摘要
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。
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.
出处
《医学分子生物学杂志》
CAS
CSCD
2012年第3期189-193,共5页
Journal of Medical Molecular Biology
基金
国家自然科学基金（No.307702098,801070374）
