Screening Lung Cancer Serous Specific Peptide And The Tumor Marker Via Phage-display Peptide Library

Aim: To find the lung cancer specific peptide for serological screen and clinic classifies and to identify the target molecular to which the peptide bond via phage-display technology. Method: 24 serous specimens form lung cancer patients and physical examinates were collected respectively for the four-round subtractive screening with Ph.D.12TM phage display peptide kit. The highest specific monoclone were picked out based on the results of serological and cellular ELISA. The peptide labeled with FITC was synthesized according to the DNA sequencing of the optimal one, and tested with serous specimens, cell lines(including A549, HCI-H1299, WI-38) and tissue section via fluorescent assay in vitro. The peptide labeled with hydrophilic CdSe/ZnS quantum dots was injected to nuke mice grafted with lung cancer cell to evaluat the intracorporal targeting property via immunofluorescent assay. T7 phage-display lung cancer cDNA library was screened with biotin-linked peptide which was immobilized to the avidin-coated ELISA plate. After three rounds of screening, 20 monoclones were randomly selected for DNA sequencing and the information of the phage-displaying proteins were obtained from NCBI gene data base, www.ncbi.nlm.nih.gov/BLAST. The target molecular was defined according to the occurance frequence in the 20 T7 monoclones. Results: Among 30 randomly selected monoclones, 5 postive monoclones were found after 4-round screenings according to serous ELISA. The total diagnositc rate of the optimal one,ZZ-8,reached 45.8%. The detection rate of squamous cell carcinoma reached 87.5%, lung adenocarcinomar reached 33.3% and adenosquamous carcinoma of lung reached 50.0%. In the basis of ELISA, monoclone ZZ-8 also showed desirable specificity to cancer cells that it selectively bond to cancer cell line A549 and NCI-H1299, but not to normal cell line WI-38. Peptide sequence of ZZ-8 was concluded to be HSLKSTVDALWW by DNA sequencing. The total diagnositc rate of FITC-ZZ-8 was proved to be 41.7%. The detection rate of squamous cell carcinoma reached 75.0%, lung adenocarcinomar reached 66.7% and adenosquamous carcinoma of lung reached 25.0%. FITC-ZZ-8 was indicated that strongly boud to cancer cell line and lung cancer tissue section while relatively weaker to normal cell line and tumor-adjacent tissue section. The peptide labeled with QD was indicated that it mainly distributed to tumor, liver and kideny while hardly to other organs in vivo. After three screening of T7 lung cancer cDNA library, 20 T7 phage monoclone were selected randomly for DNA sequencing analysis and 9 proteins were identified after searching in database, www.ncbi.nlm.nih.gov/BLAST. Among all the proteins, CD63 was the one with the highest occurance frequence, which accounted for 25.0%. Conclusion : The synthetic peptide ZZ-8, whose amino acid sequence was HSLKSTVDALWW, showed specificity to lung cancer serum protein, lung cancer cell and tomur tissue. Its binding target probably was CD63, which could be the lung cancer marker for clinic dignosis.