Creation of proteinaceous insecticide from Bacuillus thuringlensis δ-endotoxin and single chain variable fragment

苏云金芽孢杆菌δ-内毒素和单链可变片段制备蛋白质杀虫剂

• 批准号: 09558076
• 负责人: SATO Ryoichi
• 金额: $ 5.7万
• 依托单位: Tokyo University of Agriculture & Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09558076/
• 关键词: Bacillus thuringiensis; δ-endotoxin; single chain variable; insecticide; アミノペプチターゼN; δ内毒素; 殺虫剤

Creation of proteinaceous insecticide was challenged using Bacillus thuringiensis δ-endotoxin and single chain variable fragment of monoclonal antibody as materials.Monoclonal antibody was constructed against membrane protein of the midgust from longicorn beetle. Acalolepta luxuriosa and single chain variable fragment was made from the antibody. single chain variable fragment and Bacillus thuringiensis δ-endotoxin was connected by geen engineering and binding capability of the chimera-proteins were confirmed. From now on we will assess the insecticidal activity of the proteins using Acalolepte luxuriosa.On the other hand, receptor of the Cry1Aa δ-endotoxin on the midgut of the silkworm, Bombyx mori was researched to know indispensable nature for the activity of the target molecule of the δ-endotoxin since it seems most important for the activity of the chimera-protein to select appropriate molecules as targets of the single chain variable. As a result, it was shown that Cry1Aa and Cry9Da utilize aminopeptidase N as a receptor on the midgut cells of the silkworm. Cry1Aa bound to N terminal region of aminopeptidase N consisting of about 60 amino acid residues.

以苏云金芽孢杆菌δ-内毒素和单克隆抗体单链可变区片段为原料，构建了抗天牛中突菌膜蛋白的单克隆抗体。用该抗体制备了广翅无翅夜蛾的单链抗体。通过基因工程将单链可变区片段与苏云金芽孢杆菌δ-内毒素连接起来，并证实了嵌合蛋白的结合能力。另一方面，对家蚕中肠Cry 1Aa δ-内毒素受体进行了研究，以了解δ-内毒素靶分子活性的本质，因为选择合适的分子作为单链可变区的靶分子对嵌合蛋白的活性至关重要。结果表明，Cry 1Aa和Cry 9Da利用氨肽酶N作为家蚕中肠细胞上的受体。Cry 1Aa与氨肽酶N的N端结合，由约60个氨基酸残基组成。

项目成果

A.Shinkawa: "Binding of phylogenetically distant Bacillus thuringiensis Cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's"Current Microbiol.. 39. 14-20 (1999)

A.Shinkawa：“系统发育上相距较远的苏云金芽孢杆菌 Cry 毒素与家蚕氨基肽酶 N 的结合表明 Cry 毒素的重要性”《当前微生物学》.. 39. 14-20 (1999)

K.Nakanishi: "Bacillus thuringiensis insecticedal protein Cry 1Aa binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary"Biochim Biophys Acta. 1432. 57-63 (1999)

K.Nakanishi：“苏云金芽孢杆菌杀虫蛋白 Cry 1Aa 与宿主昆虫氨肽酶 N 的高度保守区域结合，从而导致其进化”Biochim Biophys Acta。

K. Yaoi: "cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori"Biochim biophys Acta. 1444. 131-137 (1999)

K. Yaoi：“来自家蚕的苏云金芽孢杆菌 Cry1Aa 毒素结合 120 kDa 氨肽酶 N 的 cDNA 克隆和表达”Biochim Biophys Acta。

A. Shinkawa: "Binding of phylogenetically distant Bacillus thuringiensis Cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's conserved structure in"Current Microbiol.. 39. 14-20 (1999)

A. Shinkawa：“系统发育上相距较远的苏云金芽孢杆菌 Cry 毒素与家蚕氨基肽酶 N 的结合表明 Cry 毒素保守结构的重要性”Current Microbiol.. 39. 14-20 (1999)