Establishment and identification of B16 cell line stably expressing human survivin gene / 第二军医大学学报

ABSTRACT

Objective: To construct the eukaryotic expression vector of human survivin gene and stably transfect it into mouse melanoma B16 cell line. Methods: The full length human survivin cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo; the restriction enzyme position and 6 his tag were added to obtain recombinant plasmid pIRES-neo-SUR-(his) 6, which was then transfected into B16 cells by Lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, and the transcription and expression of the human survivin gene were identified by RT-PCR, Western blotting analysis and immunofluorescence assay. Results: The result of restriction enzyme digestion and the sequence analysis showed that the recombinant of pIRES-neo-SUR-(his)6 was successfully constructed. RT-PCR results showed survivin gene (about 530 bp) was amplified from the total RNA in the group stably transfected with pIRES-neo-SUR-(his)6. Western blotting analysis showed the expression of survivin protein in pIRES-neo-SUR-(his)6 transfection group, but not in the control group. FACS and immunofluorescence assay showed high fluorescence signal in pIRES-neo-SUR-(his)6 transfection group, with the fluorescence positive rate being 91.38% when No. 5 monoclonal antibody was used; no fluorescence signal was found in the control group( transfected with pIRES-neo). Conclusion: We have successfully constructed the eukaryotic expression vector of human survivin pIRES-neo-SUR-(his)6, and established a B16 cell line stably and highly expressing human survivin gene.