Screening And Identiifcation For Esophageal Carcinoma Cell Specific Binding Peptides Using Phage Display Peptide Library

Esophageal cancer is one of the most malignant tumors of digestivesystem.In our country, Henan and Shanxi provinces is one of the areas withhightest of esophageal cancer morbidity and mortality,and this trend is on therise. Esophageal cancer begin hidden and the screening and treatment is nobasic improvement.when esophageal cancer diagnose, the cancer is usuallyadvanced,and poor prognosis with the number of five-year survival rate notmore than15percent and symptomatic persons less than10percent. At presentthe treatments of esophageal cancer are operation,chemotherapy andradiotherapy.But a single treatment is difficult to achieve the good effect, andthe recurrence rate is very high. The combination of the treatments do not gainpopularity because of obvious side effects and ineffective.Therefore, to find anew way to the early diagnosis and high targeted therapy is becoming a newdirection to the cancer treatment.In recent years, the tumor biological treatment is extensive researchingand applying in clinical, the success of the monoclonal antibodies become animportant research direction to biological treatment. The monoclonal antibodiescoupling with targeted drugs accurately combine to tumor cells on the targetspecific molecular, and play the targeted therapy effect.Therefore, to find a highaffinity ligand as the carrier,which can combine with the esophageal cellsurface molecular specific, can effectively screen out of the early esophagealpatients, reduce the side effects of the drugs targeted therapy, improve the localdrug concentration with the tumor cells, and enhance the clinical value of theearly esophageal diagnosis and targeted therapy.In1985, Smith proposed the concept of phage display random peptide library technology,and in1990Huse successfully constructed the first phagecombinatorial library, for the early detection of cancer and targeted therapyprovides a new method. Using the characteristics of phage antibody smallmolecular weight,organization penetrating and quickly get to target antigens,we attempt to construct the antibody libraries of esophageal cancer, and screenthe esophageal Eca109cell.This experiment is designed to screen the polypeptides specifically bindingto esophageal cancer Eca109cells from phage display peptide library and toprovide a theoretical basis for early diagnosis of esophageal cancer or targeteddrug delivery in chemotherapy.With the esophageal carcinoma Eca109cells as the target cells and normalesophageal epithelial cells as the control cells for subtraction biopanning fromphage display peptide library.The positive phage clones were identified by cellELISA and immunofluorescence experiments.DAB experiments confirm thattwo positive phage clonings do not bind to normal esophageal tissue. Theamino acid sequences of the identified peptides were deduced by DNAsequencing,and analyse the homology of the amino acid sequence.This experiment results is that after three rounds of subtraction biopanningfrom phage display peptide library, we gradually raise the standard of thescreening positive cloning phage, and keep phages inputs,recovery rate from2.1×10-8to3.4×10-5, improving about103times, it showed that the phagesbinding to esophageal cancer cells Eca109were enriched. From the twentyrandomly selected clones, nine positive phage clones showing specific bindingto esophageal cancer cells Eca109. Cell ELISA confirmed two specificity of thephage binding to the esophageal cancer cells Eca109but not binding to normalesophageal epithelial cells. Immunohistochemistry confirmed two specificity ofthe phage binding to the esophageal cancer cells Eca109but not binding tonormal esophageal epithelial cells. DAB experiments confirm that two positive phage clonings do not bind to normal esophageal tissue.The DNA of the abovetwo phage positive clonings are extracted, sequencing analysis shows that:inthe two peptide sequence, nonpolar aliphatic amino acids is41.67%, polaraliphatic amino acids is33.33%,and there is no homology of the amino acidsequence and on known proteins are identified by BLAST analysis.This experiment conclusions is that： Specific peptides binding toesophageal canceer cells Eca109has been obtained from phage display peptidelibrary.Two positive phage clones were identified by cell ELISA andimmunofluorescence experiments,which bind to the esophageal cancer cellsEca109but not bind to normal esophageal epithelial cells. DAB experimentsconfirm that two positive phage clonings do not bind to normal esophagealtissue.The DNA of the two phage positive clonings are extracted, sequencinganalysis shows that: low homology between the peptides sequences displayedby the phages and on known proteins are identified by BLAST analysis.Theexperiment of the two positive cloning might become the carrier which to be anew way of targeted therapy to inhibit esophageal cancer.The next work is tosynthesize the short peptide which coded by the amino acid sequence of phagepositive clonings, and apply to living experiments to confirm its affinity for thespecificity of the esophageal cancer cells. It provides a basis for early diagnosisof esophageal cancer or targeted therapeutic agents in chemotherapy.