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

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1 212bp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50 000,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48h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。

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-7cells 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-7cells,respectively.The purification of proteins was determined using Ni-NTA column.The activity of AURKA was analyzed by autoradiography.The proliferation of MCF-7cells 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-7cells,the number of alive MCF-7cells 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-7cells.The overexpression of AURKA can promote the proliferation of MCF-7cells