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：
SATO Ryoichi
金额：
$ 8.45万
依托单位：
Tokyo University of Agriculture and Technology
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：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558068/
关键词：
Bacillus thuringiensis Phage display Directed evolution Delta -endotoxin 進化光学

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.

我们使用两种方法在Crylaa毒素表面引入封闭分子，分析了APN结合位点。在针对CRY1AA毒素构建的7种单克隆抗体中，1B10和2C2抑制了Cry1aA毒素与BMAPN1的结合，这表明它们的结合位点（Eppitopes）位于毒素的BMAPNL结合位点附近。为了确定抗体的真实表位，在冰冻A上的521arg或582Val引入半胱氨酸取代，然后在较小的阻断分子N-（9- acridinyl）MaleImide（NAM）中引入与Arg521cys和Val582cys的-SH的n-（9- acridinyl）MaleImide（NAM）。结合测定表明，两种阻断抗体结合了Val582cys毒素，但不结合val582cys-nam毒素，这表明两种抗体的表位位于域III的Val582cys附近。此外，NAM与Val582Cys共价效果影响了BMAPN1与Val582cys毒素的结合，而不是与之结合的BTR175，并降低了Bombyx Mori幼虫中Val582cys毒素的毒性。这些结果表明，Cry1aa上的BMAPN1结合位点位于582VAL附近，BMAPN1充当Bombyx mori幼虫中Cry1aA毒素的受体，我们克隆了4个cDNA，我们对氨基肽酶N同形成型的4 cDNA进行了反对的氨基酸序列，并显示了对氨基酸的构造，并显示了四个氨基酸序列，并显示了四个氨基酸序列，并将其构造的四个粘结剂进行了反式抗体，并显示了四个cdnas，并显示了氨基酸的构造。 Cry1aA和Cry1ab主要结合同工型1（BMAPN 1）。最后，我们准备了10^9个噬菌体，这些噬菌体表达在域2和3中随机突变的Cry1aA毒素。此外，我们成功地制定了一种基本方法，以选择对cadherin样受体（BTR175）的高亲和力毒素的噬菌体（BTR175）的噬菌体，从使用Cry1aa和Cry1ab作为模型材料的噬菌体的混合物中的混合物，以表达phages on Phages on phages on phages phages。