【摘要】 目的：筛选与Neuritin相互作用的蛋白并鉴定，探讨Neuritin促进神经突起生长的可能分子机制。方法：利用酵母双杂交技术筛选与Neuritin相互作用的蛋白；构建重组质粒pcDNA3.1-His-Neuritin和pcDNA3.1-Myc-Syap1,转染至HEK 293细胞，利用免疫共沉淀(co-immunoprecipitation, CO-IP)明确Neuritin与Syap1(synapse-associated protein 1)的相互作用；过表达和低表达Neuritin,利用Western blot检测Neuritin对Syap1表达水平的影响；利用间接免疫荧光观察Neuritin过表达对Syap1转运以及对SH-SY5Y细胞轴突形态的影响。结果：对酵母双杂交阳性克隆进行测序和序列比对分析，共获得57条与Neuritin互作的蛋白，与Neuritin功能高度一致的Syap1列为候选蛋白；正向与反向CO-IP结果表明Neuritin和Syap1具有特异性相互作用；过表达和低表达Neuritin后，Western blot结果表明Neuritin是Syap1的负向调控因子；Neuritin过表达可以促进SH-SY5Y细胞中Syap1蛋白向轴突终末转运，并可促进轴突延长。结论：Neuritin与Syap1具有特异性相互作用，是Syap1的负向调控因子，并且过表达Neuritin可以促进Syap1蛋白向轴突终末转运和神经突起延长。更多还原

【Abstract】 Objective: To screen and identify the proteins interacting with Neuritin and explore the possible molecular mechanism by which Neuritin promotes neurite outgrowth. Methods: The proteins interacting with Neuritin were screened by the yeast two-hybrid technique. Recombinant plasmids pcDNA3.1-His-Neuritin and pcDNA3.1-Myc-Syap1 were constructed and then transfected into HEK 293 cells, and the interaction between Neuritin and Syap1 was determined by CO-IP. The effect of Neuritin expression levels on Syap1 expression levels was detected by Western blot analysis. Indirect immunofluorescence was used to observe the effects of Neuritin overexpression on Syap1 transport and axon morphology of SH-SY5Y cells. Results: A total of 57 proteins interacting with Neuritin were obtained by sequencing and sequence comparison analysis of the yeast two-hybrid positive clones. Syap1, which has a function that is highly consistent with Neuritin, was listed as a candidate protein. The forward and reverse CO-IP results showed that Neuritin and Syap1 had a specific interaction. Following the overexpression and underexpression of Neuritin, Western blot results showed that Neuritin was a negative regulator of Syap1. Neuritin overexpression can promote the terminal transport of the Syap1 protein in SH-SY5Y cells to the axon, and thereby promote axon elongation. Conclusion: Neuritin has specific interaction with Syap1 and is a negative regulator of Syap1. Overexpression of Neuritin can promote the terminal transport of Syap1 protein to the axon and neurite prolongation.更多还原