Delivery Strategies In Mice For MAPs With A B-Cell Epitope Of M3-M4 Loop In The Primary Subunit Of NMDA Receptor

Excitotoxicity, associated with many CNS diseases such as cerebrovascular disease, head injury, brain neoplasm and following syndromes (cardio-brain syndrome, gastron-intestinal bleeding, et al), anxiety, depression, pain, epilepsy and dementia, were triggered primarily by massive Ca2+ influx followed by overactivation of glutamate receptor channels of the N-Methyl-D-aspartate (NMDA) subtype. Efficient prevention and cure measures should interrupt the neurotoxicities induced by the over-activated NMDA receptor (NR) in local brain effectively and timely. Although approaches to treat these disorders with antagonists of NR had 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, it is necessery to develop new therapeutic strategies.Vaccine-based strategies and antibody-therapy are novel explorations in the field. In the study, we mainly study the delivery strategies for MAPsNR1a—a mutiple antegen peptides with NE1(a B-cell epitope of M3-M4 loop in NR1a). We aslo screen phage display antibody libraries with MAPsNR1a. Main methods and results as followed:1.Synthesis, identification and evaluation of MAPsNR1a vaccine in miceOcta-branch antigen peptide conjugates (MAPsNR1a) was synthesized by solid- phage method. Mice were immunized with MAPsNR1a by intranasally (i.n.), intraperitoneally (i.p.) and subcutaneously (s.c.) and the antibody against MAPsNR1a were found in the last two routes. The immune safety was detected by behavioral observations, Morris water maze, step-through test, step-down test and HE staining of nasal mucosa. The results demonstrate that MAPsNR1a immunized by i.p. and s.c. were safety and effective. A new strategy for autoimmunological prevention and cure on excito- tocitic CNS diseases would be established.2.Cloning and identification of MAPsNR1a binding human single-chain antibodies from phage display antibody librariesThe NE1 antibodies have protection against excitotocity. Fc of monoclonal antibody from mouse could produce many side effects in human body and the cost is also very high with traditional preparation. So affinity panning was used to select NE1 binding clones from Human Single Fold scFv Libraries I+J. Bingding, washing and reinfection were ensued sequentially, after 4 rounds panning positive clones were verified to show higher affinity to MAPsNR1a by ELISA. And the I clones cloned in J phagemid particles and MAPsNR1a-binding ScFv clones were obtained. The research would provide a foundation for the functional study and preparation of protective ScFv against excitotoxicity.