PREPARATION AND CHARACTERIZATION OF 2 HUMAN CARCINOEMBRYONIC ANTIGEN FAMILY PROTEINS OF NEUTROPHILS, CD66B AND CD66C, IN SILKWORM LARVAE

As a step to investigate the cell adhesion mechanism and physiological roles of two CD66 antigens in human neutrophils, carcinoembryonic ant igen gene family member 6 (CGM6, CD66b) and nonspecific cross-reacting antigen (NCA, CD66c), we prepared their soluble recombinant forms in silkworm larvae. Each cDNA fragment for CGM6 and NCA was ligated into the transfer vector pBK283 after modification to encode the protein la cking the membrane anchor. The resulting vectors were introduced to th e Bombyx mori nuclear polyhedrosis virus, with which silkworm larvae w ere infected. Recombinant proteins secreted into the hemolymph of larv ae at concentrations up to 1.3 mg/ml were purified by cation exchange followed by gel filtration or antibody affinity chromatography. The sm aller apparent masses of the antigens compared with those of the nativ e antigens appeared to be primarily due to incomplete glycosylation. B oth recombinant antigens are quite similar to the corresponding native antigens in terms of the antigenic reactivity against a panel of CD66 monoclonal antibodies. In addition, the recombinant CGM6 and NCA exhi bited cell binding activity against CHO cells expressing NCA and CGM6, respectively. Thus the two biologically active recombinant CD66 antig ens prepared in large quantities in silkworm larvae should be useful f or their functional studies, and our present system will be available for the production and purification of other carcinoembryonic antigen family members, whose biological functions are also unknown. (C) 1996 Academic Press, Inc.