Abstract
ObjectivesTo develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products.ResultsVegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis (Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25–500 pg/ml, with a sensitivity of 10.24 pg/ml.ConclusionsThe established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.
Highlights
Some insect pathogenic microbiology synthesizes a large number of insecticidal proteins
The vip3Aa20 gene was amplified from genetically modified (GM) MIR162 genomic DNA and subcloned into the pET28a expression vector for overexpression and purification
His-Vip3Aa20 was successfully expressed in E. coli BL21 cells
Summary
Some insect pathogenic microbiology synthesizes a large number of insecticidal proteins. These insecticidal proteins form inclusion bodies (such as Cry and Cyt proteins) or are secreted into the cultural medium (such as Vip and Sip proteins). Cry proteins are widely used in agricultural pest control (Ashouri 2004; Clive 2007; de Maagd et al 2003; Estruch et al 1997; Shelton 2012; Tabashnik et al 2015). In 1996, Vip proteins were screened from the culture supernatant of Bacillus thuringiensis (Estruch et al 1996) and found to be toxic to a wide range of Lepidoptera species, some of which show tolerance or low susceptibility to Cry proteins. The Vip3Aa protein has been found to be toxic to most Lepidoptera insects. Vip3Aa genes have been successfully transferred into cotton and maize, as patented in the United States in 2009 (Adamczyk and Mahaffey 2008; Kurtz et al 2007)
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