PTPN6重组蛋白的真核表达纯化及功能鉴定

Recombinant protein PTPN6 eukaryotic expression,purification and functional identification

[目的]构建真核表达载体pENTER-PTPN6,在HEK293T细胞中表达并纯化,并初步探讨PTPN6对肺癌细胞系A549细胞增殖能力的影响。[方法]构建真核表达载体pENTER-PTPN6,利用jetPRIME分别转染HEK293T和A549细胞,用Western Blot检测PTPN6在细胞中的表达情况,并通过磁珠纯化PTPN6蛋白,最后利用CCK-8法检测PTPN6对A549的增殖影响并绘制细胞生长曲线。[结果]Western Blot结果显示转染细胞内有PTPN6的高表达,磁珠纯化得到高纯度的目标蛋白,而且转染后的肺癌细胞A549增殖能力较对照组显著下降。[结论]成功构建了真核表达质粒pENTER-PTPN6并在真核表达系统中表达和纯化,重组PTPN6具有抑制A549细胞的增殖的作用,为进一步探讨PTPN6基因的功能及寻找治疗肺癌新方向奠定了基础。

[Objective]To construct eukaryotic expression vector pENTER-PTPN6 and observe the effect of PTPN6 protein on the proliferation of lung cancer cell line A549. [Methods]The eukaryotic expression vector pENTER-PTPN6 was constructed and transfected into cell line HEK293 T and A549 via jetPRIME expression of PTPN6 in cells was detected with the help of Western Blotting and purified with magnetic beads,finally the cell growth curve was gained through the Cell Counting Kit-8 method. [Results]Western Blotting confirmed that PTPN6 was highly expressed in transfected cells and purified by magnetic beads. What＇s more,PTPN6 significantly depressed the proliferation of A549 cells compared with the control group.[Conclusion]The eukaryotic expression vector pENTER-PTPN6 was successfully constructed and expressed effectively in eukaryotic expression system. The cell growth curve showed that PTPN6 significantly inhibited the proliferation of A549 cells,which laid the basis for further research of biological function of PTPN6 and new treatment of lung cancer.