Expression pattern of gangliosides in inhibition of colorectal carcinoma cells growth by EpCAM mAb

다국어 초록 (Multilingual Abstract)

Insect cell expression system using baculovirus has several benefits from high capacity, flexibility, safety to humans and glycosylation capability. Thus, the baculovirus insect cell system has been widely used for production of recombinant protein. In this study, the insect cell expression system was designed to produce anti-cancer mAb CO17-1A, which recognizes the antigen GA733 highly expressed on the surface membrane of colorectal carcinoma cells. The heavy (HC) and light chain (LC) genes of the mAb CO17-1A were cloned under the control of two different promoters, P10 and PPH, respectively, on baculovirus expression pFastBacTM Dual vector. The gene expression cassettes carrying HC and LC genes were transferred into a parent bacmid in E.coli (DH10Bac). The bacmid was transfected to Sf9 insect cells to generate the baculovirus expressing mAb CO17-1A. Western blot and immunoflouorescence analyses confirmed expression and secretion of mAb CO17-1A in the virus infected insect cells. The optimum condition for mAb expression was optimized at 24, 48 and 72hr after the virus infection with MOI (optimum virus concentration) ranging (0.2, 1 and 5). Expression of mAb CO17-1A in insect cells significantly increased at both 48 and 72hr after transfection with the MOI 1. HPLC chromatography revealed that the insect-derived mAb CO17-1A had insect specific glycan structures. Cell ELISA showed that the purified mAb from insect cell cultured media had a specific binding activity to SW948 human colorectal cancer cell. These results indicated that the baculovirus insect cell system is able to express, assemble, and secrete functional full size monoclonal antibody with insect specific glycosylation.