Abstract: A series of toxin protein are created in Scorpion's body, anti-neuroexcitation peptide Ⅲ is one of them. The recombinant expression vector pPIC9K-ANEPⅢ was constructed, the plasmid pNJUTRXA-ANEPⅢ was transformed into BL21. The target protein was induced by IPTG, and the expressed protein was evaluated by Tricine-SDS-PAGE. The result indicated that the size of the fusion protein was about 8000 kD. The recombinant plasmid pPIC9K-ANEPⅢ was transformed into Pichia pastoris GS 115 by electro transformation, one of His⁺Mut⁺ strain which can survive on the 2.0 mg/mL G418 plate for expression was screened out. After inducing by 0.5% methanol, the expressed protein has been evaluated by Tricine-SDS-PAGE, the size of the secreted protein was about 7800 kD, and the highest expression level appeared at 72 h. Prokaryotic and eukaryotic expression product of ANEPⅢ gene was extracted, the insect resistance test was carried out after preliminary purification. The result showed that the prokaryotic expression product did not show biological activity, but the eukaryotic expression product had better biological activity. 2nd instar larvae of Lymantria dispar were feeded by eukaryotic expression product, which concentration was 1 mg/mL, the corrected mortality was 37.9% after 5 days, and the larval feeding capacity descended to 50%. The same experiment performed on larvae of Tenebrio molitor by eukaryotic expression product, its concentration was 1 mg·ml^-1, the corrected mortality of larvae was 20.0% after 5 days.

Key words: anti-neuroexcitation peptide, vector construction, protein expression, bioassay