Screening Of Specific Ligands For Bladder Cancer Cell Markers Using Phage Display Technology

Bladder cancer is a kind of nasty tumor on the surface of bladder mucosa,which is the most common nasty tumor in urinary system.It is considered to be a highly immunogenic cancer.Compared with other kinds of cancer,its mutation rate is higher.Because the early diagnosis of bladder cancer can improve the survival rate of patients,there is an urgent need for urine based biomarkers in medicine for non-invasive diagnosis of bladder cancer.Urinary bladder cancer antigen(UBC),as a typical bladder cancer marker,is a class of cytokeratin fragments secreted in urine with higher expression in urothelial carcinoma and therefore served as the target protein in this study.At present,all methods to detect UBC content have certain limitations,and there are real problems such as low sensitivity of the detection method,tedious operation and expensive price,which urgently establish a new method of low-cost,high specificity and sensitive biological detection.First of all,we used UBC as the target protein and carried out four rounds of screening by phage display technology to successfully obtain recombinant phage that specifically bound to UBC,then the phage was tested for binding activity,ss DNA extraction,sequencing,translation and polypeptide sequence alignment to determine the three entry standard ligand sequence.Then through molecular simulation docking technology,using Chembio office software to simulate the three-dimensional structure of the three ligand sequences,and using Auto Dock Tools software to simulate the binding site analysis and evaluation,and finally use Py Mol software to obtain the position of the peptide and the target protein.The combination of simulated imaging and the screening of two peptide sequences,and the two sequences were subjected to bio-membrane interference experiments,and a sequence with higher affinity was obtained and synthesized,namely: K-H-F-T-H-N-H-H-P-I-T-W.The cell immunofluorescence experiment was used to identify the peptide and the human bladder epithelial cells.Cancer cells have strongly binding,but not in normal cells and other cancer cells.Finally,Silver nanoparticles(AGNPS)were used as immunoprobe and magnetic bead peptide probe for sandwich detection of UBC antigens by combining peptide ligands and surface enhanced Raman scattering(SERS)technique.A surface enhanced Raman scattering detection way for UBC antigen was developed to realize quantitative detection in clinical urine samples.According to the threshold value of 10 μg/L,the detection limit of UBC was6.25 μg/L.Six cases had 100 % concordance among clinical urine testing and clinic pathological diagnosis.In this study,based on the specific ligand peptide of UBC,a new way of SERS has been established.It provides a new method for low-cost,high-specificity,and high-sensitivity rapid detection of UBC,and it also provides a new idea for clinical detection of tumor markers.