast cancer.