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Function and optimization of colorectal cancer vaccine candidate spGA733-FcK and spGA733-Fc expressed in baculovirus-insect cell system
The antigen GA733 is a cell-surface highly expressed glycoprotein on most human colorectal carcinomas. GA733 can be characterized as a cancer vaccine. In this study, GA733 was fused to the human immunoglobulin IgG Fc fragment to become recombinant gene GA733-Fc. Based on this, 4 recombinant genes were constructed as follows: GA733-Fc with signal peptide sequence and fusion of ER retention sequence (KDEL) (spGA733-FcK), GA733-Fc with signal sequence (spGA733-Fc), GA733-Fc fused to ER retention sequence (GA733-FcK) without signal peptide and GA733-Fc without signal peptide. Baculovirus-insect cell expression system is widely used for the high level production of recombinant proteins especially for glycoproteins. Constructed 4 recombinant genes were cloned to baculovirus express vectors. DH10Bac E.coli.-mediated transformation was used to generate recombinant bacmid DNA. Recombinant DNA was confirmed by PCR. Insect cell was transfected by bacmid to produce the recombinant baculovirus infects insect cell to produce recombinant protein. Western blot and sandwich ELISA showed the expression of recombinant proteins. Each cell lines (sf9 and HighFive) differed in recombinant proteins production level and protein secretion capability. N-Glycosylation analysis showed the function of signal peptide and ER retention sequence (KDEL). Taken together, baculovirus-insect cell system can be used to express recombinant GA733-Fc glycoproteins.
