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目的研究单极纺锤体蛋白激酶1(Mps1)对蛋白酶体α7亚基(PSMA7)蛋白稳定性的影响。方法通过重组PCR方法将663位天冬氨酸突变为丙氨酸,构建Mps1激酶活性缺失的突变体(pc DNA3-Flag-Mps1KD),并通过免疫印迹实验检测其是否能成功表达。采用免疫印迹实验检测Mps1及Mps1KD对PSMA7蛋白稳定性的影响;通过实时荧光定量PCR(RT-PCR)技术检测Mps1及Mps1KD对PSMA7 RNA表达水平的影响。结果免疫印迹结果证实构建的突变质粒Mps1KD能正确表达且野生型Mps1可明显增强PSMA7稳定性,而Mps1KD则不能;加入Mps1激酶活性抑制剂后,降低PSMA7稳定性;RT-PCR检测结果发现Mps1及Mps1KD对PSMA7 RNA表达水平无影响。结论成功构建了Mps1激酶活性缺失突变体真核表达质粒pc DNA3-Flag-Mps1KD,并在293T细胞中得到了表达;研究还发现Mps1通过其激酶活性可增强PSMA7蛋白稳定性,但对其RNA表达水平无影响,为进一步研究Mps1对蛋白酶体功能影响及对肿瘤发生发展奠定基础。
Objective To investigate the effect of monopolar spindle protein kinase 1 (Mps1) on the proteasome α7 subunit (PSMA7) protein stability. Methods Mutant 663 aspartic acid was mutated to alanine by recombinant PCR to construct pcDNA3-Flag-Mps1KD mutant which lacked Mps1 kinase activity and its expression was confirmed by Western blotting. The effect of Mps1 and Mps1KD on the stability of PSMA7 protein was detected by Western blotting. The effect of Mps1 and Mps1KD on the expression of PSMA7 RNA was detected by real-time fluorescence quantitative PCR (RT-PCR). Results The results of Western blot showed that Mps1KD could be expressed correctly and wild-type Mps1 significantly enhanced the stability of PSMA7 but not Mps1KD. The stability of PSMA7 was decreased by adding Mps1 kinase inhibitor. The results of RT-PCR showed that Mps1 and Mps1KD had no effect on PSMA7 RNA expression. Conclusion The eukaryotic expression plasmid pcDNA3-Flag-Mps1KD with Mps1 kinase-deficient mutant was successfully constructed and expressed in 293T cells. It was also found that Mps1 enhanced the stability of PSMA7 protein through its kinase activity, but its RNA expression No effect on the level of Mps1 for further study of proteasome function and lay the foundation for tumor development.