Screening Mimicry Peptide Of IL-2Rα And Researching Into Its Effects On Skin Allograft Rejection In Mice

IL-2 is an important cytokine in immune response that secreted by activated T lymphocyte. IL-2R which is composed ofα,βandγpeptide chains must bind with IL-2 to have biological effects.αpeptide chain is important for the specific binding of IL-2R and IL-2, and the signal can be delivered into the cell byβandγpeptide chains after IL-2 had binded with high affinity IL-2R that hasαpeptide chain.Bcauseαpeptide chain only expressed on T lymphocytes of patients such as organ transplantation,malignant tumour and some AD( autoimmune disease),but not expressed on testing T lymphocytes,which make possibilities to treat some diseases especially organ-graft refection.Cellullar immunologic response caused by T lymphocytes especially CD4+T and CD8+T is a main part of acute rejection.Because CD8+T lymphocytes need helps of cytokines secreted by CD4+T lymphocytes to make effects that CD4+T lymphocytes are more important for graft rejection.The mechanism of action of CsA which has made enmormous economic retums and social effects is inhibition of IL-2 and IL-2R, but its application was restricted by its renal toxicity and price.Besides mouse mAb of anti-human IL-2Rαcombined with other immunodepressants could prolong the survival time of graft,but adverse reactione of mouse mAb cut down the therapeutic efficacy.So it is imperative to manufacture a new immunosuppressant of IL-2/IL-2Rα.Investigations have showed that small peptide reasonably engineered could specially imimate or interrupte inter-recognition of protein,so peptide library based on this theory provided technique ensure on blockage drugs of peptide.Our study mainly utilizated phage display technique to screen the mimitope of IL-2Rαand synthetized the peptide based on above results as immunosuppressant which has higher specificity and permeability,lower side effects.Immunosuppressive activities of the small peptide were detected by experiments in vivo and in vitro and effects on skin allograft rejection in mice were studied at the same time .we want to develop it into an peptide immunosuppressant for clinical application. The project includes four parts as following:PartⅠScreening the mimotope of IL-2Rα1. Preparation of the target molecule(mAb-5G1)Ascites from BALB/c mice immunized by 5G1 hybridoma cell were purified by ammonium sulfate precipitation and column chromatography of DEAE-Sephadex A-50.The relative molecular weight of the purified antibody is 150kD which detected by 8%SDS-PAGE and its purity is beyond 98%.mAb-5G1 could specially bind with MT-2 cell(which highly expresses IL-2Rα),which was detected by ELISA.So mAb-5G1 can be made as a target molecule for biopanning the phage library.2. Screening phage cyclical 9-mer peptide libraryThe phage cyclical 9-mer peptide library was screened for 5 turns,while mAb-5G1 was made as target molecule.We obtained the higest affinity phage by lowing concentration of target molecule,decreasing action time of taget molecule and peptide library,increasing time and number of washing.According to the recovery rate specific phage clones that could bind with mAb-5G1 were evidently enriched.3. Identification the antigenicity of specific phage clonesAfter 5 turns screening 20 phage clones randomly picked were made to bind with mAb-5G1 and BSA,then determined the titre of eluant.Results indicated that 5 clones could bind with mAb-5G1 tightly,while poorly with BSA. Character of such 20 phage clones to bind with mAb-5G1 was determined by ELISA and dot-blot,and results was the same as above.4. DNA analysis of positive phage clonesResults indicated that two DNA sequence is the same among the 5 clones.Conservation sequence(Lys-X-{X}-Lys-Gly) deduced by those relative amino acid sequences and non-homology was between such sequences with human IL-2Rαeither DNA or amino. So the sequence is the mimotope of IL-2Rα.Part II Synthesis and analysis mimicry peptide of IL-2Rα1. Synthesis of mimicry peptide (CP)The cyclical seven peptide-CP(amino sequence: Cys-Asn-Ala-Leu- Lys-Arg-Lys-Gly-Cys) was chemosynthesized based on conservation sequence and amino frequency of human IL-2Rαprotein.The relative molecular weight of CP is 990.2 and its purity is beyond 95%.2. Toxicity analysis of CPToxicity of CP is determined by co-culture with mouse splenic lymphocyte.Results indicated that every concentration of CP were avirulent to splenic lymphocyte which survival rate was 100%,while survival rate of CsA and cyclophosphamide at 100nmol/L was 98% and 95%.3. Imitation the conjuntion of CP and human IL-2 by computerResults indicated that CP formed three-dimensional struture and stably conjugated with IL-2.There were three hydrogen bond between CP and IL-2.Part III Immunological activity analysis of CP1. Assays of ConA-induced splenocyte cycle in miceEffects of CP on splenocyte cycle in mice were detected by FACS .Results indicated that CP could block the splenocyte cycle at G0/G1 stage;Percentage of cells at G0/G1 stage went up from 22.58% to 55.53% and blockage effect could be enhanced by the combination CP and CsA.Such results matched with the following detected results of immunological activities and also demonstrated that CP as mimic peptide of IL-2Rαhad immunosuppressive action.2. Assays of lymphproliferation responseCP could dose-dependently inhibite mouse splenic lymphocyte proliferation induced by ConA ;Depressant effect could be enhanced by the combination CP and CsA. The inhibition rate of CP(31.3μg/ml )+CsA was 60.7±5.1%,which doubled CP's ,and the same as CsA+DEX.Human PBL proliferation and mouse MLR indicated the same results. CP and ZA scarcely inhibited mouse splenic lymphocyte proliferation induced by LPS.Because LPS mainly acts on B lymphocyte ,there were non-direct effects for CP on humoral immunoresponse. The result of lymphoblast proliferation induced by IL-2 indicated that the reaction could be dose-dependently inhibited and also verified that CP was mimicry peptide of IL-2Rα.3. Assays of delayed type hypersensitivity(DTH)CP could inhibite DTH in mice and suppressive effect could be enhanced by the combination CP and CsA(inhibition rate: 35.1±1.9%). CP also could inhibite hyperplasia of spleen and thymic.The results indicated that CP could inhibite activation and proliferation of CD4+ Th1 cells,which matched with the result of skin allograft rejection in mice.4. Assays of graft versus host rejection(GvHR)CP had a little effect on GvHR in rats,but suppressive effect could be enhanced by the combination CP and CsA(inhibition rate: 43.7±4.3%) and higher than CsA alone(inhibition rate: 37.5±2.2%). Part IV Effects of CP on skin allograft rejection in mice CP could inhibite skin allogragt rejection in mice and induce host tolerance to skin graft,such as lower reactivity to specific stimulation(donor's spleen lymphocyte) and nonspecific stimulation(ConA) ;the survival time of skin graft prolongation;more lampros of organization and blood vessel in skin graft,et al.Inhibition could be enhanced by the combination CP and CsA.Results indicated that transplantation tolerance might be induced by clone anergy,number of CD4+ T decrease,retrieve of Th1 type cytokines polarization etc.In this study,we utilizate mAb-5G1 as target to screen phage library.After 5 turns we obtain 5 positive phage clones that had high affinity to target molecule.Conservative amino acids sequence:Lys-X-{X}-Lys-Gly was deduced by DNA sequences.CP had immunosuppressive activity determined by experiments and could inhibite skin allograft rejection in mice. CP may be developed for a novel immunosuppressant .However, many problems still need to be solved. There is a long way to go before practical use in clinic.