Abstract: 【Objective】Vip3A, which is produced and secreted by Bacillus thuringiensis at the vegetative stage, has a wide spectrum of activities against Lepidopteran insects. The receptor of Vip3A toxin on the midgut of sensitive insect has been not identified so far. The phage display technology was used to try to screen the binding peptide of Vip3Aa10 in this article, which may provide a clue for the discovery of the mode of action of Vip3A.【Method】pET28a-Vip3Aa10 plasmid was transformed into E. coli BL21 (DE3) cells, which was induced by IPTG. The expressed Vip3Aa10 was isolated by affinity chromatography, followed by digestion with trypsin and further purification with ion exchange chromatography. The specific binding phages of Vip3A were screened from phage display peptide library with the Vip3Aa10 as bait after four rounds screening with gradual stringent condition. The gene fragment inserted into phage was amplified by PCR with the genomic DNA of screened phage as template, followed by gene sequencing and amino acid deducing. The peptide synthesized by chemistry method was incubated with brush border membrane vesicles (BBMV) together with Vip3Aa10. And the influence of peptide on the interaction of Vip3Aa10 with BBMV was determined by Western blotting. For bioassay, the peptide was loaded with Vip3Aa10 on the surface of artificial diet. After the surface of diet was dry, one first instar Spodoptera exigua larva was placed in each well. Larvae mortality was scored after 6 days. 【Result】E. coli BL21(DE3) containing pET28a-Vip3Aa10 plasmid was induced by IPTG at 25℃. The expressed Vip3Aa10 was soluble, which was isolated by affinity chromatography, activated by trypsin and further purified by ion exchange chromatography, successively. The peptides were isolated through four rounds screening with activated Vip3Aa10 as bait. Nine peptides were screened from Ph.D.-12, including three abundant peptides, which were named P12-1, P12-2 and P12-3, respectively. Five peptides were screened from Ph.D.-7, among which there was one abundant peptide, named P7-1. Binding assay result showed that P12-2 and P7-1 could inhibit the interaction of Vip3Aa10 with BBMV to different extents. Bioassay result showed that P7-1 could significantly reduce the insecticidal activity of Vip3Aa10 against S. exigua. 【Conclusion】The peptide P7-1 could inhibit the interaction between Vip3Aa10 and BBMV significantly, and reduce the insecticidal activity of Vip3Aa10 up to 35%.

Key words: Bacillus thuringiensis, vegetative insecticidal protein, binding peptide, screening, activity analysis