| 唐敏怡,莫广财,区家铭,王 帅,吴晓丽,赵子建,李芳红.硒结合蛋白2的表达及与FKBP38蛋白的相互作用[J].,2024,(17):3205-3210 |
| 硒结合蛋白2的表达及与FKBP38蛋白的相互作用 |
| Expression of Selenium Binding Protein 2 and Their Interaction with FKBP38 Protein |
| 投稿时间：2024-03-17 修订日期：2024-04-23 |
| DOI：10.13241/j.cnki.pmb.2024.17.002 |
| 中文关键词: SELENBP2蛋白 重组pcDNA3.1-6×HIS-mSELENBP2质粒 FKBP38蛋白 免疫共沉淀 肝脏 |
| 英文关键词: SELENBP2 protein Recombinant pcDNA3.1-6×HIS-mSELENBP2 plasmid FKBP38 protein Co-immunoprecipitation Liver |
| 基金项目:广东省重点领域研发计划项目（2019B020201015） |
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| 中文摘要: |
| 摘要 目的：构建重组pcDNA3.1-6×HIS-mSELENBP2质粒并探讨SELENBP2蛋白与FKBP38蛋白的相互作用。方法：首先以小鼠肝脏总RNA为模板，根据小鼠SELENBP2基因CDS序列设计的引物通过RT-PCR扩增SELENBP2蛋白编码序列，通过基因工程技术进行限制性核酸内切酶酶切、与pcDNA3.1-6×HIS载体连接并转化入JM108感受态大肠杆菌扩增，挑选阳性单克隆大肠杆菌并提取质粒后得到重组pcDNA3.1-6×HIS-mSELENBP2质粒。重组质粒经测序分析鉴定后，使用Lipofectamine 3000转染试剂转染至小鼠肝细胞--AML12细胞中培养30 h，采用Western blot法检测蛋白表达情况。使用AlphaFold-Multimer预测SELENBP2蛋白与FKBP38蛋白相互作用的可能性及可能相互作用的结构域。最后，将重组质粒转染入过表达FKBP38蛋白的AML12细胞中，培养30 h后收集细胞蛋白样品，进行免疫共沉淀实验。结果：重组pcDNA3.1-6×HIS-mSELENBP2质粒测序结果显示插入的SELENBP2蛋白编码序列与GenBank中SELENBP2基因CDS序列相同，且转染重组质粒后，AML12细胞能有效表达SELENBP2-HIS融合蛋白。AlphaFold-Multimer结果显示SELENBP2蛋白与FKBP38蛋白相互作用的可能性较高，且作用于FKBP38蛋白的TPR结构域。免疫沉淀实验结果显示，在AML12细胞中，SELENBP2蛋白与FKBP38蛋白存在相互作用。结论：重组pcDNA3.1-6×HIS-mSELENBP2质粒构建成功并能在AML12细胞中正确表达，为进一步研究SENELBP2蛋白的功能奠定了基础。SELENBP2蛋白与FKBP38蛋白相互作用，为SELENBP2蛋白的研究进一步拓宽了思路，为深入研究SELENBP2蛋白在肝脏中的生物学功能提供了新方向。 |
| 英文摘要: |
| ABSTRACT Objective: This study aims to construct pcDNA3.1-6× HIS-mSELENBP2 recombinant plasmid and explore the interaction between SELENBP2 and FKBP38 proteins. Methods: Firstly, total RNA from mouse liver tissue was isolated as a template and designed the primers according to the CDS sequence of the mouse SELENBP2 gene to synthesize the SELENBP2 protein coding sequence through RT-PCR. The SELENBP2 gene sequence was recycled by restriction endonuclease and connected by ligase with pcDNA3.1-6× HIS plasmid vector, and then transformed into a JM108 competent Escherichia coli for amplification. After selecting positive monoclonal E. coli and extracting plasmids, the pcDNA3.1-6× HIS-mSELENBP2 recombinant plasmid was obtained using genetic engineering technology. Then, after sequencing analysis and identification, the recombinant plasmid was transfected into AML12 cells using Lipofectamine 3000 transfection reagent and cultured for 30 h to detect protein expression using Western blot analysis. Using AlphaFold Multimer to predict the possibility of interaction between SELENBP2 and FKBP38 proteins, as well as the possible domains of interaction. Finally, the recombinant plasmid was transferred into AML12 cells which over expressed FKBP38 protein and after 30 h of cultivation, cell protein samples were collected for immunoprecipitation test. Results: The sequence analysis results of pcDNA3.1-6×HIS-mSELENBP2 recombinant plasmid showed that the inserted SELENBP2 protein coding sequence was in accordance with the expected sequence as the CDS sequence of the mouse SELENBP2 gene in GenBank report. After transfection of pcDNA3.1-6×HIS-mSELENBP2 recombinant plasmid into AML12 cells, the SELENBP2 HIS fusion protein can be effectively expressed. The results of AlphaFold Multimer showed that the possibility of interaction between SELENBP2 and FKBP38 proteins is high, and it acted on the TPR domain of FKBP38 protein. There is an interaction between the SELENBP2 protein and the FKBP38 protein in AML12 cells according to the co-immunoprecipitation experiment. Conclusion: The pcDNA3.1-6×HIS-mSELENBP2 recombinant plasmid has been successfully constructed and can be correctly expressed in AML12 cells, laying the foundation for further research on the function of the SENELBP2 protein. The interaction between SELENBP2 and FKBP38 proteins further broadens the thinking of the study of SELENBP2 protein and provides a new direction for the further studying of the biological function of SELENBP2 protein in the liver. |
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