Cloning and characterization of vegetative insecticidal protein (Vip3A) from Thai isolated Bacillus thuringiensis

Abstract

Vip3A is a group of vegetative insecticidal proteins produced during vegetative growth phase and secreted into the culture media by some strains of Bacillus thuringiensis (Bt). Vip3A proteins produced by different Bt strains show variable toxicity and specificity to different insects. Twenty Bt isolates collected from various locations in Thailand were screened for a new Vip3A by PCR using primers specific to vip3 gene. Most of Bt isolates were found to carry vip3Aa gene. Only Bt isolates number 22 and 107 were found to carry vip3A genes similar to vip3Af and vip3Ad, respectively. Both genes were cloned into pET28b to obtained 6xHis-tag fusion proteins. The fusion proteins were highly produced in Escherichia coli and were purified using HisTrap FF column. Insect bioassays revealed that Vip3Ad from Bt#107 was not toxic to Spodoptera exigua and Spodoptera litura larvae since the mortality was only 8% and 2%, respectively when both species were fed with Vip3Ad up to 40 mg/cm². Vip3Af from Bt#22 caused mortality to 20% in S. exigua and 60% in S. litura when feeding both insects with the toxin at 2 mg/cm². Although the newly discovered Vip3Ad and Vip3Af were not as good as the benchmark protein (Vip3Aa) when tested against S. exigua and S. litura, both toxins might have higher activity against other insects.