[Construction and expression of eukaryotic expression vectors of full-length, amino-terminus and carboxyl-terminus Raf gene.].

BACKGROUND:Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. METHODS:Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. RESULTS:The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. CONCLUSIONS:Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-CRaf were successfully constructed and can be expressed in 293T cells.