Construction And Expression Of DNA Vaccine Encoding Xenogeneic G250 Complex Antigen For Renal Cell Carcinoma

G250(MN/CAⅨ) is a tummor related antigen of renal cancer.Specially expressed in renal cell carcinoma(RCC),it has been used with a wide range of renal cell carcinoma immunotherapy.The sduty is to construct a eukaryotic expression plasmid of tG250 fusion gene which encoding the most cytotoxic T lymphocyte epitopes of human G250(CAⅨ) and part CAⅨgene of mouse and monkey,and detect the expression in eukaryotic cell COS7.Firstly,Through literature research,to determine the extracellar domain of G250 which major epitopes focused,and find the corresponding gene sequence in Genbank; to find monkey and mouse gene sequences of CAⅨand compare three gene sequences through DNAMAN software.Then the domain G250 epitopes concentrated was retained,the rest was substituted by part CAⅨgene of monkey and mouse.So xenogeneic G250 designed compared with the extracellular domain of human G250 sequence after the removal of signal peptide,the homology reached 89%.Secondly, xenogeneic G250 fusion gene was constructed by gene synthesis and PCR,then inserted into a eukaryotic expression vector pCI-Fc-GPI that included the gene of the signal peptide of human Igκ,human IgG-Fc and GPI,and then inserted into another eukaryotic expression vector pVAX1-IRES-GM/B7 which included IRES and human GM-CSF as well as B7.1 gene.Lastly,the recombinant plasmid pVAX1-sig-tG250-Fc-GPI-GM/B7(pVAX1-tG250FcGB) was transfected to the COS7 cells,and the expression was detected by FACS(Fluorescence Activated Cell Sorting) and IMF(immunofluorescence).Results:The sequence of tG250 fusion gene was consistent with that of design.PCR and enzyme digestion analysis showed that the recombinant plasmid pVAX1-tG250FcGB was constructed successfully.The expression of this plasmid had been demonstrated by FACS and IMF.Conclusively,The tG250 fusion gene has been constructed successfully.The recombinant plasmid pVAX1-tG250FcGB has been expressed successfully in the COS7 cells.These results have provided necessary basises to further study the functions of this vaccine,and provided a new technical strategy for immunotherapy of renal cell carcinoma.