Screening And Identification Of The Peptides Specifically Binding To Lung Cancer By Using Phage Display In Vivo

Lung cancer divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), non-small cell lung cancer accounts for about 80 percent of the total number of lung cancer. Lung cancer is one of the world's malignant tumor becase of its highest morbidity and mortality, mainly due to lung cancer onset of occultd and the screening and treatment is no fundamental improvement. when lung cancer symptoms that the cancer is usually advanced, and poor prognosis with a total of five-year survival rate not more than 15 percent and symptomatic persons less than 10 percent. However, in the early diagnosis of lung cancer patients, the prognosis of surgical treatment in a marked improvement than advanced lung cancer, and the survival rate up to 70 percent. Therefore, actively looking for early diagnosis of lung cancer-specific markers is the most important problem to improve of lung cancer treatment. At the same time, for advanced lung cancer, to accelerate the development of targeted therapy drugs are an effective means to overcome the current highly toxic cancer chemotherapy, low-efficacy, easy-resistant.Objective: lung cancer cells A549 as the study target, using phage display technology in vivo panning and identificated peptides which combined specific with the lung cancer, which lay the foundation for the early diagnosis and targeted therapy of lung cancer. Methods: First of all, the lung cancer cells A549 were inoculated into nude mice when the tumors to a certain degree of value-added, then,using phage display technology in vivo, peptide library was injected into tumor-bearing vein in nude mice, and then get lung cancer with combination of tissue-specific exogenous phage peptides, to do so in vivo screening of three rounds.Then, randomly selected a group of monoclonal phage from the third round panning, using ELISA and Cell-mediated immunity chemistry to identificated the affinity to A549 lung cancer cells.Amplification of positive clones picked, then extracted phage single-stranded DNA according to the method of molecular cloning, which send to sequencing companies ,then the phage peptides exogenous nucleotide sequence were obtained according to sequencing results, and then the sequences were Bioinformatic analysised.Next to the specific identification of peptides, first chemical synthesis peptides, and fluorescence labeling FITC. Using fluorescent-labeled peptides to identified the specificity, affinity, in vivo distribution of peptide with lung cancer cells and tissue. Cell Immunofluorescence identification of peptide's affinity on lung cancer cell A549, NCI-H1299, hepatoma cell 7402, HepG2, normal lung cell WI-38, and normal liver cell LO2. Tissue Immunofluorescence identification of peptide's affinity on lung cancer tissue , lung normal tissue. The use of fluorescent peptides in the distribution of tumor-bearing nude mice in the experimental identification of peptides targeting distribution in the tumor-bearing nude mice.Results: After three rounds panning in vivo, the phage which combined with the tumor tissue is 104-fold enriched. Then randomly selected 30 monoclonal phages from the third round screening, ELISA were used to identified the affinity of the phages to lung cancer cells A549. ELISA results shows that 27 phage clones have high-affinity to A549 in all the 30 phage clones, in which, P/N> 2.5 more than 17 clones, and 17 in the above-mentioned clones, P/N> 3.0 have four clones , namely, 1, 12, 14 and 20 clones, at the same time, Cell-mediated immunity chemical were used to identify the affinity of the clones which P/N> 3.0 to A549 , compared with normal control group, DAB staining was significantly positive. Subsequently, 17 phage clones which identified by ELISA with the results P/N> 2.5 were amplified and sequenced, then the phage peptides exogenous nucleotide sequence were obtained according to sequencing results, of which 14 phage clones were inserted into the 6 different exogenous peptide nucleotide sequence, named zp1, zp2, zp3, zp4, zp5, zp6, the other 3 phage clones show that the loss of peptide sequence (empty phage).Bioinformatics analysis showed that the polypeptide of the amino acid sequence zp2 literature at home and abroad, has yet to report, and its function is unknown, so we intend to carry out zp2 peptide in system. Next peptide zp2 length was chemical syntheticed, and fluorescent labeling FITC (zp2-FITC). Cell Immuno- -fluorescence experiments showed that zp2-FITC has a strong affinity on A549, and also on NCI-H1299 , and that affinity was weak on 7402 and HepG2 ,However normal lung cells WI-38, normal liver cells LO2 don't have. Tissue Immuno- -fluorescence experiments showed that zp2-FITC on the combination of clinical lung cancer tissues was significantly higher than the normal control group. Experiments showed that the distribution of nude mice, zp2-FITC in tumor-bearing nude mice more tumor site distribution, better targeting, and secondly, in the liver, kidney organizations have distribution, however, in nude mice brain, heart, lungs, etc. normal tissues and their distribution is not.Conclusion: (1) successfully established tumor-bearing nude mice model; (2)Panning the lung cancer-specific phage in vivo by using the above-mentioned models; (3) Using ELISA and cell-mediated immunity chemical identified 27 phage clones has a high degree of specificity and affinity to A549; (4) After DNA sequencing, obtaining more than 6 peptide sequence, respectively, named zp1, zp2 ... zp6, by bioinformatics analysis, zp2 study has not yet been reported, in the subject zp2 to be take for systematic studies; (5) Using immunocytochemistry and immunohistochemistry identified peptide zp2 has high specificity and affinity to human lung cancer cells and tissues,and certificated peptide zp2 has significant tumor targeting;...