| 非泛素化蛋白降解体系靶向敲减KRAS癌蛋白的初步研究* |
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| 引用本文： | 马怡晖,庞霞,许晶晶,夏培苡,高汉青. 非泛素化蛋白降解体系靶向敲减KRAS癌蛋白的初步研究*[J]. 中国肿瘤临床, 2016, 43(21): 937-942. DOI: 10.3969/j.issn.1000-8179.2016.21.868 |
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| 作者姓名： | 马怡晖 庞霞 许晶晶 夏培苡 高汉青 |
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| 作者单位： | 作者单位:郑州大学第一附属医院病理科(郑州市450052) |
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| 基金项目： | 本文课题受国家自然科学基金项目(编号81401936)资助This work was supported by the National Natural Science Foundation of China (81401936) |
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| 摘 要： | 目的:尝试构建能与KRAS癌蛋白相互作用的“RBD+ODC”融合蛋白,经由“鸟苷酸脱羧酶/ 抗酶”系统（onithine decarboxylase/antizyme,ODC/AZ）在蛋白水平直接实现对KRAS癌蛋白的降解。方法:应用分子克隆的方法构建AZ、ODC 、“ RBD+ODC”、突变体“RBD+ODC”、突变体KRAS等真核表达质粒,分别瞬时转染人HEK 293T 和胰腺癌细胞PANC-1,应用Westernblot法在蛋白水平直接检测外源性和内源性KRAS表达水平的改变,应用Co-IP 法检测到“RBD+ODC”能够与KRAS癌蛋白相互作用。结果:成功构建了AZ、ODC 、“ RBD+ODC”、突变体“RBD+ODC”、突变体KRAS等多个真核表达质粒,相较于对照组,“ RBD+ODC”能够在蛋白水平直接实现对外源性和内源性KRAS癌蛋白的“敲减”,并且能够与KRAS有效结合。结论:靶向敲减KRAS癌蛋白的非泛素化蛋白降解体系的构建,为下一步功能试验奠定了基础。
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| 关 键 词： | 蛋白敲减? 结合结构域 功能结构域? 鸟苷酸脱羧酶 抗酶 |
| 收稿时间： | 2016-07-25 |
A preliminary study on targeted degradation of KRAS oncoprotein by using a ubiqui-tin-independent,proteasome-mediated degradation pathwayODC/AZ |
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| Affiliation: | Department of Pathology, 1st Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China |
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| Abstract: | Objective:To construct chimeric fusion proteins,RBD+ODC,which can bind to KRAS oncoprotein, and thenknockdownthe KRAS oncoprotein in PANC-1 cells via theODC/AZpathway. Methods:Five eukaryotic expression plasmids, including AZ, ODC,RBD+ODC,mutantRBD+ODC,and mutant KRAS, were constructed by molecular cloning and then transfected into HEK293T and PANC-1 cell lines transiently. The protein levels of KRAS in HEK293T and PANC-1 cel s were detected by Western blot. The interaction of KRAS andRBD+ODCwas detected by Co-IP. Results:Five eukaryotic expression plasmids were constructed successful y. Compared with the controls,RBD+ODCcanknockdownthe endogenous and ectogenous KRAS at the posttranslational level in HEK293T and PANC-1 cel s separately. Furthermore,RBD+ODCcan bind to KRAS effectively. Conclusion:The construction of ubiquitin-independent, proteasome-mediated degradation system targeted KRAS oncoprotein established the foundation for future studies. |
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| Keywords: | protein knockdown binding domain function domain ODC AZ |
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