The fusion protein of scorpion neurotoxin BjαIT and Galanthus nivalis agglutinin (GNA) enhanced the injection insecticidal activity against silkworms, but only has lethal activity against newly hatched larva when administered orally.

Abstract

Fusing insect derived neurotoxic peptides with Galanthus nivalis agglutinin (GNA) has been shown to enhance the insecticidal activity of the neuropeptides, especially when administered orally. This study produced a recombinant scorpion insect specific neurotoxin BjαIT, GNA, and a fusion protein BjαIT/GNA using Pichia pastoris as an expression host. Recombinant rBjαIT/GNA was found to be easily degraded during expression in yeast which and produced a main protein product with a molecular weight of approximately 14 kDa. Cytotoxicity results showed that rBjαIT, rGNA, and rBjαIT/GNA had no toxicity to mammalian NIH/3T3 cells. Adding rBjαIT or rBjαIT/GNA at a concentration as low as 1 ng/mL to insect cell culture medium inhibited the proliferation of insect Sf9 cells, with rBjαIT exhibiting stronger cytotoxicity, while 20 ng/mL rGNA did not inhibit the proliferation of Sf9 cells. Silkworm larval injection results showed that rBjαIT/GNA was the most toxic of the three proteins, followed by rBjαIT, and rGNA. When rBjαIT/GNA was injected at a concentration of 0.129 nmol/g body weight 46.7% of silkworm died within 48 h. Feeding newly hatched silkworms with rBjαIT/GNA at a leaf surface concentration of 40 µg/cm² resulted in 76.7% mortality within 24 h. However, rBjαIT/GNA showed almost no oral insecticidal activity against second instar silkworms. The results indicated that rBjαIT/GNA has stronger injection insecticidal activity and feeding insecticidal activity than rBjαIT and rGNA individually, making it more suitable for biological control.