Approach for creation of proteinaceous insecticide from Bacilhus thuringiensis delta-endotoxin by directed evolution using phage display system
利用噬菌体展示系统定向进化从苏云金芽孢杆菌δ-内毒素中制备蛋白质杀虫剂的方法
基本信息
- 批准号:12558068
- 负责人:
- 金额:$ 8.45万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (B)
- 财政年份:2000
- 资助国家:日本
- 起止时间:2000 至 2002
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
We analyzed APN-binding sites using two methods that introduced blocking molecules on the surface of CrylAa toxin. Of seven monoclonal antibodies constructed against Cry1Aa toxin, 1B10 and 2C2 inhibited the binding of Cry1Aa toxin to BmAPN1, suggesting that their binding sites (epitopes) are located close to the BmAPNl binding site of the toxin. To determine the true epitopes of the antibodies, cysteine substitutions were introduced at 521Arg or 582Val on CrylAa and then a smaller blocking molecule, N-(9-acridinyl)maleimide (NAM), was covalently bound to the -SH of Arg521Cys and Val582Cys. The binding assay showed that both blocking antibodies bound the Val582Cys toxin, but not the Val582Cys-NAM toxin, suggesting that the epitopes of the two antibodies were located adjacent to the Val582Cys of Domain III. In addition, NAM covalently 〓ound to Val582Cys affected BmAPN1 binding to the Val582Cys toxin, but not BtR175 binding to it, and reduced the toxicity of Val582Cys toxin in Bombyx mori larvae. These results suggest that the BmAPN1 binding site on Cry1Aa is located near 582Val and that BmAPN1 functions as a receptor for Cry1Aa toxin in Bombyx mori larvae.Next, we cloned 4 cDNAs of aminopeptidase N isoforms, showed primary structures from their deduced amino acid sequences, constructed antibodies against four each recombinant isoforms, and then clearly showed that Cry1Aa and Cry1Ab mainly bind to isoform 1 (BmAPN 1). Finally, we prepared 10^9 phages which express Cry1Aa toxin randomly mutated in domain 2 and 3. In addition, we succeeded to make a fundamental method to select high affinity toxin- expressing phages to cadherin-like receptor (BtR175) from the mixture of the phages using Cry1Aa and Cry1Ab as model materials to be expressed on the phages.
我们用两种在CrylAa毒素表面引入封闭分子的方法分析了APN结合位点。在构建的7个抗Cry1Aa毒素的单抗中,1B10和2C2抑制了Cry1Aa毒素与BmAPN1的结合,表明它们的结合部位(表位)靠近毒素的BmAPN1结合部位。为了确定抗体的真实表位,在CrylAa的521Arg或582Val引入半胱氨酸取代,然后将较小的封闭分子N-(9-acridinyl)Maleimide(NAM)共价结合到Arg521Cys和Val582Cys的-SH上。结合实验表明,两种封闭抗体均与Val582Cys毒素结合,但不与Val582Cys结合,表明两种抗体的表位均位于Val582Cys结构域Ⅲ的Val582Cys附近。此外,NAM与Val582Cys共价结合〓影响BmAPN1与Val582Cys的结合,但不影响BtR175与Val582Cys的结合,从而降低了Val582Cys毒素对家蚕幼虫的毒性。这些结果表明,Cry1Aa上的BmAPN1结合位点位于582Val附近,BmAPN1是家蚕幼虫Cry1Aa毒素的受体。接下来,我们克隆了4个氨基肽酶N亚型的cDNAs,从它们推导的氨基酸序列中显示了一级结构,并构建了针对4个重组亚型的抗体,清楚地表明Cry1Aa和Cry1Ab主要结合在亚型1(BmAPN1)上。最后,我们制备了10^9个表达在结构域2和3上随机突变的Cry1Aa毒素的噬菌体。此外,我们还成功地从这些噬菌体的混合物中筛选出高亲和力的针对类钙粘素受体(BtR175)的表达噬菌体,并以此为模型材料在噬菌体上表达。
项目成果
期刊论文数量(22)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Rajeev K.Upadhyay: "Advances in Microbial Control of Insect Pests"Kluwer Academic/Plenum Publishers. 330 (2002)
Rajeev K.Upadhyay:“害虫微生物控制的进展”Kluwer 学术/全会出版社。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Kazuko Nakanishi: "Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella - their classification and the factors that determine their binding specificity to Bacillus thuringiensis CrylA toxin"FEBS Letter. 501. 215-220 (2002)
Kazuko Nakanishi:“来自家蚕和小菜蛾中肠的氨基肽酶 N 亚型 - 它们的分类以及决定它们与苏云金芽孢杆菌 CrylA 毒素结合特异性的因素”FEBS Letter。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Hirotaka Hara: "A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae."FEBS Letter. 538. 1-6 (2003)
Hirotaka Hara:“钙粘蛋白样蛋白在家蚕幼虫的中肠上皮细胞上充当苏云金芽孢杆菌 Cry1Aa 和 Cry1Ac 毒素的受体。”FEBS 信函。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Rajeev K.: "Upadhyay, Advances in Microbial Control of Insect Pests"Advances in Microbial Control of Insect Pests. 330 (2002)
Rajeev K.:“Upadhyay,害虫微生物控制的进展”害虫微生物控制的进展。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Kazuko Nakanishi: "Aminopiptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella-their classification and the factors that determine their binding spcificity to Bacillus thuringiensis Cry1A toxin"FEBS Letter. 519. 215-220 (2002)
Kazuko Nakanishi:“来自家蚕和小菜蛾中肠的氨基肽酶 N 亚型 - 它们的分类以及决定它们与苏云金芽孢杆菌 Cry1A 毒素结合特异性的因素”FEBS 信函。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
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SATO Ryoichi其他文献
Approximate Analysis of EM Wave Scattering From Two Cracks on a Ground Plane
地平面上两个裂纹的电磁波散射的近似分析
- DOI:
- 发表时间:
2021 - 期刊:
- 影响因子:0
- 作者:
SATO Ryoichi;SHIRAI Hiroshi - 通讯作者:
SHIRAI Hiroshi
SATO Ryoichi的其他文献
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{{ truncateString('SATO Ryoichi', 18)}}的其他基金
Evolvement of "durability mechanics" for the evaluation of shrinkage effect on performance of reinforced concrete beams
用于评估收缩对钢筋混凝土梁性能影响的“耐久性力学”的演变
- 批准号:
24246077 - 财政年份:2012
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (A)
DEVELOPMENT OF A PRACTICAL TENSILE CREEP MODEL BASED ON SIMPLE EXPERIMENTS
基于简单实验的实用拉伸蠕变模型的开发
- 批准号:
24656272 - 财政年份:2012
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
Generation of a new category, "Proteinaceous Insecticide" based on mechanisms which cause specificity diversity of Bacillus thuringiensis insecticidal protein.
基于苏云金芽孢杆菌杀虫蛋白特异性多样性的机制,产生了一个新类别“蛋白质杀虫剂”。
- 批准号:
24310054 - 财政年份:2012
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Development of Accurate Detection/Identification Method of Stricken Residential Buildings Using Radar Porlarimetry
雷达偏振测量受灾住宅精确检测/识别方法的研制
- 批准号:
22510188 - 财政年份:2010
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Realization of a secure and safe proteinaceous-insecticide based on the directed evolution of the insecticidal toxin from soil bacterium, Bacillus thuringiensis
基于土壤细菌苏云金芽孢杆菌杀虫毒素的定向进化,实现安全可靠的蛋白质杀虫剂
- 批准号:
21310051 - 财政年份:2009
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Durability Mechanics for High Shrinkage Concrete Members
高收缩混凝土构件的耐久性力学
- 批准号:
21246071 - 财政年份:2009
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (A)
Detection and Classification of Landslide and/or Stricken Residential Area in Mountainous Region Using Radar Polarimetry
利用雷达旋光法对山区滑坡和/或受灾住宅区进行检测和分类
- 批准号:
19510183 - 财政年份:2007
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Development of Expansive-High Strength Concrete and Systematic Evaluation of Structural Behavior of Reinforced Concrete Beams
膨胀高强混凝土的开发及钢筋混凝土梁结构性能的系统评价
- 批准号:
16360220 - 财政年份:2004
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Directed evolution of the Bacillus thuringiensis insecticidal toxin for the creation of proteinaceous insecticide.
苏云金芽孢杆菌杀虫毒素的定向进化,用于制造蛋白质杀虫剂。
- 批准号:
15380228 - 财政年份:2003
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Research for a basic anti-microbial recognition system of the animal using silkworm, Bombyx mori
家蚕动物基本抗菌识别系统的研究
- 批准号:
10680601 - 财政年份:1998
- 资助金额:
$ 8.45万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
相似国自然基金
phage display联合基因转染技术对Bcl-2蛋白质结构和功能的研究
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