杜氏盐藻葡萄糖-6-磷酸异构酶在真核细胞中的表达

目的:分别构建杜氏盐藻葡萄糖-6-磷酸异构酶(DsGPI)绿色荧光蛋白表达载体和真核表达载体。方法:PCR扩增得到上、下游分别添加EcoRⅠ和BamHⅠ酶切位点的DsGPI基因,双酶切后与绿色荧光蛋白表达载体pEGFP-C1连接,得到重组载体pEGFP-C1-DsGPI,转染狗肾细胞MDCK,观察绿色荧光蛋白在细胞中的定位;同理将上、下游分别添加BamHⅠ和EcoRⅠ酶切位点的DsGPI基因与真核表达载体pcDNA3.1(+)连接,得到pcDNA3.1-DsGPI,转染食管癌EC9706细胞,分别应用RT-PCR法和Westernblot法检测DsGPI mRNA和蛋白的表达。结果:DsGPI在MDCK细胞中定位于细胞核,在EC9706细胞中被有效表达。结论:成功构建了DsGPI的绿色荧光蛋白表达载体和真核表达载体。

Expression of Dunaliella salina glucose-6-phosphate isomerase in eukaryotic cells

Aim:To construct Dunaliella salina glucose-6-phosphate isomerase(DsGPI) green fluorescent protein expression vector and eukaryotic expression vector.Methods:The complete cDNA sequences of DsGPI was cloned into the plasmid pEGFP-C1 to create a recombinant plasmid pEGFP-C1-DsGPI by using BamHⅠ and EcoRⅠ sites and identified.Localization of the EGFP fusion proteins was examined by fluorescence microscopy after the recombinant plasmids were transfected in MDCK cells.Recombinant plasmids pcDNA3.1(+)-DsGPI were constructed with the same method and transfected in EC9706 cells,DsGPI mRNA was detected by RT-PCR and DsGPI protein were examined by Western blot.Results:EGFP fusion proteins were mainly localized in the nucleus area in MDCK cells and expression of DsGPI increased in EC9706 cells.Conclusion:DsGPI green fluorescent protein expression vectors and eukaryotic expression vectors has been constructed successfully.