Study On Epitope Of NR1 In The Essential Subunit Of NMDA Receptor

The N-methyl-D-aspartate(NMDA, NR) subunit of ionotropic glutamate receptor is a prominent ligand-gated ion channel. NMDA receptor channels play important roles in various physiological functions such as synaptic plasticity, synapse formation underlying memory and learning during development. They are also associated with various pathological states including neurological disorders and psychiatric disorders. Excitotoxicity caused by overactivation of NMDA receptor may be a common pathological condition to these diseases. Although approaches to treatment of these disorders with antagonists of NMDA receptor (NR) have been tested to be effective in animal models, successful therapy in humans was limited by the severe side effects of complete blockade for their space window and time window. Therefore, there is a necessary to develop new therapeutic strategies. Vaccine-based strategy is a novel exploration in this field. In this study, we first prepared and purified the R1JHL monoclonal antibody against NR1 membrane protein, and then with the help of this specific antibody we screened a random phage displayed dodecapeptide library. The study provide experimental foundation for the further determining of the B cell epitope of JHL monoclonal antibody against NR1 membrane protein and immunotherapeutic strategy against excitotoxicity. Main methods and results are as followed:1. Preparation and purification of JHL monoclonal antibody: R1JHL hybridoma cells were injected into the abdominal cavity of BalB/C mice to prepare the R1JHL monoclonal antibody anti-NR1. The antibodies were purified by Protein A-Sepharose CL-4B affinity chromatography and the purity of the purified antibodies were detected by 15% SDS-PAGE. Western-blot confirmed that the R1JHL monoclonal antibody could specifically recognize the NR1 membrane protein extracted from rat brain tissues.2. Screening of NMDAR1 epitope: To determine the B cell epitope of a monoclonal antibody against NR1, a random phage displayed dodecapeptide library was screened with the R1JHL monoclonal antibody against NR1. After four rounds of biopanning, the peptide sequences of positive clones were determined and analyzed by DNA sequencing and ELISA assay. A positive clone was found (HYNLSSDRKVRL). There was a consentient sequence DRK in NR1. This result demonstrated that DRK in NR1 might be the B cell epitope recognized by R1JHL.Above all, we purified the R1JHl monoclonal antibody by affinity chromatography. A random phage displayed dodecapeptide library was screened with R1JHL monoclonal antibody anti-NR1 and obtained a positive clone which has the consentient sequences (DRK) with NR1. Our study will provide a foundation for the further determining of the B cell epitope of JHL monoclonal antibody against NR1 and immunotherapeutic strategy against excitotoxicity.