Abstract
A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VHmarkedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305kDa goat antibody–enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.
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