Construction of a eukaryotic expression vector hVEGF_(165)-fused hirudin/pcDNA3.0 and its expression / 第二军医大学学报

ABSTRACT

Objective:To clone the fused gene of hVEGF_(165) and hirudin,construct a eukaryotic expression vector hVEGF_(165)-FH/pcDNA3.0 and express the vector in human endothelial cell strain. Methods: hVEGF_(165) cDNA fragment was amplified by PCR and fused hirudin peptide was synthesized by primer annealing. The fused hVEGF_(165 ) and hirudin gene was cloned into vector pcDNA3.0 to construct a new vector hVEGF_(165)-FH/pcDNA3.0 and the recombinant plasmid was confirmed by restriction enzyme and auto-sequencing. The correctly cloned plasmids were subjected to rapid in vitro transcription and translation identification. Liposome mediated, recombinant plasmids were used to transfect human endothelial cell strain and the products were identified. Results: hVEGF_(165)-FH was successfully fused and formed a 711 bp target fragment. The recombinant vector hVEGF_(165)-FH/pcDNA3.0 was successfully transformed into human endothelial cell strain which expressed the fusion protein. Conclusion: The recombinant vector hVEGF_(165)-FH/pcDNA3.0 can express fused protein in human endothelial cell strain, which paves a way for further study on the activity of the fusion protein and gene therapy.