摘要: |
目的:构建带myc/his标签的pcDNA3.1/TDP-43及其截断体质粒,探讨myc/his-TDP-43融合蛋白对tau外显子10可变剪接的影响。方法:采用PCR法扩增TDP-43基因及其各截断体,将扩增产物和载体pcDNA3.1双酶切后回收,连接载体和目的片段,获得重组质粒。在HEK-293FT细胞中转染pcDNA3.1/TDP-43·myc·his及其截断体质粒,Western 印迹法检测相关蛋白的表达。TDP-43或siTDP-43和tau迷你基因SI9/SI10共转,RT-PCR检测TDP-43对tau外显子10可变剪接的影响。结果:成功构建了pcDNA3.1/TDP-43·myc·his全长及各截断体质粒,并在HEK-293FT细胞中检测到其蛋白表达。Myc/his-TDP-43融合蛋白可呈剂量依赖性促进4R-tau的表达(P<0.05或0.01)。结论:Myc/his TDP-43融合蛋白可促进4R-tau的表达,携带myc/his标签不影响TDP-43对tau外显子10可变剪接的促进作用,为后续研究奠定了基础。 |
关键词: 反式激活反应DNA结合蛋白 tau蛋白 可变剪接 阿尔茨海默病 |
DOI:10.12025/j.issn.1008-6358.2018.20180215 |
分类号:Q 786 |
基金项目:大学生创新创业计划国家级项目(201710304031Z),江苏省高校自然科学基金面上项目(16KJB310013),南通市市级科技计划项目(MS12015058),南通大学博士科研启动基金(15B30). |
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Effect of myc/his-TDP-43 fusion protein on alternative splicing of tau exon 10 |
MIAO Shi-chen,QU Shu-ting,LU Ying,TANG Xiao,REN Liang-liang,GU Jian-lan*
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School of Medicine, Nantong University, Nantong 226001, Jiangsu, China
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Abstract: |
Objective:To construct plasmids of TDP-43 and its truncations to pcDNA3.1 vector with myc/his tag and explore the effect of myc/his-TDP-43 fusion protein on the alternative splicing of tau exon 10. Methods:The cDNA of TDP-43 and its truncations were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 after endonuclease cleavage. The recombinant plasmids were transfected into HEK-293FT cells and the expression of TDP-43 were detected by Western blots. TDP-43 or siTDP-43 was co transfected with tau mini gene, SI9/SI10, and the alternative splicing products of tau exon 10 was detected by RT-PCR. Results:pcDNA3.1/TDP-43·myc·his and the truncation plasmids were constructed successfully. Myc/his TDP-43 and truncation fusion proteins were expressed in HEK-293FT cells. Myc/his-TDP-43 fusion protein induced the expression of 4R-tau. Conclusions:Myc/his-TDP-43 fusion protein promoted 4R-tau expression. The truncation plasmids of TDP-43 might be helpful for the future study of the function of different domains of TDP-43 in tau pathology and alternative splicing of tau exon 10 in neurodegenerative diseases. |
Key words: TDP-43 tau protein alternative splicing Alzheimer disease |