Cloning, expression and characterization of recombinant cry1AB for the development of immune-based detection methods
2011
Chaicharoen, A., Kasetsart Univ., Kamphaeng Saen Campus, Nakhon Pathom 73140 (Thailand). Center for Agricultural Biotechnology | Hongprayoon, R., Kasetsart Univ., Kampaengsaen Campus, Nakhom Pathom 73140 (Thailand). Dept. of Plant Pathology
The insecticidal crystal proteins encoded by cry gene of Bacillus thuringiensis have been used for control both as biopesticides and in transgenic plants. In this study, the cry1Ab gene was cloned and sequenced from the genome of transgenic maize MON 810 by polymerase chain reaction. The open reading frame of cry1Ab gene consisted of 2,459 base pairs, encoding a protein of 806 amino acids. DNA sequencing result confirmed 99% homology with that of the accession number AY326434 in GenBank and it shared 98% identity with homologous pesticidal crystal protein Cry1Ab by BLAST analysis. The cry1ab gene was inserted into Escherichia coli expression plasmit pET to produce pTAb. Recombinant Cry1Ab protein was expressed following IPTG induction and verified by Western blot analysis using anti-his tag. Analysis of the fusion proteins by SDS-PAGE showed a molecular weight of 110 kDa. The result of this study opens up the possibility of preparing large-scale production of recombinant cry1Ab protein. Subsequently, the protein will be used in order to produce antibody for immune-based methods employed in the detection of relating GMO materials.
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