苏云金芽孢杆菌Vip3Aa10毒素结合肽的筛选及活性测定

摘要

[Objective]Vip3A, which is produced and secreted by Bacillus thuringiensisat 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 werescreened 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%.%[目的]Vip3A是由苏云金芽孢杆菌(Bacillus thuringiensis)在对数生长期产生并分泌到胞外的对鳞翅目昆虫有较广杀虫谱的毒素,其在敏感昆虫中肠上的结合受体尚未得到鉴定.利用噬菌体展示技术,论文试图从肽库中筛选到能与 Vip3A 毒素特异性结合的多肽,为研究 Vip3A 毒素的杀虫模式提供线索.[方法]将构建的pET28a-Vip3Aa10表达质粒转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达后,用亲和纯化的方法制备Vip3Aa10,并用胰蛋白酶活化和离子交换层析进一步纯化.以活化的Vip3Aa10毒素为诱饵,经"吸附-洗脱-扩繁"4轮条件逐渐严格的淘选,从噬菌体随机肽库中筛选能特异性地与活化的Vip3Aa10结合的噬菌体.以筛选到的噬菌体基因组DNA为模板,经PCR扩增和测序,获得插入到噬菌体上的外源基因的序列,并推导出相应多肽的氨基酸序列.将化学合成的多肽与活化的Vip3Aa10一起与甜菜夜蛾(Spodoptera exigua)刷状缘膜囊泡共育后,用免疫印迹方法检测多肽对活化的Vip3Aa10与BBMV结合的影响.将合成的多肽与活化的Vip3Aa10一起涂布在饲料表面,晾干后,每孔放1只1龄的甜菜夜蛾幼虫.6 d后统计昆虫死亡情况.[结果]在25℃条件下用IPTG诱导含pET28a-Vip3Aa10质粒的大肠杆菌BL21(DE3)可以表达出可溶性的Vip3Aa10蛋白.经亲和纯化、胰蛋白酶活化以及离子交换层析后,可得到较纯的活化的Vip3Aa10.以活化的Vip3Aa10为诱饵,经过4轮条件逐渐严格的淘选,从十二肽库中筛选出9条多肽,其中3条多肽的丰度较高,分别命名为P12-1、P12-2和P12-3,从七肽库中筛选出5条多肽,其中1条多肽的丰度较高,命名为P7-1.结合试验和杀虫活性测定试验结果表明,P12-2和P7-1多肽能不同程度地抑制Vip3Aa10与甜菜夜蛾刷状缘膜囊泡的结合,以及抑制Vip3Aa10的杀虫活性.[结论]用噬菌体展示技术筛选的多肽P7-1能显著性地抑制Vip3Aa10与刷状缘膜囊泡的结合,可降低35%以上的Vip3Aa10的杀虫活性.