Construction And Identification Of Tumor DNA Vaccine Based On Survivin

Objective:Immunotherapy is a promising strategy for the treatment of tumors.DNA vaccine is one important aspect of tumor immunotherapy.Survivin is found to be a tumor associated antigen and it is over-expressed in many tumors.The present study aims to construct the eukaryotic expression vector of mouse survivin fused with a hexahistidine tag and its expression verified in eukaryotic cells.The results of this study should establish a good basis for the investigation of tumor DNA vaccine based on survivin.Methods:The primers were designed according to the mouse survivin gene,and the cDNA of survivin was cloned by RT-PCR.The eukaryotic expression vector for secreting type of survivin was then constructed.After sequencing analysis,the plasmid was amplified in E.coli.The recombinant plasmid was transfected into HeLa cell to express the target protein.Then the recombinant protein was enriched by metal ion chelating chromatography from the culture supernatant and identified by Western blotting analysis.Results:The cDNA encoding survivin was cloned from the total RNA of mouse spleen, and DNA sequence analysis showed that the cloned cDNA was identical to the published sequence.Then,the eukaryotic expression vector encoding survivin which fused with an IL-2 signal sequence at amino terminus and a hexahistidine tag at carboxyl terminus was successfully constructed and verified by sequencing analysis. After expansion in E.coli,the recombinant vector was expressed in HeLa cells.The recombinant survivin protein enriched by HisTrap HP column was assessed by Western blotting,indicating that the protein was expressed in secreting form in HeLa cells.Conclusion:The cDNA of mouse survivin has been cloned successfully,and the eukaryotic expression vector for the secreting survivin protein containing a interleukin 2 signal sequence is constructed.This study would be useful for research of tumor DNA vaccine which targeted to survivin in future.