| 英文摘要
| Alzheimeru0027s disease is an irreversible and progressive neurodegenerative disorder with unclear causes. It is characterized by the formation of amyloid plaques and neurofibrillary tangles, decrease of interneuronal connections and apoptosis of nerve cells in the brain. The vast majority of Alzheimeru0027s disease belongs to late-onset and occurs after the age of 60. Although late-onset Alzheimeru0027s disease has not yet been found to have a specific causative gene, studies have shown that some risk factors may increase the risk of disease progression, such as APOE (apolipoprotein E) and RAGE (receptor of product of advanced glycation end products). To identify peptides that bind to APOE or RAGE and design peptide drugs which can modulate the disease-associated activities of these risk factors, it is desirable to obtain pure APOE and RAGE first. Plasmid constructs encoding fusion proteins of glutathion S-transferase and APOE/RAGE were used to overexpress GST-APOE and GST-RAGE fusion protein in Escherichia coli, which were later purified using affinity chromatography and size exclusion chromatography. However, the expression level of GST-APOE was too low to be purified; RAGE, on the other hand, formed inclusion bodies, probably due to its being a transmembrane protein with hydrophobic side chains aggregated and entangled out of the membrane. Although we tried to denature/renature inclusion bodies to obtain functioning RAGE, as protein conformation seemed to be changed during the renaturation process, the attempt to obtain purified protein used directly for screening purpose was unsuccessful. GST-protein-peptide interaction was therefore monitored by indirect antigen-antibody specific interaction instead.
In order to screen for interacting peptides efficiently, a bacterial surface display random peptide library combined with flow cytometric sorting technology was applied. Bacterial cell surface display technology uses recombinant DNA technology to express and localize foreign functional proteins on the surface of specific bacterial cells. In this dissertation, a random 15 peptide library was designed to screen out RAGE-interacting peptides, whose binding ability were preliminarily verified by dot-blot analysis. The identities of the candidate RAGE-interacting peptides were determined by sequencing analysis and subject to further studies. |
