大鼠脊髓神经元细胞核中TET3与OGT相互作用的分析与鉴定

doi:10.20047/j.issn1673-7210.2023.09.01

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Abstract 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.

Key words： Spinal cord Neurons Ten-eleven translocation protein O-linked-β-N-acetylglucosamine transferase

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	WANG Liping* XU Ting* QIU Sheng

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WANG Liping* XU Ting* QIU Sheng. Analysis and identification of the interaction between TET3 and OGT in the nuclei of rat spinal cord neurons[J]. 中国医药导报, 2023, 20(9): 7-11.

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