Construction and identification of pIRES2-EGFP-hBMP-2 bicistronic eukaryotic expression vector / 中国组织工程研究

ABSTRACT

BACKGROUND: Bone morphogenetic protein (BMP) and its derived scaffold or vector here been widely used in experiment and gradually applied in the clinic. However, the tracing method challenges the therapeutic effect, The discovery and application of enhanced green fluorescent protein (EGFP) can solve this problem.OBJECTIVE: To construct the bicistronic eukaryotic vector pIRES2-EGFP-hBMP-2 and to observe its expression in human embryo kidney (HEK) 293 cells.METHODS: hMBP-2 gene was extracted from human osteosarcoma by RT-PCR and inserted into PMD18-T vector. Following the DNA sequence verification, it was then sub-cloned into the eukaryotic vector pIRES2-EGFP. After restriction enzyme analysis the pIRES2-EGFP-BMP-2 was transfected into HEK 293 cells. Then its expression was observed using fluorescence microscopy and Western blot.RESULTS AND CONCLUSION: hBMP-2 gene was amplified and the eukaryotic vector plRES2-EGFP-hBMP-2 was constructed successfully. At 48 hours after tranfection of pIRES2-EGFP-hBMP-2 into HEK 239 cells, approximately 30% of cells emitted green fluorescence under a fluorescence microscope. Western blot results demonstrated that there was a specific BMP strap at Mr46×10s side, which indicated that the target gene could be expressed in constructed pIRES2-EGFP-hBMP-2 vector.