【摘要】 目的克隆人DC-SIGN基因,并构建含有该目的基因的重组逆转录病毒载体,获得稳定表达DC-SIGN分子的L929基因转染细胞。方法利用RT-PCR的方法从人外周血单核细胞来源的树突状细胞(DC)总RNA中克隆出人DC-SIGN基因,通过双酶切(EcoRI,BamHI)装入逆转录病毒载体pGEZ-Term中,脂质体法共转染包装细胞293T,用含有完整病毒颗粒的293T细胞的培养上清感染L929细胞,72h后,加入Zeocin进行筛选,挑选出能稳定表达DC-SIGN蛋白的L929细胞株。结果构建了用于表达的含DC-SIGN基因的重组逆转录病毒载体,经转染包装细胞293T后,获得具有感染能力的重组DC-SIGN逆转录病毒和转染L929细胞,继而经RT-PCR、流式细胞仪表型检测,筛选出了稳定表达人DC-SIGN蛋白的L929转基因细胞。结论构建了含人DC-SIGN基因重组逆转录病毒载体和稳定表达人DC-SIGN蛋白的细胞株,为该基因功能的后续研究和单克隆抗体的研制奠定了基础。更多还原

【Abstract】 Objective To clone human DC-SIGN gene and construct recombinant retrovirus vector carrying the target gene which can be expressed stably in mammal cell line L929.Methods Human DC-SIGN gene was amplified by RT-PCR using the mRNA of dendritic cells derived from human peripheral blood monocytes as template and then confirmed by DNA-sequence analysis.Digested with the restriction endonucleases EcoRI and BamHI,the human DC-SIGN gene was inserted into retrovirus vector pGEZ-Term.The recombinant retrovirus vector together with its two helper virus vectors was cotransfected into the package cell 293T in the context of LipfectAMINE.Then the supernatant of 293T was used to infect L929 cells.After 72 hours,L929 cell line stably expressing human DC-SIGN protein was selected in the presence of Zeocin(500μg/ml).Results The full-length of human DC-SIGN gene was cloned and the recombinant retrovirus vector was constructed,The L929 cell line expressing human DC-SIGN was successfully transfected and selected.Results of RT-PCR and flow cytometry indicated that L929 transgenic cells can stably express human DC-SIGN protein on the membrane of cells.Conclusion Cloning of human DC-SIGN gene and construction of the recombinant retrovirus vector and L929 transgenic cell line stably expressing DC-SIGN protein can contribute to further biological function research and monoclonal antibody preparation.更多还原