| Research background:In the nervous system,synaptic vesicle recycling is the bridge of information transmission between neurons.Synaptic vesicles complete a of synaptic vesicles cycle by exocytosis and endocytosis.Research has shown that the function of neuron synaptic of epilepsy patients is apparent anomaly. This pathological synaptic increase nerve impulse transmission frequency from several or dozens of times per second in normal up to dozens to hundreds of times per second by rapid cycling and regeneration of synaptic vesicles, which causes epileptiform discharge spread rapidly. Because of releasing neurotransmitters by synaptic vesicles is a complex process which is mediated by a variety of proteins and regulated precisely. Therefore, more and more researches focus on proteins involved in the process and the role of each other.Dynamin-1 is a kind of G protein, a member of the GTPase family. It is composed of four functional domains:N-terminal GTPase domain, pleckstrin homology domain (PH), GTPase effector domain (GED) and proline-and arginine-rich domain (PRD). It is different with other members in the family such as dynaminⅡ,Ⅲ, dynamin-1 is specific expressed in the synaptic site of the neurons, brain and neuroendocrine cells.It is a presynaptic membrane protein. At present a large number of studies have shown that dynamin-1 play an important role in synaptic vesicle recycling and vesicle transport in the Golgi body. Therefore, the reseach of related effect proteins of dynamin-1 help us to investigate the mechanism of dynamin-1 in synaptic vesicle recycling and also help us to find an effective target of clinical treatment of epilepsy.Objective:The research of dynamin-1 in synaptic vesicle recycling in the mechanisms and pathogenesis of epilepsy, the use of molecular cloning of dynamin-1 in the structure domain, GST Pull-down joint mass spectrometry to understand the interaction of dynamin-1 protein.Method:Firstly we constructed prokaryotic expression plasmid of four functional domains in dynamin-1 (pGEX-4T-2-PH,pGEX-4T-2-PRD,pGEX-4T-2-GED,pGEX-4T-2-GTPase) respectively by the technology of molecular cloning, Induced expressed and purificated four recombinant plasmids by E. coli expression system and glutathione agarose creamy purified by column, obtain four fusion proteins:GST-PH,GST-PRD,GST-GED,GST-GTPase.Then we make protein-protein interactions between brain synaptosome of rats and the four fusion proteins respectively by the technology of GST pull-down, and do analysis and identification of protein patterns.Results:1.We have successfully constructed the expression of these four plasmids (pGEX-4T-2-PH, pGEX-4T-2-PRD, pGEX-4T-2-GED, pGEX-4T-2-GTPase);these four expression plasmid in E.coli coli BL21 (DE3) expression in the high condition:25℃,1 mmol/L IPTG,induced 4 h, by Glutathione Sepharose 4B affinity chromatography were purified fusion protein of high purity.2.The LC-MS mass spectrometry the protein in the synaptosome with dynamin-1 has a total of 35 interacting proteins.Conclusion:In this reaserch, by the technology of GST pull-down we obtain 35 protiens which is classified of Synaptic vesicle associated protein cytoskeleton proteins and metabolic enzymes.These proteins we obtain are related with synaptic vesicle recycling intimately.We do not know the relationship between these proteins and dynamin-1 and need further study. The study has laid the foundation for clarifying the function.regulatory mechanism of dynamin-1 and cracking the pathway/protein network of synaptic vesicle recycling.
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