真核表达载体pcDNA6/myc-hisB-GFP的构建

Construction of the pcDNA6/myc-hisB-GFP eukaryotic expression plasmid

目的构建带绿色荧光蛋白(GFP)的pcDNA6/myc-hisB的真核表达载体。方法采用亚克隆的技术扩增得到GFP的开放阅读框序列,HindⅢ和KpnⅠ双酶切后插入pcDNA6/myc-hisB,重组体经质粒PCR和酶切鉴定,并转染HEK293T细胞后观察绿色荧光的表达,同时经Western blot检测GFP和标签蛋白myc的表达。结果 PCR扩增得到特异的GFP开放阅读框序列,质粒PCR和酶切鉴定显示pcDNA6/myc-hisB-GFP构建成功,细胞转染结果表明重组体可以表达GFP和myc蛋白。结论获得带有绿色荧光蛋白的pcDNA6/myc-hisB-GFP的真核表达载体,为此载体的应用拓展了更大的空间。

Objective To construct an eukaryotic expression vector containing green fluorence protein(GFP) . Methods The open read frame sequence of GFP was obtained by subclone,digested by Hind Ⅲ and Kpn Ⅰ,and then inserted into pcDNA6/myc-hisB plasmid.The recombinant pcDNA6/myc-hisB-GFP plasmid was identified by PCR and restriction endonuclease digestion,and then was transferred with HEK 293T cells for observing the expression of GFP.The expression of GFP and myc tag was tested by Western blot. Results The specific open read frame sequence of GFP was obtained by PCR.The recombinant pcDNA6/myc-hisB-GFP plasmid was constructed successfully.The GFP was found under fluorescence microscope and myc protein was detected by Western blot after the transfection. Conclusion The pcDNA6/myc-hisB-GFP eukaryotic expression plasmid containing green fluorence protein can be constructed successfully,which can be used for studying the function of some specific proteins.