摘要
目的:构建针对RPAP3 TPR区域的慢病毒载体,观察过表达对细胞周期的影响。方法:通过生物信息学软件比较RPAP3结构域组成,推测功能;分析RPAP3核定位信号,构建瞬时表达质粒pEGFP-N2-RPAP3,激光共聚焦显微镜观察RPAP3蛋白的亚细胞定位;通过酵母双杂交和GST-Pulldown实验研究RPAP3与HSP70的相互作用及作用靶点;构建慢病毒载体pLJM.1-RPAP3,转染293T细胞,收病毒感染MCF7细胞,嘌呤霉素筛选获得稳定转染细胞系,流式细胞分析对细胞周期的影响。结果:RPAP3在多物种广泛存在,有高度保守性;蛋白存在典型核定位信号,激光共聚焦显微镜下,GFP标记的RPAP3蛋白主要分布在细胞核;酵母双杂交和GST-Pulldown实验证实RPAP3与HSP70间存在相互作用,且作用发生在RPAP3的三联TPR结构域和HSP70的GPTIEEVD末端之间;流式细胞显示RPAP3 TPR区域的高表达阻滞细胞周期且凋亡增加。结论:RPAP3与HSP70间的相互作用发生在RPAP3的三联TPR结构域和热休克蛋白70的GPTIEEVD末端之间;构建高表达细胞株发现其对细胞周期及凋亡有影响。
Objective： To construct the RPAP3 （TPR domain）-containing lentivirus, and investigate the influence of the overexpression of PRAP3 on cell cycle. Methods： RPAP3 with other homology proteins were compared by using sequence analysis tools. Then RPAP3＇s nuclear localization signal sequence was analyzed, and produced transient expressing plasmid and its subcellular localization was studied by laser scanning confocal microscope. The Yeast two-hybrid and GST-Pulldown we used To invest the coaction of RPAP3 with HSP70 and the reaction site,.localization by laser scanning confocal microscope. To invest the coaction of RPAP3 with HSP70 and the reaction site, we used the Yeast two-hybrid and GST-PuUdown. A lentivirus expression vector wAS constructed, and infected MCF7 cells. Flow cytometry was used to to investigate the effect on cell cycle. Results： RPAP3 exists among many species and was highly conserved. It has a typical nuclear localization single （RKKKKGKAKESSKKTRE） and is mainly distributed in the cell nucleus under la- ser scanning confocal microscope. The interaction was confirmed, which happens between the three-TPR domain and hsp70 terminal GPTIEEVD by Yeast two-hybrid and GST-Pulldown. The results of flow cytometry analysis showed that the overexpression of RPAP3 inhibit the cell cycle and increased apoptosis. Conclusions： RPAP3 can interact with hsp70 and hsp90 and it happens between the three-TPR domain and hsp70 terminal GPTIEEVD. Overexpression of RPAP3 influenced the cell cycle and apoptosis.
出处
《现代生物医学进展》
CAS
2012年第12期2245-2249,2244,共6页
Progress in Modern Biomedicine
基金
国家自然科学基金资助项目(30971092)
