Selection Of Peptide Specifically Binding To Breast Cancer Cells By In Vitro Phage Display

Objective:To screen the small molecular peptides binding to human breast medullary carcinoma Bcap-37 cells by phage display technology in vitro,and conduct preliminary identification.To provide a theoretical basis for early diagnosis and treatment of breast cancer.Methods:We cultured human breast medullary carcinoma Bcap-37 cells.The Bcap-37 cells were used as the target cells and human normal breast epithelial cells（NBEC）MCF-10A as the absorption cells in vitro.We used phage display technology to subtraction biopanning specific binding peptide with human breast medullary carcinoma Bcap-37 cells.Then we obtained exogenous polypeptides of specifically binding to breast cancer cells.After 4rounds of subtraction biopanning in vitro,20 monoclonal phages were randomly selected.Their affinity binding to Bcap-37 cells were identified by ELISA.The DNA of positive phage clones were extracted and sequenced.The amino acid sequences were deduced and analysed.The breast cancer cells specific binding peptide and control peptide were synthetized by solid phage synthesis method.Using MTT and Wound-Healing experiment to study the effects of synthetic peptide with proliferation and migration on Bcap-37 cells.Results:1.We successfully cultured human breast medullary carcinoma Bcap-37 cells and normal breast epithelial cells MCF-10A in vitro.2.After four rounds of biopanning in vitro,the phages recovery rate of binding to human breast medullary carcinoma Bcap-37 cells were gradually increased from 2.0×10^-7at the first round to 1.6×10^-4 at the fourth round subtraction biopanning.3.After the fourth round of screening,20 monoclonal phages were selected randomly,which their affinity binding to Bcap-37 were identified by ELISA.Then ELISA results suggested that 11 clones（1,3,5,6,8,12,14,16,17,18,20）were strongly positive.4.The DNA of the positive monoclonal phages were extracted and sequenced,four amino acid sequences were obtained.The sequence LPRNTNL has the highest repetition rate,analysing the homology of amino acid sequence LPRNTNL,suggested that there are some similar amino acid sequences were found in the present date base.5.The polypeptides LPRNTNL and sv LPRNTNL were prepared by solid phage synthesis method.The MTT and Wound-Healing experiment results showed that the LPRNTNL had no significantly effect on the proliferation and migration of Bcap-37 cell.Conclusion:Through four rounds of subtractive screening,we successfully obtained the small molecular peptide sequences that can specifically bind to Bcap-37 cells by in vitro phage display technology.This research may provide a theoretical basis for the early diagnosis and targeted therapy of breast cancer.