Prokaryotic Expression, Purification And Identification Of The M3-M4 Loop In The Essential Subuint Of Human NMDA Receptor

The N-methyl-D-aspartate(NMDA) subtype of ionotropic glutamate receptor is a prominent ligand-gated ion channel. NMDA receptor channels play important roles in various physiological function such as synaptic plasticity and synapse formation undertaking memory,learning during development. They are also important for a variety of pathological states including acute and chronic neurological disorders, psychiatric disorders, and neuropathic pain syndromes. Excitotoxicity caused by supraphysiological stimulation of NMDA receptor may be a common pathological condition to these diseases. Because of their central involvement in the cascade leading to neuronal death following a variety of cerebral insults, pharmacological NMDA receptor antagonists have been evaluated for potential clinical use. These drugs are effective in many experimental animal models of disease,and some of these compounds have moved into clinical trials. However, the initial enthusiasm for this approch has waned because their side-effects. So an alternative approach to antagonize NMDA receptor must be found quickly.After analyzing the physicochemical characteristic, antigenicity and autoimmunogenicity of polypeptides in human NR1 related to activation of NMDA receptor, the extracellular M3-M4 segments of NR1 was determined as the readily interfered target by immunization to control the activation of NMDA receptor. So a new feasible vaccination strategy targeting this segment would be expected to build with therapeutic potential for excitotoxic brain injury.This approach would open up a newavenue for neurological and phychiatnc disorders such a5 stroke .trauma, epilepsy and amentia.The M3-M4 segment of human NR1 consists of 163 ammo acids with the relative molecular weight 18800 In order to achieve further immunogenicity and application studies. this article acquire M3-M4 loop peptide by gene engineering technique. Our work mainly includes:1.Obtain a cDNA fragment encoding M3-M4 loop in human NR1 by RT-PCR using the total RNA extracted from a human brain glioma as template2.Redesign optimally the PCR primer according to a computer-aided high expression model for the foreign gene in pBV220 vector. The prediction of RNA secondary structure and the calculation of the codon adaptation were respectively finished through RNAfold and Goldkey software3 Use EcoR I and BamH 1 to digest the PCR product and the vector pBV220,link the above two products. construct the expression system: pBV-NR1L3.Transform competent DH5a with pBV-NR1L3. use Sangers method to sequence the pBV-NR1L3.4.Induce pBV-NR1L3 plasmid to express the objective protein by shifting the culture temperature from 37℃ to 42℃ Confirm optimal induction time and expression form through SDS-PAGE and confirm the percentage of expressed protein accounting for total bacteria protein by gel scanning.5.Purify the recombinant protein by preparative SDS-PAGE. Elution protein from gel by electrophoresis.After the protein was resumed, the pure powder was obtained by frost-dryness method.6.Characterize the expression product by a series of identifications, such as apparent molecular weight, immunoreactivity and peptide mass fingerprinting.In this work.we have successfully constructed the prokaryotic expression system pBV-NR1L3. The obtained gene was highly expressed with pBV220 vector by redesigning PCR primer. The result of gel scanning showed that the M3-M4 loop peptide accounted for 29% of total bacteria protein. The expressed product was purified and identified. The purity was up to 95% and the results of identification were the same as those anticipated. This study provided immunogen enough for further work. There would be a great meaning in building immune therapeutic strategy against excitotoxic brain injury.