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

我们采用两种方法在CrylAa毒素表面引入阻断分子，分析了apn结合位点。在抗Cry1Aa毒素的7个单克隆抗体中，1B10和2C2抑制了Cry1Aa毒素与BmAPN1的结合，这表明它们的结合位点（表位）位于Cry1Aa毒素的BmAPNl结合位点附近。为了确定抗体的真正表位，在CrylAa上的521Arg或582Val处引入半胱氨酸取代，然后将一个较小的阻断分子N-(9-acridinyl)马来酰亚胺（NAM）共价结合到Arg521Cys和Val582Cys的- sh上。结合实验表明，两种阻断抗体均能与Val582Cys毒素结合，但不能与Val582Cys- nam毒素结合，这表明两种抗体的表位位于III结构域的Val582Cys附近。此外，发现NAM共价〓能影响BmAPN1与Val582Cys毒素的结合，而不影响BtR175与Val582Cys毒素的结合，从而降低了Val582Cys毒素对家蚕幼虫的毒性。这些结果表明，BmAPN1在Cry1Aa上的结合位点位于582Val附近，BmAPN1在家蚕幼虫中作为Cry1Aa毒素的受体发挥作用。接下来，我们克隆了4个氨基肽酶N异构体的cdna，通过推导出的氨基酸序列显示了它们的初级结构，构建了针对4个重组异构体的抗体，并明确了Cry1Aa和Cry1Ab主要与1异构体（bmapn1）结合。最后，我们制备了10^9个表达Cry1Aa毒素在结构域2和3随机突变的噬菌体。此外，我们成功地建立了一种从噬菌体混合物中选择高亲和力表达毒素的噬菌体（BtR175）的基本方法，以Cry1Aa和Cry1Ab为模型材料在噬菌体上表达。

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.