Construction And Identification Of Bicistronic Eukaryotic Vector PIRES2-EGFP-hBMP-2

Objective:To construct the eukaryotic vector pIRES2-EGFP-hBMP-2 and to observe its expression in HEK 293 cells.Methods:hMBP-2 gene was cloned 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 with fluorescence microscope and Western Blot.Results:hBMP-2 gene was successfully cloned and identified by restriction enzyme analysis. Construction of pIRES2-EGFP-BMP-2 was verified, then the expression of EGFP and BMP-2 in HEK 293 cells was proved by fluorescence microscope and Western Blot.Conclusion:It validated that HEK 293cells could be transduced by Lipofectamime 2000 efficiency and the eukaryotic vector pIRES2-EGFP-hBMP-2 could successfully express target protein in it. The present study demonstrated that the two gene sequences encoding BMP-2 and EGFP could be translated in protein respectively. These HEK239 cells with pIRES2-EGFP-BMP-2 provides a promizing tool for the further investigating the repairing mechanism of BMP-2 in bony and cartilaginous diseases.