吉林大学学报（医学版）2017，Vol.43Issue(2)：266-270,5.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
Expression and purification of human Aurora A in Pichia pastoris and MCF-7 cells
摘要
Abstract
Objective:To construct the eukaryotic expression vectors of human Aurora A(AURKA), and to detect the expression and purification of AURKA in Pichia pastoris X33 and MCF-7cells.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 XcellTM 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 cou nting 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关键词
人源激光激酶A/毕赤酵母X33/MCF-7细胞/细胞增殖Key words
human Aurora A/Pichia pastoris X33/MCF-7 cells/cell proliferation分类
生物科学引用本文复制引用
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(2):266-270,5.基金项目
吉林省科技厅科研基金资助课题(20130206010YY) （20130206010YY）
