| Excitotoxicity, associated with stroke and neurodegeneration, is triggered primarily by massive Ca2+ influx arising from overactivation of glutamate receptor channels of the N-Methyl-D-aspartate (NMDA) subtype. Although approaches to treatment of these disorders with antagonists of NMDA receptor (NR) have been testified to be effective in animal models, successful therapy in humans was limited by the severe side effects of complete NR blockade for their space window and time window. Therefore, there is a necessity to develop new therapeutic strategies. Vaccine-based strategies and antibody-therapy are novel exploration in this field. In this study, we mainly investigate the antigenicity, immunogenicity of NRla (NR key subtype) and the protection of antibody against NR1 in excitotoxicity, which provide experimental foundation for immnotherapeutic strategy against excitotoxicity. Main methods and results are as followed:1 Epitope analysis of agonist-binding region of NRlaPhysicochemical properties and antigenicity of two agonist-binding regions of NRla were analyzed through bioinformatics: domain P1 containing 151 amino acid residues preceding the first transmembrane domain of the human NRla, domain P2 with 144 residues following the third transmembrane domain .Four parameters including Hopp-Woods and Kyte hydrophilicityjanin accessibility, Karplus-Schulz flexibility, and Welling antigenicity were used to determine the antigenic sites, and Prosite programme and Chou-Fasman method were employed to analyze their related sequence motif and the secondary structures. Finally, comparison of the comprehensive predictions withsome of the available experimental information was made. Compared with P1, P2 may be of higher antigenicity. The antigenic sites in P2 were dipersed rather uniformly, including or nearing receptor-activating sites: they are FLVLDRPEERI, RLRNPSDKFIYAT, YRHMEKHNYES, RDNKLHAFIW, ASQKCDLVTT, KDSPWKQNVS, ELKSHENGF. P2 may be more easily used as a molecular target than P1 in immunization intervention to control the activation of NMDAR.2 Specific epitope analysis in M3M4 loop targetTo determine the B cell epitope of a monoclonal antibody against the M3-M4 loop of NMDAR1, a random phage displayed dodecapeptide library was screened with the monoclonal antibody MAB363 against the M3M4 loop of NMDAR1. After four rounds of biopanning, the peptide sequences of positive phage clones were determined and analyzed by DNA sequencing. ELISA and competitive inhibition assay. A positve clone was found (clonel: VHTNPSTWQPIL). The binding between clone 1 and MAB363 were competitively inhibited by the recombined M3M4 loop expressed by E.Cpli DH5 a ; The binding between M3M4 and MAB363 could be competitively inhibited by one of solid-synthesized epitope peptides: RLRNPSKD. There was a same sequence among them: NPS. Deleted with NPS, the_ peptide could not inhibit the binding of MAB363 to M3M4. These results demonstrate that NPS in M3M4 loop is the B cell epitope recognized by MAB363, which may be important for developing a practical immunization strategy against excitotoxic brain injury.3 Immunogenicity of M3M4 loop recombinant peptide antigen of NR1, neuroexcitotoxicity protective effects and their mechanisms study of antiserum against M3M4(1) To explore the immunogenicity of M3M4 loop recombinant peptide vaccine, M3M4 recombinant peptide was injected subcutaneously to immunize Balb/C mice(H-2 ), CD4/CD8 T lymphocytes subsets, the cytokine levels in serum and the liter of specific humoral response were detected with FACS, indirect ELISA and ELISA separately. Results: compared with control mice, the percentage of CD4+ and CD8+ T lymphocytes were increased significantly, Th1 and Th2 cytokines levels -TNF-a ,IFN- r ,IL-4,IL-6 were significant increased significantly in serum. Titer of specific antibody against M3M4 was above 1:1000. Specific antibody against epitope(NPS) could also be detected in serum. Conclusion: M3M4 recombinated peptide antigen has immunogenicity and could induce Th...
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