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【目的】利用原核表达小菜蛾(Plutella xyllostella)中肠膜结合碱性磷酸酯酶(membrane-bound alkaline phosphatase,mALP)并经Ligand blot验证其具有与Cry1Ac毒素结合的能力;通过同源建模和分子对接研究Cry1Ac-mALP的结合模式,预测毒素和受体结合区域及关键氨基酸位点(热点残基),为了解毒素-受体互作机制及分子改造增强Cry毒素活性的研究打下基础。【方法】针对小菜蛾mALP全长设计引物,并以小菜蛾c DNA为模板扩增mALP基因,双酶切后用T4连接酶连接至pET-26b原核表达载体,将构建的pET-26b-mALP载体转化Trans1-T1克隆感受态,挑取克隆并提取质粒后进行PCR、双酶切和测序验证,将验证无误的重组质粒转化E.coliBL21(DE3)表达感受态细胞,进行诱导表达。将诱导表达后的mALP转至PVDF膜上,通过Western blot和Ligand blot分别验证mALP是否成功表达以及是否具有与Cry1Ac毒素结合的能力。对mALP进行同源建模、分子动力学模拟以及模型评价,获得的mALP最佳三维结构与Cry1Ac毒素利用Patch DOCK和Fire Dock程序进行分子对接试验,对确定的最佳毒素-受体复合物进行结合区域和结合氨基酸位点分析,并通过计算机辅助的丙氨酸突变扫描试验确定毒素和受体参与的关键氨基酸残基。【结果】扩增出小菜蛾mALP基因并克隆至pET-26b原核表达载体,转化E.coli BL21(DE3)表达感受态后挑取阳性克隆提取质粒后进行PCR、双酶切和测序均显示构建正确。通过原核表达和Western blot验证成功表达了mALP蛋白,并经Ligand blot试验证实了原核表达的mALP具有和Cry1Ac毒素结合的能力。利用同源建模成功获得了mALP的三维结构,通过Patch DOCK和Fire Dock分子对接程序,获得毒素和受体的对接复合物,通过溶剂可及表面积变化计算和Ligplot分析,确定毒素结构域Ⅱ和结构域Ⅲ均参与了受体结合,并且毒素和受体均以疏水结合和氢键结合模式参与结合,最后通过热点残基预测发现Cry1Ac毒素和mALP中分别有3个氨基酸残基(376ASN、443SER和486SER)和4个氨基酸残基(452ARG、499THR、502TYR和513TYR)是参与互作的关键氨基酸位点。【结论】经原核表达的小菜蛾mALP同样具有与Cry1Ac毒素结合的能力,并利用分子模拟技术预测了小菜蛾mALP三维结构及与Cry1Ac毒素结合模式。
【Objective】 Prokaryotic expression of mALP in the midgut membrane of Plutella xyllostella and its ability to bind to Cry1Ac toxin were confirmed by Ligand blotting. Molecular Docking To study the binding pattern of Cry1Ac-mALP, predict the toxin-receptor binding domain and key amino acid residues (hot spots), and lay a foundation for understanding the mechanism of toxin-receptor interaction and molecular engineering to enhance Cry toxin activity. 【Method】 The mALP gene was designed based on the mALP length of Plutella xylostella. The mALP gene was amplified from c DNA of P. xylostella by double digestion and ligated into pET-26b prokaryotic expression vector with T4 ligase. The constructed pET-26b-mALP Transformants were transformed into competent clones of Trans1-T1. The clones were picked and cloned. The plasmids were extracted and purified by PCR, double enzyme digestion and sequencing. The recombinant plasmids were transformed into E.coli BL21 (DE3) competent cells for expression. The induced expression of mALP was transferred to PVDF membrane. Western blot and Ligand blot were used to verify the successful expression of mALP and its ability to bind to Cry1Ac toxin. Homology modeling, molecular dynamics simulation and model evaluation of mALP were performed. The best three-dimensional structure of mALP obtained and Cry1Ac toxin were subjected to molecular docking experiments using Patch DOCK and Fire Dock programs. The best toxin-receptor complex identified Binding domain and binding amino acid site analysis and key amino acid residues involved in toxins and receptors were determined by computer-aided alanine mutation scanning assays. 【Result】 The mALP gene of Plutella xylostella was amplified and cloned into the prokaryotic expression vector pET-26b. The recombinant plasmid was transformed into E. coli BL21 (DE3) for expressing competent cells. The positive clones were picked out for PCR. The double digestion and sequencing showed that the mALP gene was constructed correct. The mALP protein was successfully expressed by prokaryotic expression and Western blot, and the ability of prokaryotic mALP to bind to Cry1Ac toxin was confirmed by Ligand blot. The three-dimensional structure of mALP was successfully obtained by homology modeling. By docking the program with Patch DOCK and Fire Dock, the docking complex of toxin and receptor was obtained. Through the calculation of solvent-accessible surface area change and Ligplot analysis, the toxin domain II and Domain Ⅲ are all involved in receptor binding, and both toxin and receptor are involved in the binding by means of hydrophobic binding and hydrogen bonding. Finally, three amino acid residues of Cry1Ac toxin and mALP were predicted by hot spot prediction (376ASN, 443SER And 486SER) and four amino acid residues (452ARG, 499THR, 502TYR and 513TYR) are the key amino acid sites involved in the interaction. 【Conclusion】 The mALP of the diamondback moth, Plutella xylostella, expressed in prokaryotic cells was also able to bind to Cry1Ac toxin. The mALP three-dimensional structure of the diamondback moth Plutella xylostella and its binding to Cry1Ac toxin were predicted by molecular simulation.