Construction and identification of pIRES2-EGFP-hTGF-β3 plasmid

Abstract

Objective To construct the pIRES2-EGFP-hBMP-2 plasmid carrying human bone morphogenetic protein-2 (BMP-2) gene.Methods BMP-2 gene was amplified using reverse transcriptionpolymerase chain reaction (RT-PCR) in peripheral blood lymphocytes,then the pIRES2-EGFP-hBMP-2plasmids were constructed.End nuclease cutting,PCR,and sequencing were done to identify the plasmids,and concentration and purity were detected by using ultraviolet spectrophotometer.Results Mter end nuclease cutting of the pIRES2-EGFP-hBMP-2 plasmids,the fragment length of empty vector and hBMP-2 gene was 5.3 kb and 366 bp respectively.Using PCR amplification,the fragment length of hBMP-2 gene was 366 bp.Using ultraviolet spectrophotometer,the concentration of plasmid carrying empty vector and hBMP-2 gene was 382 and 383 mg/L respectively.The plRES2-EGFP-hBMP-2 plasmids carrying correct sequence were confirmed by the identification of sequencing.Conclusion We constructed plRES2-EGFP-hBMP-2 plasmid successfully and established foundation to study its induction of new bone formation using human BMP-2 gene therapy. Key words: Bone morphogenetic proteins-2 gene; Eukaryotic expression vector