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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。
Objective To clone human DC-SIGN gene and construct recombinant retroviral vector containing the target gene to obtain L929 gene transfected cells stably expressing DC-SIGN. Methods Human DC-SIGN gene was cloned from human peripheral blood mononuclear cell (DC) total RNA by RT-PCR and inserted into retroviral vector pGEZ by EcoRI and BamHI -Term, 293T cells were co-transfected with 293T cells by lipofectamine. L929 cells were infected with the culture supernatant of 293T cells containing the complete virus particles. After 72 hours, Zeocin was added for selection. L929 cells stably expressing DC-SIGN protein Cell lines. Results Recombinant retrovirus vector containing DC-SIGN gene was constructed and transfected into 293T cells. The competent recombinant DC-SIGN retrovirus and transfected L929 cells were obtained, and then transfected into L929 cells by RT-PCR , Flow cytometry phenotype detection, screened L929 transgenic cells stably expressing human DC-SIGN protein. Conclusion The recombinant retroviral vector containing human DC-SIGN gene and the cell line stably expressing human DC-SIGN protein were constructed, which laid the foundation for the further study on the function of this gene and the development of monoclonal antibody.