Screening Of Mimic Peptides Binding Specifically The First And The Second Extracellular Loop Of CC Chemokine Receptor 5 (CCR5) From A Phage Display Peptide Library, And The Study On The Therapeutic Effect Of Mimic Peptides In EAE Mice

Purpose:To obtain mimic peptide binding specifically with the first and the second extra-cellular loop of CC chemokine receptor 5(CCR5), and apply it to mice model of the (experimental autoimmune encephalomyelitis (EAE)), then, observing the treatment effect after applied mimic peptides to EAE mice.Methods:Phage display peptide library was used to screen peptides bonding specifically with the first and the second extra-cellular loop of CCR5. Through the assays of ELISA, the binding activity of the selective phage clones were identified and their DNA sequences were analyzed, then mimic peptide was synthesized and was injected into abdominal cavity of the EAE mice. Spinal cord tissues in each group were obtained, the pathologic changes of the spinal cord tissues were studied by H&E staining in EAE control and mimic peptide groups.Results:1. Screening phage clones with CCR5 bonding specifically by Ph.D.-7TM, ratios of the input-output of the phage step up gradually. It illustrate that enrichments of the phage with CCR5 are obvious.2. Twenty phage clones are determined from the thirteen times by ELISA. The result shows eighteen clones are positive, it shows that phages with CCR5 bonding specifically enrich efficiently.3. The amino acid sequences of the 10 positive phage clones boning specifically with CCR5 are determined separately, The results show that polypeptide sequence in the second extra-cellular domain such as SLPLPKP,STFTTTL,QTSSAAL accounts for 6,6,7 separately, while that polypeptide sequence in the third extra-cellular domain all of them is TPITQLL.4. EAE animal model is established successfully, and apply the four peptides to EAE model mouse. The result shows that mouse in EAE grouping start to onset in 13 days, and reach peak on 25 days approximately, the attack rate is 80%.The average scores of the clinical score is 3. However invasion time obviously delay in the simulate peptide grouping, and reach peak on 40 days approximately.5. By H&E staining, it reveal that multiple inflammatory cells infiltrating in the structure of the spinal cords EAE mice. The neuroglial cells and horizontal cells take the majority of the inflammatory cells. There are many inequality domains of the size which is lack of myelin in white substances, and there is a direct association with severity extent of the clinical symptom of the EAE mice and level in the domain of the demyelination.Conclusion:Early treatment of EAE mice with CCR5 mimic peptides effectively ameliorated the development of disease and provide a significant protective effect compared to control mice. Further histological analysis demonstrated that spine cord infiltration of monocytes and lymphocytes decreased significantly in CCR5 mimic peptides treated mice, while abundant inflammatory cells were seen in the issues of the EAE mice, and there is evident demyelination change in white matter. Our datas show that CCR5 mimic peptide has protective effects in EAE, indicating that CCR5 mimic peptide could be a candidate drug in the treatment of EAE and clinical treatment of human being MS. In conclusion, all of the four short peptides respectively embody the role of suppressing and delaying the development of the EAE in different levels. The average inhibition ratio is 43%, the effect is significant(P<0.05). Thus the research shows that simulation peptide bonding competitively with CCR5 play important role during the course of EAE.