Bacillus thuringiensis VipAal Expression and Purification from Ecoli to be Determined in Seeds and Leaves of Genetically-Modified Corn Plants

摘要

Alternatives to Bacillus thuringiensis (Bt) pesticide crystal protein (Cry), such as vegetative phase insecticidal proteins (Vip), to combat insect in genetically-modified plants has being investigated to provide an insect resistance management additional tool. Likewise, this study aimed expressing and purifying Bt-Vip3Aal from an Escherichia coli strain for generating polyclonal rabbit antibodies to be then employed for Vip3Aal detection in genetically-transformed corn plants. To perform this subject, Vip3Aal was expressed in the JM-109 strain and purified by metal-chelate affinity chromatography. Next, a Dot-blot, Western-blot and an enzyme-linked immunosorbent assay (ELISA) using Vip3Aal-specific polyclonal rabbit antibodies were carried out to detect Vip3Aal in genetically-modified corn plant seeds and leaves, respectively. FR*Btl corn plants transformed with CrylFal gene served as negative control. As results, Vip3Aal production method yielded up to 16 ug of purified Vip3Aal per fermentation supernatant milliliter, 50.4+-4.0% recovery and 97.2+-2.7% of SDS-PAGE purity. Vip3Aal amino acid sequence corresponded totally with expected sequence and Vip3Aal shown a high insecticidal (93.5+-8.8%) at 2 ig mL~(-1) and immunogenicity capacity in rabbits (1:30000+-4142). Vip3Aal quantification assays evidenced high specificity and sensitivity (1 ng mL~(-1) (Dot-blot) and 1 ng mL~(-1) (detection limit) 30 ng mL~(-1) (quantification limit)) in the ELISA. Quantification results were further corroboratedthrough a biological assay using Spodoptera frugiperda and plant materials as insect diet supplements, where death rate ranged 68.7-7.0% in a doses dependent manner. Thus, these immunoassays can be successfully used to detect Vip3Aal, in the seeds and leaves allowing discrimination between modified-and non-modified-genetically corn seeds and plants.