Construction and expression of a bifunctional single-chain antibody against Bacillus cereus spores

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 tertia...

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Bibliographic Details
Published in:Applied and environmental microbiology 1998-07, Vol.64 (7), p.2490-2496
Main Authors: Koo, K. (North Carolina State University, Raleigh, NC.), Foegeding, P.M, Swaisgood, H.E
Format: Article
Language:English
Subjects:
BACILLUS CEREUS
Bacteria
BACTERIAL SPORES
Biological and medical sciences
Biotechnology
Cloning
Escherichia coli
ESPORAS BACTERIANAS
Food industries
Food Microbiology
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
IMMUNOBLOTTING
Methods. Procedures. Technologies
Microbiology
Miscellaneous
PCR
POLYMERASE CHAIN REACTION
PROTEINAS RECOMBINANTES
PROTEINE RECOMBINANTE
RECOMBINANT ANTIBODIES
RECOMBINANT PROTEINS
Rodents
SPORE BACTERIENNE
Synthetic digonucleotides and genes. Sequencing
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Summary: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
ISSN:0099-2240
1098-5336
DOI:10.1128/aem.64.7.2490-2496.1998