人NKG2D真核表达载体的构建及其在COS-7细胞中的瞬时表达

目的：构建人NKG2D重组真核表达载体，并研究其在COS-7细胞中的表达。方法：应用RT-PCR，自健康人外周血单个核细胞(PBMC)中获取NKG2D cDNA，应用基因重组技术构建含目的基因的T载体克隆后，构建含目的基因的真核细胞表达质粒，并经酶切和测序证实。然后应用脂质体介导将重组真核表达质粒转染COS-7细胞．用RT-PCR和流式细胞术(FCM)检测转染细胞NKG2D表达。结果：重组真核表达载体中插入片段序列与GeneBank登录的cDNA序列一致。NKG2D在COS-7细胞中获得表达。结论：成功地构建了重组表达栽体pcDNA3．1-NKG2D，并实现了在COS-7细胞中的瞬时表达，为研究NKG2D的生物学活性奠定了基础。

Construction of recombinant eukaryotic expression vector of NKG2D gene and its expression in COS-7 cells]

OBJECTIVE: To construct recombinant eukaryotic expression vector of NKG2D, and to examine its expression in COS-7 cells. METHODS: Human NKG2D cDNA was obtained from peripheral blood mononuclear cells using RT-PCR, and then the target gene was cloned into pMD18-T vector. A eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique, and was confirmed by double restriction enzymes digestion and DNA sequence analysis. The recombinant plasmid with encapsuled lipofectamine was transfected into COS-7 cells, and the transfected COS- The 7 cells containing expressive target gene were confirmed by RT-PCR and flow cytometry. RESULTS: The sequence of NKG2D cDNA obtained from the recombinant eukaryotic expression vector was identical with that published on GeneBank. The NKG2D gene was expressed successfully in COS-7 cells. CONCLUSION: The recombinant expression plasmid containing NKG2D gene is constructed successfully. After being transfected to COS-7 cells, the NKG2D protein is expressed by the engineering COS-7 cells line, which lays a foundation for further studying biological activity of NKG2D.