Construction and Expression of Eukaryotic Expression Vector Encoding Xenogeneic G250 Complex Antigen for Renal Cell Carcinoma

Objective:To construct a eukaryotic expression plasmid of tG250 fusion gene which encoding the most cytotoxic T lymphocyte epitopes of human G250 and part G250 of mouse and monkey, and detect its expression in eukaryotic cell COS7.Methods:tG250 fusion gene was constructed by gene synthesis and PCR, and was 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.The recombinant plasmid pVAX1-sig-tG250-FC-GPI-GM / B7 was transfected to the COS7 cells, and the expression was detected by fluorescence activated cell sorting(FACS) and immunofluorescence(IMF).Results:The sequence of tG250 fusion gene was consistent with that of design.PCR and enzyme digestion analysis showed that the recombinant plasmid pVAX1-sig-tG250-FC-GPI-GM / B7 was constructed successfully.The expression of this plasmid had been confirmed by FACS and IMF.Conclusion:The recombinant plasmid pVAX1-sig-tG250-FC-GPI-GM / B7 has been constructed and expressed successfully in the COS7 cells.These results are necessary and basic for construction of DNA vaccine targeting G250 and research on the anti-tumor effects in the future.