人DC-SIGN基因表达质粒的构建及其表达

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BamH Ⅰ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.

Vector construction and eukaryotic expression of the human DC-SIGN

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.