人KIR2DL1-Ig融合蛋白在COS-7细胞的表达

目的：获得人KIR2DL1分子(killer lg—like receptor 2DL1)胞外区与人IgG Fc段的融合蛋白。方法：从人外周血单个核细胞中提取总RNA，通过RT—PCR扩增编码KIR2DL1胞外段cDNA，经Nhe Ⅰ和BamH Ⅰ双酶切后，定向插入真核细胞表达载体CD51negl中。构建的重组真核表达载体CD51negl—KIR2DL1。经酶切分析和测序鉴定后，通过DEAE—dextran／ehloroquine法转染COS—7细胞：瞬时表达后，取培养上清液经亲和层析、ELISA、SDS—PAGE及Western印迹，鉴定融合蛋白的表达及其免疫学活性。结果：序列测定证实，该重组表达载体含有正确的KIR2DL1胞外区基因序列。重组真核表达载体CD5lnegl—KIR2DL1转染COS—7细胞后，ELISA法检测细胞培养上清中有融合蛋白的表达。SDS—PAGE结果显示，该融合蛋白的相对分子质量(Mc)约为73000。Western印迹结果证实，该蛋白能被特异性单克隆抗体(mAb)EB6所识别。结论：KIR2DL1—Ig融合蛋白表达载体成功构建并在COS—7细胞中获得功能性表达，为KIR2DL1的功能及其配体MHC的研究奠定了基础。

Expression of human KIR2DL1-Ig fusion protein in COS-7 cells

AIM: To express, purify and identify the KIR2DL1 IgG Fc fusion protein. METHODS: Extracellular region of KIR2DL1 cDNA was amplified by RT PCR from peripheral blood mononuclear cells, and cloned this fragment into fusion expression vector CD5lnegl. The recombinant vector CD5lnegl KIR2DL1 was obtained after restriction endonuclease digestion and sequencing. COS 7 cells were transfected with this eukaryotic expression vector CD5lnegl KIR2DL1 constructed in our Laboratory through DEAE dextran/chloroquine method. The expressed KIR2DL1 IgGFc fusion protein in COS 7 cell culture surpernatant was identified by ELISA with mAb EB6 and HRP anti hIgFc mAb and Western blot. RESULTS: Restriction endonuclease digestion and sequencing indicated that the CD5lnegl KIR2DL1 had been constructed successfully. The fusion protein could be detected by ELISA in COS 7 cell culture surpernatant. Western blot analysis also showed that the fusion protein could react to both EB6 and anti hIgFc mAb. There was only one specific band at the position of the relative molecular mass ( M r) 73 000, and it was equivalent to the expected value. CONCLUSION: KIR2DL1 IgG Fc fusion protein expressed in COS 7 cells successfully and the protein can mimic the natural KIR2DL1 protein and is used as a potential tool in the recognition of its ligands.