Flag-Tinagl1真核表达载体的构建及功能验证

Construction and functional verification of tubulointerstitial nephritis antigen like 1 eukaryotic expression vector

目的构建带Flag标签的肾小管间质性肾炎抗原样蛋白1(Tinagl1)基因真核表达载体,检测其对肝癌细胞HepG2 ERK/AKT磷酸化、生长和迁移的影响。方法以人乳腺cDNA文库为模板,PCR扩增Tinagl1基因片段,将其插入pcDNA3.0载体,经双酶切和测序验证后,将重组质粒转染到肝癌HepG2细胞中,采用Western印迹检测重组蛋白对ERK和AKT磷酸化水平的影响,生长曲线和划痕实验检测其对肝癌细胞HepG2生长和迁移的影响。结果双酶切和Western印迹结果表明,pcDNA3.0-Flag-Tinagl1真核表达质粒构建成功,Tinagl1可降低ERK和AKT的磷酸化水平;生长曲线和划痕实验表明Tinagl1抑制肝癌细胞HepG2生长和迁移。结论构建了带Flag标签的Tinagl1真核表达载体,并能抑制肝癌细胞HepG2中ERK/AKT磷酸化、生长和迁移,为进一步研究Tinagl1在肝癌细胞中的功能奠定了基础。

Objective To construct a eukaryotic expression vector of tubulointerstitial nephritis antigen like 1(Tinagl1)labeled with flag tag effect its effect on ERK/AKT phosphorylation,proliferation and migration of liver cancer cells.Methods Tinagl1 gene was amplified from the mammary cDNA library by PCR and cloned into the pcDNA3.0-Flag vector.Human HepG2 cells were transfected with pcDNA3.0-Flag-Tinagl1.Western blotting was performed to detect the protein expression and the effect on the phosphorylation of ERK/AKT.CCK-8 assay was used to determine its effect on the growth of cervical cancer cells.Scratch test was used to detect the effect of Tinagl1 on the migration of HepG2 cells.Results A pcDNA3.0-Flag-Tinagl1 eukaryotic expression vector was confirmed by double digestion identification and Western blotting analysis.Expression of Tinagl1 could down-regulate the phosphorylation level of ERK and AKT.Growth curves and scratch tests proved that Tinagl1 could inhibit the proliferation and migration of HepG2.Conclusion A eukaryotic expression vector pcDNA3.0-Flag-Tinagl1 has been constructed and proved to have an inhibitory effect on the phosphorylation of ERK/AKT,the growth and migration of the cells.