Screening For Derivative Peptides Of C5a Receptor And Preliminary Study On Its Function

The complement C5a ,the product in complement system activation or of phagocytic cells, is an important mediator and chemotactic factor .It is a powerful pre-inflammatory factor in acute lung inflammation(ALI),sepsis,meningitis, rheumatic arthritis(RA) and glomerular nephritis. Therefore it is possible that in humans these diseases can markedly be treated by blockade of C5a .At present most inhibitors of C5a anaphylatoxin competed with C5a for binding to C5a receptor(C5a R) can effectively depress the activity of C5a . In this study,we paid more attention to C5aR and screened some new antagonists which were the derivative peptides of C5aR and can bind directly to C5a for blockade of C5a. We also had a preliminary study on their bioactivity and obtain the binding domains of C5a like receptor (C5L2) by multiply parameter analysis.The main purpose of our study is to screen and evaluate new inhibitors of human complement C5a . There are three parts in this study:1. Panning peptides binding to C5a anaphylatoxin Phage display was used to screen peptides which can bind to C5a. we firstly used Ph.D.-12 phage display peptide library to screen positive clones. Panning was carried out by incubating a library of phage-displayed peptides with a plate coated with the target peptide rhC5a ,then washing away the unbound phages ,and eluting the specifically-boud phages. The eluted phages were then amplified and taken through additional bingding/amplification cycles to enrich the pool in favor of binding sequences.After panning 3 rounds,individual clones were characterized by DNA sequencing.We randomly picked 151 clones for DNA sequencing. According to experimental results of Ph.D.-12 phage display peptide library, we used Ph.D.-c7c phage display peptide library to pan positive clones with the same method in order to screen more peptides and recognize precisely interaction sites between C5a and C5aR. In this case we randomly picked 138 clones for DNA sequencing .