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：《国际呼吸杂志》2011年第15期1169-1173,共5页International Journal of Respiration

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Sal I和BamH1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人载体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 Sal I and BamH I , 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：Q78[生物学—分子生物学]