摘要
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 study the effect of monopolar spindle 1( Mps1) on the stability of proteasome subunit alpha type-7( PSMA7). Methods Using recombinant PCR,Mps1 kinase dead mutation( KD mutation) eukaryotic expression plasmid( pc DNA3-Flag-Mps1KD) was constructed by changing Asp663 into Ala. The effect of Mps1 and Mps1 KD on the stability of PSMA7 was detected with Western blotting. Real-time fluorescent quantitative PCR( RT-PCR) technology was used to detect the effect of Mps1 and Mps1 KD on PSMA7 RNA expression level. Results The plasmid pc DNA3-FlagMps1 KD expressed Mps1 KD protein in 293 T cells. Further study showed that Mps1 increased the stability of PSMA7 significantly while Mps1 KD mutation did not. The stability of PSMA7 was decreased by the inhibitor of MPS1( reversine).RT-PCR results found that Mps1 did not affect PSMA7 RNA expression level. Conclusion Mps1 KD mutation plasmid is constructed successfully. Mps1 enhances the stability of PSMA7 by means of its kinase activity. These results can contribute to further study on the effect of Mps1 on proteasome and on tumor development.
出处
《军事医学》
CAS
CSCD
北大核心
2016年第5期392-395,共4页
Military Medical Sciences
基金
国家自然科学基金资助项目(81472497
81572620)
山东省自然科学基金资助项目(ZR2015HM003)