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

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。

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.