人DC-SIGN基因表达质粒的构建及其表达  

Vector construction and eukaryotic expression of the human DC-SIGN

作　　者：邵长周[1] 张浩茹[1] 瞿介明[2] 何礼贤[1] 

机构地区：[1]复旦大学附属中山医院呼吸科,上海200032 [2]复旦大学附属华东医院呼吸科,上海200040

出　　处：《中华哮喘杂志（电子版）》2011年第4期1-4,共4页Chinese Journal of Asthma(Electronic Version)

基　　金：国家自然科学基金(30772019);上海市重点学科建设项目(B115)

摘　　要：目的构建含有人DC-SIGN基因的真核表达载体,并在293T细胞中表达目的蛋白。方法人血培养获得树突状细胞(DC),以DC的mRNA逆转录cDNA为模板,应用PCR体外扩增获得目的 DNA,用限制性内切酶SalⅠ和BamHⅠ分别酶切VRC4409载体和PCR产物,通过连接、转化及克隆的筛选将目的基因克隆入载体VRC4409,构建载体VRC4409-DC-SIGN,经PCR鉴定及测序分析证实,脂质体介导转染293T细胞,蛋白质印迹分析其表达DC-SIGN蛋白。结果重组质粒经双酶切和测序鉴定证实VRC4409-DC-SIGN构建成功,DC-SIGN蛋白在293T细胞中成功表达。结论成功构建质粒VRC4409-DC-SIGN并在293T细胞中表达目的蛋白,为进一步研究DC-SIGN的生物学功能奠定了基础。Objective To constructed recombinant eukaryotic plasmid VRC4409-DC-SIGN,then to express the protein DC-SIGN in 293T cells.Methods Dendritic cells（DC） was obtained from the human blood culture.DC-SIGN gene was amplified from cDNA of DC.The DC-SIGN gene and vector VRC4409 were digested by SalⅠand BamHⅠ,and the DC-SIGN gene fragments and the plasmids of VRC4409 were ligated with T4 DNA rapid ligase to form the recombinants VRC4409-DC-SIGN.After restriction analysis and sequencing,the plasmid VRC4409-DC-SIGN was transfected into 293T cells in the mediation of liposome.The expression of DC-SIGN was analyzed by western blot.Results The recombinant plasmids VRC4409-DC-SIGN were confirmed by restriction enzyme assay and sequencing.The DC-SIGN protein were successfully expressed in 293T cells.Conclusions The eukaryotic expression vector VRC4409-DC-SIGN was correctly constructed and the DC-SIGN protein was successfully expressed in 293T cells.This will facilitate the following study on DC-SIGN.

关 键 词：DC-SIGN 293T细胞 蛋白表达 

分 类 号：R512.91[医药卫生—内科学]