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Expression and subcellular Localization of Recombinant sdf-1 and Its mutant intrakine in Transfected COS-7 Cells

Chinese Journal of Biomedical Engineering(English Edition) 2006年第2期15卷 69-77页

作者： CHEN Hong-yuan GUO Zhi-gang TAN-yi MA Wei-feng CAI Shao-xi DU-jun CAI Shao-hui Key laboratory for Biomechanics & Tissue Engineering of the State Ministry of Education College of Bioengineering Chongqing UniversityChongqing 400044 Key laboratory for Biomechanics & Tissue Engineering of the State Ministry of EducationCollege of Bioengineering Chongqing UniversityChongqing 400044 Key laboratory for Biomechanics & Tissue Engineering of the State Ministry of EducationCollege of Bioengineering Chongqing UniversityChongqing 400044 Department of Clinical Pharmacology Pharmacy School of Jinan UniversityGuang zhong 510080 Key laboratory for Biomechanics & Tissue Engineering of the State Ministry of EducationCollege of Bioengineering Chongqing UniversityChongqing 400044 Key laboratory for Biomechanics & Tissue Engineering of the State Ministry of EducationCollege of Bioengineering Chongqing UniversityChongqing 400044 School of Pharmaceutical ScienceSun Yat-Sen UniversityGuangzhou 510612 Department of Clinical PharmacologyPharmacy School of Jinan UniversityGuang zhong 510080

Objective:This paper is to explore a method of transferring human sdf-1 and its mutant sdf-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion *** could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the ***:Amplify the target gene with PCR from the constructed plasimid sdf-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 sdf-1KDEL,pcDNA3.1 sdf-154KDEL,pEGFPsdf-1KDEL and pEGFPsdf-154KDEL 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-1His and sdf-154His were confirmed by Western blot,and the sdf-1EGFP and sdf-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein sdf-1 KDELHis and sdf-1 54 KDELHis expressed in COS-7 *** localization analysis showed that sdf-1KDELEGFP and sdf-154KDELEGFP were located mainly in endoplasmic ***:Four expression vectors pcDNA3.1 sdf-1KDEL, pcDNA3.1sdf-154KDEL, pEGFPsdf-1KDEL and pEGFPsdf-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .

关键词： intrakine,sdf-1,mutant,subcellular localization

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