| Lung cancer is one of the most common malignant tumor in the worldwide, and it's morbidity and mortality are both on the first rank of all malignant tumors. In China, it's morbidity and mortality showed a rapid increase during the past 20 years. Chinese Cancer Prevention and Control Program (2004-2010) showed that lung cancer has become the most common cancer in China. So far, the five-year survival rate of lung cancer is less than 15% from diagnosis to death. One of the major reason is that the lung cancer patients who have been definited are often already in advanced stage or metastasis. Therefore, early diagnosis and early treatment are the preferred strategy for the treatment of lung cancer. Phage display peptide library as a powerful tool can be used to explore the interaction during proteins. It establish direct connections between large numbers of random peptides and DNA. Beacause of convenient and effective, it has been widely used in screening and researching anticancer drugs and tumor biomarkers.The lung squamous carcinoma cell line NCI-H1299 was used as the antigen and lung normal cell line MRC-5 was used as control for subtraction biopanning from Ph.D.-12TM phage display peptide library. After 3 rounds of panning, 106 phage clones have been received. The enrichment of phages which are binding with NCI-H1299 is about 100-fold. Then, 120 phage clones were randomly selected from the 106 phage clones and amplified. Meanwhile, some phage clones were picked up from the original library as the control. The affinity and specificity with NCI-H1299 of phage clones were identified by ELISA and immunocytochemical staining. ELISA results showed that 45 phage clones had a high affinity to NCI-H1299(P/N>2), the positive rate was 37.5%. Besides, 24 of 45 showed more affinity to NCI-H1299 (P/N>2.2) and DAB staining results confirmed they could specifically bind on the surface of NCI-H1299.The 24 phage positive clones were amplified and sequenced. 10 different nucleotide sequence were obtained and named zs1, zs2…zs10 separately. Among these sequence, zs6 repeated 13 times. It was the only sequence which repeated most frequently and the repeat rate was 54.2%. It encod a 12 peptide consisted by the 3 Thr (T), 2 Leu (L), 2 His (H), 1 Ala (A), 1 Asp (D), 1 Arg (R), 1 Pro (P), 1 Trp (W), the amino acid sequence is **AD***HRPW*. zs6 has not yet been reported in any literature. There is no envidence showed there is any relationships between zs6 and tumors including lung cancer.Subsequently, the peptide zs6 and fluorescein isothiocyanate(FITC)-conjugated peptide zs6 were synthesized and purified. First, immunofluorescence assay was used to identify the specificity and affinity of peptide [zs6]FITC to lung cancer cells and human lung cancer tissue. The results showed that peptide [zs6]FITC has high affinity with lung squamous carcinoma cell line NCI-H1299 and lung adenocarcinoma cell line A549, but almost no affinity with normal lung cell line MRC-5. Meanwhile, the affiniy of peptide [zs6]FITC with human lung cancer tissue was significantly higher than normal tissues. At the same time, human lung cancer tissue microarray experimental results show that the positive rate of peptide [zs6]FITC using for identification of human lung cancer tissue was 24.8%. Then, tumor-bearing nude mice experiment was used to observe the distribution of peptide [zs6]FITC in vivo. It suggest that peptide zs6 could bind specifically to lung tumor in vivo and there were no distribution in nude mice brain, heart, lung and other normal tissues. But the peptide [zs6]FITC still can be found in the major metabolic organ, such as liver, kidney.In order to search the binding site of peptide zs6 with lung cancer, Biotin- conjugated peptide zs6 was synthesized and purified. Peptide [zs6]Biotin was immobilized on ELISA plate which has coated with avidin. Then, peptide zs6 was panning with human lung cDNA library. After 4 rounds screening, several phage clones were picked and amplified. Plasmid of phage were extracted and amplified by PCR, then the PCR products were purified and sequenced. Bioinformatics analysis results revealed that the peptide-specific lung cancer antigens are Envoplakin, ATPase and 987bp unknown proteins. (1)At present, there is no reports about the relationships between Envoplakin and lung cancer. Bioinformatic analysis found that Envoplakin have a higher expression in non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma) and a lower expression in small-cell lung cancer. Thus, Envoplakin may be used as a diagnostic markers of non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma). (2) ATPase exist in a variety of life forms. So the study of it do not have specific values; (3) Bioinformatics analysis showed that 987bp sequence has no homologous with the human genome. It indicated that we may have found a new tumor-specific antigen. If the expression of it have difference between lung cancer and normal tissues, it may used as a new target for diagnosis and treatment of lung cancer and lay a foundation for targeting therapy. |
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