人源极光激酶A在毕赤酵母和MCF-7细胞中的表达及纯化

doi:10.13481/j.1671-587x.20170211

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (02): 266-270.doi: 10.13481/j.1671-587x.20170211

人源极光激酶A在毕赤酵母和MCF-7细胞中的表达及纯化

于洋^1,2, 于丽娜³, 谢萍⁴, 王福启², 麻薇⁵, 施维²

1. 广州体育学院运动与健康系运动生理学教研室, 广东广州 510500;
2. 吉林大学生命科学学院分子酶学工程教育部重点实验室, 吉林长春 130012;
3. 广州医科大学附属口腔医院牙周病科广州口腔病研究所口腔医学重点实验室, 广东广州 510140;
4. 吉林出入境检验检疫局技术中心, 吉林长春 130062;
5. 吉林大学中日联谊医院心血管内科, 吉林长春 130033

收稿日期:2016-07-15 出版日期:2017-03-28 发布日期:2017-03-31
通讯作者: 施维,教授,博士研究生导师(Tel:0431-85155216,E-mail:shiw@jlu.edu.cn) E-mail:shiw@jlu.edu.cn
作者简介:于洋(1981-),女,黑龙江省佳木斯市人,讲师,理学博士,主要从事细胞、运动免疫和运动生理方面的研究。
基金资助:
吉林省科技厅科研基金资助课题（20130206010YY）

Expression and purification of human Aurora A in Pichia pastoris and MCF-7 cells

YU Yang^1,2, YU Lina³, XIE Ping⁴, WANG Fuqi², MA Wei⁵, SHI Wei²

1. Section of Exercise Physiology, Department of Sport and Health, Guangzhou Institute of Physical Education, Guangzhou 510500, China;
2. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China;
3. Department of Periodontics, Stomatology Hospital, Guangzhou Medical University, Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Guangzhou 510140, China;
4. Center of Technology, Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun 130062, China;
5. Department of Cardiology, China-Japan Union Hospital, Jilin University, Changchun 130033, China

Received:2016-07-15 Online:2017-03-28 Published:2017-03-31

摘要/Abstract

摘要： 目的：构建人源极光激酶A（AURKA）真核表达载体，检测其在毕赤酵母X33和MCF-7细胞中的表达及纯化。方法：将双酶切PCR扩增的目的基因与真核表达载体连接，构建重组质粒。电转法转染重组质粒，筛选后采用SDS-PAGE和Western blotting法检测AURKA蛋白的表达，Ni-NTA柱法纯化表达的蛋白；放射自显影法检测AURKA的生物活性；细胞计数法检测转染重组质粒后MCF-7细胞增殖情况。结果：2种重组质粒经PCR均扩增出1212 bp目的基因，表明真核表达载体构建成功。重组AURKA蛋白相对分子质量约为50000，且可磷酸化其底物组蛋白H3，表明重组AURKA具有活性。与转染空质粒和野生型MCF-7细胞比较，转染AURKA 24 和48 h后MCF-7活细胞数量明显增加。结论：AURKA在毕赤酵母和MCF-7细胞中成功表达和纯化，且其过表达可促进细胞增殖。

关键词: 毕赤酵母X33, MCF-7细胞, 人源激光激酶A, 细胞增殖

Abstract: Objective: To construct the eukaryotic expression vectorsof human Aurora A(AURKA), and to detect the expression and purification of AURKA in Pichia pastoris X33 and MCF-7 cells. Methods: The AURKA was amplified by polymerase chain reaction (PCR). The genes were digested with XhoⅠ and XbaⅠ, and respectively ligated into pPICZαA and pcDNA3.1(+) to construct the recombinant plasmids pPICZαA- AURKA and pcDNA3.1(+)-AURKA which were transfected into Pichia pastoris X33 and MCF-7 cells by Gene Pulser Xcell^TM Electroporation System. The expressions of AURKA protein were detected by SDS-PAGE and Western blotting methods after screening in Pichia pastoris X33 and MCF-7 cells, respectively. The purification of proteins was determined using Ni-NTA column. The activity of AURKA was analyzed by autoradiography. The proliferation of MCF-7 cells after transfected with AURKA was detected by cell counting method. Results: The 1 212 bp genes were amplified from the pPICZαA-AURKA and pcDNA3.1(+)-AURKA by PCR, which indicated that two eukaryotic expression vectors were successfully constructed. The relative molecular mass of recombinant AURKA protein was approximately 50 000. The recombinant AURKA phosphorylated the histone H3 by autoradiography, which manifested that it had activity. Compared with the cells transfected with empty plasmid and WT MCF-7 cells, the number of alive MCF-7 cells was significantly increased after transfected with AURKA for 24 and 48 h. Conclusion: AURKA is successfully expressed and purified in Pichia pastoris X33 and MCF-7 cells. The over-expression of AURKA can promote the proliferation of MCF-7 cells

Key words: human Aurora A, MCF-7 cells, cell proliferation, Pichia pastoris X33

中图分类号:

于洋, 于丽娜, 谢萍, 王福启, 麻薇, 施维. 人源极光激酶A在毕赤酵母和MCF-7细胞中的表达及纯化[J]. 吉林大学学报(医学版), 2017, 43(02): 266-270.

YU Yang, YU Lina, XIE Ping, WANG Fuqi, MA Wei, SHI Wei. Expression and purification of human Aurora A in Pichia pastoris and MCF-7 cells[J]. Journal of Jilin University Medicine Edition, 2017, 43(02): 266-270.

参考文献

[1] Kitzen JJ, de Jonge MJ, Verweij J. Aurora kinase inhibitors [J]. Crit Rev Oncol Hematol, 2010, 73(2):99-110.
[2] Yan M, Wang C, He B, et al. Aurora-A kinase:A potent oncogene and target for cancer therapy [J]. Med Res Rev, 2016, 36(6):1036-1079.
[3] Meraldi P, Honda R, Nigg EA. Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53^-/- cells [J]. EMBO J, 2002, 21(4):483-492.
[4] Elling RA, Tangonan BT, Penny DM, et al. Mouse Aurora A:expression in Escherichia coli and purification [J]. Protein Expr Purif, 2007, 54(1):139-146.
[5] Cheetham GM, Knegtel RM, Coll JT, et al. Crystal structure of aurora-2, an oncogenic serine/threonine kinase [J]. J Biol Chem, 2002, 277(45):42419-42422.
[6] Eyers PA, Churchill ME, Maller JL. The Aurora A and Aurora B protein kinases:a single amino acid difference controls intrinsic activity and activation by TPX2 [J]. Cell Cycle, 2005, 4(6):784-789.
[7] Marumoto T, Hirota T, Morisaki T, et al. Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells [J]. Genes Cells, 2002, 7(11):1173-1182.
[8] Satinover DL, Leach CA, Stukenberg PT, et al. Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein [J]. Proc Natl Acad Sci USA, 2004, 101(23):8625-8630.
[9] Castro A, Arlot-Bonnemains Y, Vigneron S, et al. APC/fizzy-related targets Aurora-A kinase for proteolysis [J]. EMBO Rep, 2002, 3(5):457-462.
[10] Fu J, Bian M, Jiang Q, et al. Roles of Aurora kinases in mitosis and tumorigenesis [J]. Mol Cancer Res, 2007, 5(1):1-10.
[11] Hascoet P, Chesnel F, Le Goff C, et al. Unconventional functions of mitotic kinases in kidney tumorigenesis [J]. Front Oncol, 2015, 5:241.
[12] Willems E, Lombard A, Dedobbeleer M, et al. The unexpected roles of aurora A kinase in gliobastoma recurrences [J]. Targ Oncol, 2017,12(1):11-18.
[13] Chang SS, Yamaguchi H, Xia W, et al. Aurora A kinase activates YAP signaling in triple-negative breast cancer [J]. Oncogene, 2017,36(9):1265-1275.
[14] 邹苑, 陈浩, 马洪. AURKA基因在口腔鳞癌中的表达及意义 [J]. 贵州医药, 2016, 40(4):358-359.
[15] Cui S, Zhang K, Li C, et al. Methylation-associated silencing of microRNA-129-3p promotes epithelial-mesenchymal transition, invasion and metastasis of hepatocelluar cancer by targeting Aurora-A [J]. Oncotarget, 2016, 7(47):78009-78028.
[16] 孔越, 阎爱侠. 基于极光激酶A抑制剂的计算机辅助药物设计的研究 [A]. 中国化学会第29届学术年会摘要集—第19分会:化学信息学与化学计量学 [C].北京:2014.

人源极光激酶A在毕赤酵母和MCF-7细胞中的表达及纯化

Expression and purification of human Aurora A in Pichia pastoris and MCF-7 cells

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	朱德强, 陈学军. 白花丹素对肝癌索拉菲尼耐药细胞HepG2R增殖和凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2018, 44(06): 1223-1229.
[2]	阎慧, 马淑飞, 段明华, 闫玉礼, 潘新, 李海清, 周长龙, 赵天倚. 京尼平对胃癌SGC 7901细胞体外增殖、侵袭能力和凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2018, 44(05): 924-928.
[3]	邢树刚, 张丽红, 金明华, 李百慧, 武则馨, 房波, 李伟. 杨梅黄酮对小鼠脑胶质瘤GL261细胞增殖和凋亡的影响[J]. 吉林大学学报(医学版), 2018, 44(05): 955-961.
[4]	袁虎勤, 李强. 水飞蓟素对人胃癌SGC 7901细胞增殖、迁移和侵袭的抑制作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(05): 988-993.
[5]	刘定坤, 杨楠, 金巨楼, 孙梦洁, 王倩, 吴佳, 刘志辉. 牙髓干细胞增殖、分化和迁移的研究进展[J]. 吉林大学学报(医学版), 2018, 44(05): 1100-1104.
[6]	李鑫, 马云飞, 唐庚, 韦麒, 纪红池, 田嘉安, 申延男, 王志成. 线粒体靶向KillerRed诱导的ROS增强辐射对HeLa细胞的增殖抑制作用[J]. 吉林大学学报(医学版), 2018, 44(04): 718-723.
[7]	朱效慧, 孟琴, 冯世燕. TP53在不明原因复发性流产患者绒毛组织中的表达及其对人滋养层细胞生长的影响[J]. 吉林大学学报(医学版), 2018, 44(04): 796-800.
[8]	王淼, 母润红, 李明成, 李洪杰. RNAi沉默survivin和HIF-1α基因对胃癌BGC-823细胞体外增殖和凋亡的影响[J]. 吉林大学学报(医学版), 2018, 44(04): 753-758.
[9]	吕鹏, 宁明杰, 邵玉, 张璐妮, 唐英, 王南博, 陈飞儿, 齐玲. 野黄芩苷对人脑胶质瘤U87细胞的增殖抑制作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(03): 466-470.
[10]	杨文延, 刘强, 孙志娟, 杜利清, 徐畅, 王彦, 柳杨, 王芹. 褪黑素对人非小细胞肺癌H1299细胞辐射敏感性的影响[J]. 吉林大学学报(医学版), 2018, 44(03): 532-536.
[11]	周刘祥, 林琳, 胡鑫敏, 王晓春. 内分泌腺性血管内皮生长因子对结肠癌LoVo细胞增殖和迁移的促进作用[J]. 吉林大学学报(医学版), 2018, 44(03): 516-520.
[12]	上官梦原, 赵菁, 杨艳荣, 张佳悦, 陈俊宇, 赵淑华. 五味子脂A联合卡铂对人卵巢癌Skov3细胞增殖、迁移和侵袭的抑制作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(02): 292-298.
[13]	张艳霞, 李跃辉, 张丽红, 张年萍, 闫燕艳, 杨小会, 冯湘玲. 沉默HOXA13基因对肝癌HepG₂和QGY-7703细胞恶性表型的影响[J]. 吉林大学学报(医学版), 2018, 44(02): 315-320.
[14]	于雷, 王志成, 王铁君. 咖啡因联合电离辐射对沉默Chk-1肝癌干细胞的增殖抑制和凋亡诱导作用[J]. 吉林大学学报(医学版), 2018, 44(01): 52-57.
[15]	杨冬野, 焦洋, 檀碧波, 杜志坚, 胡晓杰, 刘飞. Gli1基因在胃癌组织中的表达及其对胃癌细胞生物学特性的影响[J]. 吉林大学学报(医学版), 2018, 44(01): 63-67.