人NKG2D的基因克隆及其在CHO细胞中的表达

目的:构建重组人NKG2D真核表达载体并在CHO细胞表达重组人NKG2D。方法:用RT-PCR方法从NK-92细胞中调取NKG2D基因片段,克隆到pGEM-TEasy载体并对克隆的DNA片段进行序列分析。用限制内切酶EcoRI和BamHI消化pGEM-TEasy/NKG2D重组质粒,分离NKG2D片段,并插入真核表达质粒pEGFP-N1的相应限制酶位点,酶谱分析鉴定重组表达载体pEGFP-N1/NKG2D。然后经脂质体介导转染CHO细胞。应用荧光显微镜观测、Westernblot方法和免疫组化染色对转染细胞内pEGFP-N1/NKG2D的表达进行鉴定。结果:RT-PCR扩增获得650bp基因片段,经DNA序列分析证明所获得的DNA序列与文献报道的NKG2D序列一致。转染的CHO细胞在荧光显微镜下发出强绿色荧光,Westernblot分析显示重组蛋白能特异地与抗人NKG2D单克隆抗体结合;免疫组化检测显示,转染的CHO中有棕色颗粒,证明所构建的NKG2D真核表达载体可以在细胞中表达。结论:构建了人NKG2D的哺乳动物细胞表达载体,并成功地在CHO细胞中获得重组人NKG2D的表达。

Cloning of human NKG2D gene and its expression in CHO cells

AIM: To construct a recombinant eukaryotic expression vector of human NK cell receptor NKG2D, and express the recombinant human NKG2D in CHO cells. METHODS: A NKG2D gene fragment, with a length of about 650 bp, was amplified from the NK-92 cell line by RT-PCR and was cloned to plasmid pGEM-T Easy. Then the cloned DNA fragment was sequenced. The recombinant plasmid pGEM-T Easy/NKG2D was digested with EcoR I and BamH I, and then NKG2D fragment was isolated and inserted into the corresponding restriction site on eukaryotic expression vector pEGFP-N1. The Lipofectin was used to transfect the recombinant eukaryotic expression plasmid in CHO cells. The expression level of NKG2D gene in transfected CHO cells was detected by fluorescence microscope, RT-PCR, Western blot and immunohistochemical staining. RESULTS: The length of cDNA fragment amplified by RT-PCR was consistent with that of NKG2D. DNA sequencing of pGEM-T Easy/NKG2D revealed that the cloned DNA sequence was identical to that of reported NKG2D. Green fluorescence was seen in transfected CHO cells by fluorescence microscope. Human NKG2D mRNA was highly expressed in transfected CHO cells. Western blot and Immunohistochemical staining detection showed that NKG2D was expressed in transfected cells. CONCLUSION: A recombinant eukaryotic expression vector of human NKG2D can be constructed and it can be expressed successfully in CHO cells.