小鼠GITRL基因真核表达质粒的构建转染及其在Kupffer细胞中的表达

目的构建含小鼠GITRL基因的真核表达质粒pEGFP-N1-GITRL，体外转入小鼠Kupffer细胞。方法利用PCR方法扩增GITRL基因，克隆至pEGFP-N1载体，选择阳性克隆并进行序列测定。以脂质体化学法转染至Kupffer细胞中。结果构建了真核表达质粒pEGFP-N1-GITRL，基因测序与GenBank中发表的序列完全一致，体外瞬时转染Kupffer细胞，RT-PCR及WB检测该Kupffer细胞表达GITRL。结论成功构建了真核表达质粒pEGFP-N1-GITRL，并在小鼠Kupffer细胞成功表达，为进一步研究GITRL在Kupffer细胞中的的生物学功能提供研究基础。

Construction and Transfection of Eukaryotic Expression Plasmid Con- taining mGITRL Gene and Its Expression in Kupffer Cells

Objective To construct eukaryotic expression plasmid pEGFG-N1-GITRL contai- ning mouse GITRL gene and transfer to the Kupffer cells in mice in vitro. Methods GITRL gene was amplified by PCR, and then cloned into pEGFP-N1 vector. The positive clones were selected for sequencing. Finally the GITRL gene was transfected into Kupffer cells by liposome-chemical meth- od. Results The eukaryotic expression plasmid pEGFP-N1-GITRL was constructed successfully, and its sequences were consistent with the published once in Genbank. After transient transfection of pEGFP-N1-GITRL into the Kupffer cells in vitro, RT-PCR and WB revealed the stable expression of GITRL in Kupffer cells. Conclusion The eukaryotic expression plasmid pEGFP-N1-GITRL was successfully constructed and expressed in mouse Kupffer cells, which may provide the foundation for further study on the biological function of GITRL in Kupffer cells.