Construction and expression of a bifunctional single-chain antibody against Bacillus cereus p6ores.
The variable-region genes of monoclonal antibody against Bacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected 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 expressed in Escherichia coli under the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) had B. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.
Koo
K
K
Department of Food Science, North Carolina State University, Raleigh 27695-7624, USA.
Foegeding
P M
PM
Swaisgood
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics* / Q000378:metabolism; D001409:Bacillus cereus / Q000276:immunology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013171:Spores, Bacterial / Q000276:immunology; D014169:Transformation, Bacterial
D000907:Antibodies, Bacterial; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins