Construction of bcr-abl gene eukaryotic expressing vector and its expression in COS-7 cells

Abstract

Objective To clone and construct eukaryotic expressing vector of bcr-abl fusion gene and to express the gene in the mammal COS-7 cell lines. Methods bcr-abl fusion gene was amplified from human chronic myeloid leukemia (CML) K562 cell lines by RT-PCR and the fragment of cDNA was retrieved,purified and cloned into the pEGFP-N3 eukaryotic expressing vector. After the selection of the positive clone and by restriction enzyme analysis and DNA sequencing, the correct plasmid was transfected into COS-7 cell lines and observed the transient expression. Results A 874 bp DNA fragment was amplified by RT-PCR. The sequence analysis showed it was consistent with bcr-abl gene of GeneBank. RT-PCR, Western blotting analysis provided strong evidences that bcr-abl gene was expressed successfully in transfected COS-7 cells.Conclusion The eukaryotic expressing vector of bcr-abl fusion gene was constructed, it will lay the foundation for further study of bcr-abl gene in the diagnosis and treatment of CML. Key words: Chronic myeloid leukemia; bcr-abl; P210 protein; Gene clone; Gene expression