大鼠脊髓神经元细胞核中TET3与OGT相互作用的分析与鉴定  

Analysis and identification of the interaction between TET3 and OGT in the nuclei of rat spinal cord neurons

作　　者：王丽萍 许汀 邱晟 WANG Liping;XU Ting;QIU Sheng(Department of Anesthesiology,Dongfang Hospital Affiliated to Xiamen University(School of Medicine of Xiamen University)Fuzhou General Clinical Medical College of Fujian Medical University 900th Hospital Teaching Base of Joint Logistics Support Force for Fujian University of Traditional Chinese Medicine,Fujian Province,Fuzhou 350025,China)

机构地区：[1]厦门大学附属东方医院(厦门大学医学院)福建医科大学福总临床医学院,福建中医药大学联勤保障部队第九〇〇医院教学基地麻醉科,福建福州350025

出　　处：《中国医药导报》2023年第9期7-11,共5页China Medical Herald

基　　金：国家自然科学基金青年科学基金项目(31700740);福建省科技对外合作项目(2020I0035)。

摘　　要：目的分析与鉴定大鼠脊髓神经元细胞核中10-11易位蛋白3(TET3)与氧连氮-乙酰葡萄糖胺转移酶(OGT)的相互作用。方法原代培养大鼠脊髓神经元并提取总RNA,采用RT-PCR法获得TET3和OGT基因片段。采用DNA重组技术在TET3、OGT蛋白编码序列的羧基端融合Flag和HA标签蛋白,构建pcDNA3.0-TET3-Flag-HA和pcDNA3.0-OGT-Flag-HA重组融合蛋白质粒,经双酶切验证后分别转染大鼠脊髓神经元。48 h后提取神经元核蛋白,采用Western blot法验证融合蛋白TET3-Flag-HA和OGT-Flag-HA;采用串联亲和纯化法富集与TET3或OGT结合的蛋白复合物,经电泳分离和质谱分析筛选出与TET3或OGT相互作用的候选蛋白,并采用免疫共沉淀进行验证。结果证实重组融合蛋白质粒pcDNA3.0-TET3-Flag-HA和pcDNA3.0-OGT-Flag-HA构建成功,融合蛋白TET3-Flag-HA和OGT-Flag-HA在大鼠脊髓神经元细胞核中表达。在大鼠脊髓神经元细胞核中,与TET3相互作用蛋白是OGT,与OGT相互作用蛋白包括TET3和视网膜母细胞瘤结合蛋白5。结论TET3与OGT在大鼠脊髓神经元细胞核中形成复合体,可能参与调控相关功能蛋白的转录表达。Objective To analyze and identify the interaction between ten-eleven translocation protein 3(TET3)and O-linked-β-N-acetylglucosamine transferase(OGT)in the nuclei of rat spinal cord neurons.Methods The spinal cord neurons of rats were primarily cultured and total RNA was extracted,and TET3 and OGT gene fragments were obtained by RT-PCR.The tag proteins of Flag and HA were fused to the carboxyl end of TET3 and OGT protein coding sequences by DNA recombination technology.The recombinant fusion protein plasmids of pcDNA3.0-TET3-Flag-HA and pcDNA3.0-OGT-Flag-HA were verified by double enzyme digestion,and then transfected into the rat spinal cord neurons.The nucleoproteins of spinal cord neurons were extracted 48 hours later.The fusion proteins of TET3-Flag-HA and OGT-Flag-HA were verified by Western blot;the protein complexes bound to TET3 and OGT were enriched by Tandem affinity purification,the candidate proteins interacting with TET3 and OGT were screened by electrophoresis and mass spectrometry,and verified by co-immunoprecipitation.Results It was confirmed that the recombinant fusion protein plasmids of pcDNA3.0-TET3-Flag-HA and pcDNA3.0-OGT-Flag-HA were successfully constructed,and the fusion proteins of TET3-Flag-HA and OGT-Flag-HA were expressed in the nuclei of rat spinal cord neurons.In the nuclei of rat spinal cord neurons,the protein interacting with TET3 was OGT,and the proteins interacting with OGT were TET3 and retinoblastoma binding protein 5.Conclusion TET3 and OGT form a complex in the nuclei of rat spinal cord neurons,which may be involved in regulating the transcriptional and expression of some functional proteins.

关 键 词：脊髓 神经元 10-11易位蛋白 氧连氮-乙酰葡萄糖胺转移酶 

分 类 号：R744[医药卫生—神经病学与精神病学]