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

Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai 200025, China.

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.

PMID: 15155083 [PubMed - in process]