二化螟APN1的原核表达及其与Cry2Aa蛋白的结合特性研究

Prokaryotic expression of aminopeptidase N1 from Chilo suppressalis and in vitro binding analysis with the Cry2Aa toxin

【目的】Bt杀虫蛋白发挥杀虫活性的重要前提是Cry蛋白能够与昆虫中肠上皮细胞刷状缘膜囊(BBMVs)上的受体蛋白结合。在前期获得二化螟氨肽酶N1(Aminopeptidase N,APN1)基因全长序列的基础上,明确二化螟APN1多肽片段与Cry2Aa的结合能力。【方法】将二化螟APN1序列片段在大肠杆菌BL21(DE3)中表达,利用蛋白质单向电泳和ligand blotting技术分析二化螟APN1多肽片段与Cry2Aa的结合能力。【结果】重组载体可在表达菌株BL21(DE3)中表达一个约70 ku的蛋白,纯化后的多肽条带单一,纯度较好。Ligand blot分析结果显示,表达的二化螟APN1多肽片段可以与活化的Cry2Aa杀虫蛋白结合,且结合条带随着重组蛋白上样量的降低而减弱。【结论】APN1多肽片段可以与Cry2Aa结合,为阐明APN1基因的功能奠定基础,也为其他Bt蛋白的受体蛋白相关研究提供新的借鉴。

【Aim】The important premise of insecticidal activity of Bt toxins is that they can bind to specific receptors on the brush marginal capsule(BBMVs)of the epithelial cells in the insect midgut.Based on the full length sequence of the aminopeptidase N(APN)gene of Chilo suppressalis,the binding ability of APN1 to Cry2Aa toxin was determined.【Method】APN1 of C.suppressalis was expressed in Escherichia coli BL21(DE3).The binding ability of APN1 of C.suppressalis to Cry2Aa was analyzed by one dimensional electrophoresis and a ligand blotting assay.【Result】The recombinant vector expressed a 70 ku protein in the BL21(DE3)strain,which indicated that the prokaryotic expression vector was successfully constructed.SDS-PAGE showed a single protein band,indicating purity.Ligand blot results showed that APN1 recombinant protein could bind to Cry2Aa.The width of the binding band decreased with decreasing recombinant protein sample volume.【Conclusion】APN1 could bind to the Cry2Aa toxin,which lays a foundation for elucidating the function of APN1 gene and provides a new reference for the study of other Bt receptors.