ZBTB5基因真核表达质粒的构建及其在HEK 293T细胞中的表达

Construction of recombinant eukaryotic expression plasmid of ZBTB5 and its expression in HEK 293T cell line

目的构建ZBTB5基因的真核表达质粒,并检测其在细胞内的表达。方法以pET-28a(+)-ZBTB5质粒为模板,以ZBTB5全长编码基因特异性引物进行PCR扩增,将ZBTB5全长基因插入至pcDNA3.1-GFP真核表达载体构建重组pcDNA3.1-ZBTB5-GFP质粒。将该重组质粒双酶切及测序鉴定后,转染至HEK 293T中,荧光显微镜下及Western blot分别检测GFP和ZBTB5在细胞中的表达。同时设置pcDNA3.1和pcDNA3.1-GFP载体为对照。结果酶切及测序证实成功构建了真核表达重组载体pcDNA3.1-ZBTB5-GFP。荧光显微镜下可见GFP的表达,Western blot检测到融合蛋白ZBTB5-GFP的表达。结论成功构建真核表达质粒pcDNA3.1-ZBTB5-GFP,为探讨其进一步的功能奠定基础。

Objective： To construct the recombinant plasmid of pcDNA3.1-ZBTB5-GFP and detect ZBTB5 expression in HEK 293T cells.Methods： pET-28（a）-ZBTB5 was used as template;the full-length coding sequence of ZBTB5 gene was amplified by polymerase chain reaction（PCR） using specific primers and inserted into pcDNA3.1-GFP vector for producing the recombinant plasmid,pcDNA3.1-ZBTB5-GFP.After identifying by restriction enzyme digestion and sequencing,the plasmid was transfected into HEK 293T cell line.Expressions of the GFP and ZBTB5 in HEK 293T cell line were detected under fluorescence microscope and by Western blotting,respectively.Results： The plasmid pcDNA3.1-ZBTB5GFP was constructed successfully,after digesting by restriction enzyme and sequencing.The expression of GFP was observed under fluorescence microscope,and the fusion protein ZBTB5-GFP was proved by Western blotting.Conclusion： The recombinant eukaryotic expression plasmid pcDNA3.1-ZBTB5-GFP is constructed successfully,and it provides a basis for further functional analysis of ZBTB5.