pNTAP-MK2真核表达载体的构建及其稳定表达细胞系的建立

Construction of pNTAP-MK2 eukaryotic expression plasmid and establishment of a cell line for its stable expression

目的构建pNTAP-MK2真核表达质粒,并建立其稳定表达的HEK293细胞系。方法将MK2亚克隆至串联亲和纯化载体pNTAP质粒上,构建成重组质粒pNTAP-MK2,转化该重组质粒至感受态大肠杆菌DH5α,阳性克隆进行PCR、酶切及DNA测序验证正确后,利用脂质体PolyFect介导将其转染至HEK293细胞中,再通过G418筛选建立稳定表达TAPtag-MK2融合蛋白的HEK293细胞系;利用Western blotting和细胞免疫荧光标记法检测融合蛋白TAPtag-MK2的表达及细胞内定位情况。结果重组真核表达载体构建正确,该重组质粒能在HEK293细胞中稳定表达,表达产物TAPtag-MK2主要分布在核内。结论成功构建pNTAP-MK2真核表达载体并建立了其稳定表达的HEK293细胞系,TAP标签未对MK2定位产生明显影响。

Objectives To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandam affinity purification（TAP）-tagged MK2.Methods The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2,which was subsequently transformed into DH5？？E.coli.Afteridentification by PCR,digestion with restriction endonuclease and sequencing,the recombinant expression plasmid was transfected into HEK293 cells via liposome,and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418.The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay.Results The results of PCR,restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2.Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection.Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei.Conclusion The eukaryotic expression vector pNTAP-MK2 has been successfully constructed,and in the established cell line with stable expression of TAP tag-MK2,TAP tag does not influence the localization of exogenous MK2.