Isolation, cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis

• Published in: Biocontrol science and technology Vol. 22; no. 1; pp. 11 - 21
• Main Authors: El-Ghareeb, D.K., Osman, G.H., El Baz, A.F.
• Format: Journal Article
• Language: English
• Published: Taylor & Francis Group 01.01.2012
• Subjects: Bacillus thuringiensis; cotton leaf worm; Escherichia coli; Lepidoptera; protein expression; Spodoptera littoralis; vip3Aa
• ISSN: 0958-3157; 1360-0478
• DOI: 10.1080/09583157.2011.635288

Vegetative insecticidal protein (Vip) is a newly discovered family of toxin protein isolated from Bacillus thuringiensis (Bt). An 88.5-kDa Vip3Aa protein was secreted by a local strain of the bacterium during the vegetative growth phase. The full length of the coding region '2.3 kbp' of the vip3Aa gene was isolated from plasmid DNA, cloned in pGEM-T vector and finally cloned in pQE-30 expression vector. Nucleotide sequence revealed 98% homology with that of the previously isolated genes. Expression of the vip3Aa in Escherichia coli was carried out and the expressed protein was detected in the concentrated supernatant, not in the pellet. This indicated that vip3Aa is secreted into the culture medium. Expressed protein was purified, blotted, and assayed against the cotton leaf worm Spodoptera littoralis. The LC 50 was found to be 142.4 µ/mL while the LC 50 was 90 ppm for the wild strain. These results suggest the use of either the isolated Bt strains or the expressed vip3Aa in an integrated pest management program against lepidopteran insect pests.