Approach for creation of proteinaceous insecticide from Bacilhus thuringiensis delta-endotoxin by directed evolution using phage display system

我们使用两种方法分析了APN结合位点，在CrylAa毒素的表面上引入阻断分子。在构建的7株抗Cry1Aa毒素的单克隆抗体中，1B10和2C2抑制Cry1Aa毒素与BmAPN 1的结合，表明它们的结合位点（表位）位于毒素的BmAPN 1结合位点附近。为了确定抗体的真实表位，在CrylAa上的521 Arg或582 Val处引入半胱氨酸取代，然后将较小的封闭分子N-（9-吖啶基）马来酰亚胺（NAM）共价结合至Arg521 Cys和Val 582 Cys的-SH。结合测定显示两种阻断抗体结合Val582Cys毒素，但不结合Val582Cys-NAM毒素，表明两种抗体的表位位于结构域III的Val582Cys附近。此外，与Val 582 Cys共价结合的NAM影响BmAPN 1与Val 582 Cys毒素的结合，而不影响BtR 175与BmAPN 1的结合，从而降低Val 582 Cys毒素对家蚕幼虫的毒性。这些结果表明BmAPN 1与Cry1Aa的结合位点位于582Val附近，BmAPN 1在家蚕幼虫中可能是Cry1Aa毒素的受体。然后清楚地表明Cry1Aa和Cry1Ab主要与同种型1（BmAPN 1）结合。最后，我们制备了10^9个表达在结构域2和3中随机突变的Cry1Aa毒素的质粒。此外，我们成功地建立了以Cry1Aa和Cry1Ab为模型材料，从BtR 175的混合物中筛选对钙粘蛋白样受体（BtR 175）具有高亲和力的毒素表达载体的基本方法。

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.