CONSTRUCTION AND EXPRESSION OF A BIFUNCTIONAL SINGLE-CHAIN ANTIBODY AGAINST BACILLUS-CEREUS SPORES

The variable-region genes of monoclonal antibody against Bacillus cere us spores were cloned from mouse hybridoma cells by reverse transcript ion-PCR. The heavy- and light-chain variable-region genes were connect ed by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single- chain antibody construct was inserted into the expression vector and e xpressed in Escherichia coli under the control of the T7 RNA polymeras e T7 promoter expression system. The expressed single-chain antibody w as detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by ca lculation) had B. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.