Expression and Subcellular Localization of Recombinant SDF-1 and Its Mutant Intrakine in Transfected COS-7 Cells

INTRODUCTION The CXC chemokine stromal cell -derived factor1(SDF-1) , a member of CXC-chemokine family,is the ligand for CXCR4 and binds CXCR4 with high affinity.The interaction of SDF-1 and CXCR4 mayinduce cytoskeletal rearrangement, adhesive to endothelial cells and directional migration. SDF-1 bind-ing to CXCR4 can activiate multiple signaling pathways and produce different physiology or pathologyeffects. Compelling evidence is accumulating HIV infection and tumor metastas…

Expression and Subcellular Localization of Recombinant SDF-1 and Its Mutant Intrakine in Transfected COS-7 Cells

Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-1/54 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein. This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface. Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-Dec/PET-30a(+)with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 /SDF-1/KDEL, pcDNA3.1 /SDF-1/54/KDEL, pEGFP/SDF-1/KDEL and pEGFP/SDF-1/54/KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate, they were transferred into COS-7 cells with liposome. The exogenous expressions were observed, fusion protein SDF-1/His and SDF-1/54/His were confirmed by Western blot, and the SDF-1/EGFP and SDF-1/54/EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1/KDEL/His and SDF-1/54 KDEL/His expressed in COS-7 cells. Subcelluar localization analysis showed that SDF-1/KDEL/EGFP and SDF-1/54/KDEL/EGFP were located mainly in endoplasmic reticulum. Conclusion: Four expression vectors pcDNA3. 1/ SDF -1/KDEL, pcDNA3.1/SDF - 1/54/KDEL, pEGFP/SDF - 1/KDEL and pEGFP/SDF-1/54/KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum.