Design,Screening And The Role Of Oligopeptide Ligand For Tie2 In Targeted Gene Therapy

Gene therapy will be a novel anticancer strategy in addition to commonly used surgery,radiotherapy and chemotherapy. It could play an important role in preventing cancer recurrence and metastasis. Lack of an efficient targeting gene transfer system which can transfer gene into human cells was contributed to one of the key reasons for its having not been an efficacious anticancer therapy. So, It is very urgent for developing an cancer targeting gene transfer system.Our researches aim to construct Tie2 receptor-mediated nonvirus gene transfer system and to confirm its targetability and efficacy. It will provide evidences and practical guide for future cancer gene therapy.Section I Design and Screening of Oligopeptide ligand of Tie2【Objective】To design and screening oligopeptide ligand of defenited receptor and obtain candidate oligopeptide ligand of Tie2 receptor. 【Methods】(1) Angiopoietin-2-baesd hydropathic analysis and comparative sequence/structure analysis were used to design the peptide ligand of Tie2. (2) Cell line which had no apparent Tie2 expression was identified by RT-PCR and Western bloting; pCDNA3.0-ExTie2 plasmid was constructed and transferred into cells which had no apparent Tie2 expression; Cell line which stably expressed Tie2 was established via G418 selecting. Recombined human Tie2/Fc fusion and stable cell line were used as targets for screening 12-mer phage disply library. After 5-rounds screening , sequencing, ELISA,immunohischemostry and phage recovery assay were used to find out enriched positive phage clones.Then, peptides inserted in positive phage were chemistry synthsised. 【Results】 A 22 peptide which was designed based on structure of Ang2 protein was denoted as GA3 (WTIIQRREDGSVDFQRTWKEYK). Two peptide isolated by phage display from screening rh-Tie2 and SMCC7721-ExTie2 cells which stably expressing Tie2 were respectively added tyrosine(Y) to their C-terminit and named as GA4 (HATGTHGLSLSHY)and GA5 (NSLSNASEFRAPY). 【Conclusions】Approaches applied in designing and screening peptide ligand seem promising for developing oligopeptide ligand for receptors. The effectivity of candidate oligopeptide ligands waited for further validation.