DUSP9真核表达载体的构建及其在Hepa1-6肝细胞中的表达

Construction of pEGFP-DUSP9 recombinant plasmids and its expression in mouse Hepa1-6 hepatocytes

目的构建小鼠DUSP9基因真核表达载体,并在小鼠Hepa1-6肝细胞中表达DUSP9蛋白。方法采用RT-PCR方法,从小鼠肝组织获取DUSP9cDNA片断,经双酶切、连接重组至真核表达载体pEGFP-N1,构建pEGFP-DUSP9重组质粒,双酶切及DNA测序对该重组质粒进行鉴定。脂质体介导pEGFP-DUSP9转染Hepa1-6肝细胞,实时荧光定量PCR、Western blot检测DUSP9mRNA及蛋白表达水平的变化。结果 pEGFP-DUSP9重组质粒经双酶切及测序鉴定证明构建完全正确,成功转染Hepa1-6肝细胞并高效表达,检测到明显上调的DUSP9mRNA和蛋白表达水平。结论成功构建真核表达载体pEGFP-DUSP9,并在Hepa1-6肝细胞中表达了DUSP9蛋白,可为研究DUSP9的生理功能及了解其在糖、脂代谢、胰岛素抵抗进程中的作用奠定坚实的基础。

Objective To construct the eukaryotic expression vector of mouse DUSP9 gene and to express DUSP9 protein in mouse Hepa1-6 hepatocytes.Methods The cDNA fragment of mouse DUSP9 containing two restriction sites（ECORI,AgeI） was obtained from mouse fat tissue by using reversal transcript-polymerase chain reaction（RT-PCR）.Then,it cloned into the pEGFP-N1 vector by ECORI/ AgeI double digestion and ligation.The recombinant plasmid was identified by enzyme digest and DNA sequence analysis.The recombinant plasmids were transfected into Hepa1-6 hepatocytes by lipofectamine 2000.The expression of DUSP9 mRNA and protein was measured by real-time quantitaive PCR（SYBR GreenⅠ） and Western blot,respectively.Results Both restriction analysis and sequencing proved that the recombinant plasmid pEGFP-DUSP9 was constructed correctly.The plasmids pEGFP-DUSP9 were successfully transfected and significantly expressed in Hepa1-6 hepatocytes,the levels of mRNA and protein were obviously up-regulated.Conclusion The eukaryotic expression plasmid pEGFP-DUSP9 has been successfully constructed,and DUSP9 protein is expressed in Hepa1-6 hepatocytes.It will help further studies on the physiological function of DUSP9 gene and its effect on glucose,lipid metabolism and development of insulin resistance.