【摘要】 【目的】通过构建柑橘大实蝇（Bactrocera minax）BminOBP6的C末端截短突变体（TOBP6），比较野生型BminOBP6和突变体TOBP6对气味配体的结合能力，检测在不同pH条件下二者对气味配体结合能力的差异，为揭示BminOBP6与配体的互作机制提供参考。【方法】通过在线工具SWISS MODEL对BminOBP6进行同源建模，根据构建的3D模型确定将被切除的BminOBP6第6个α螺旋（α6）之后的C末端序列；设计特异性引物扩增BminOBP6的C末端截短突变体（TOBP6）的编码序列，即TOBP6；构建重组表达载体pET32a/TOBP6，将此重组载体与实验室保存的重组载体pET32a/BminOBP6分别转入原核细胞BL21（DE3）中，异源表达野生型BminOBP6及其突变体TOBP6；以1-NPN为荧光探针进行荧光竞争结合试验，检测BminOBP6和TOBP6在两种pH环境（pH 7.4和pH 5.0）下对1-十一醇和（+）-柠檬烯的结合能力。【结果】序列比对结果显示，BminOBP6与致倦库蚊（Culex quinquefasciatus）CquiOBP1的序列具有很高的相似性，为62.6%，远高于30%，因此选择以CquiOBP1的3D结构作为BminOBP6同源建模模板。模型显示BminOBP6的α6之后是由8个氨基酸残基（D117—P124）组成的C末端序列，即将被切除的C末端序列。在此基础上设计引物克隆获得了编码TOBP6的cDNA序列，并构建了其重组表达载体pET32a/TOBP6，该重组载体和pET-32a/BminOBP6重组载体分别转入大肠杆菌细胞中进行了异源表达。荧光竞争结合试验结果显示，在pH为7.4时，BminOBP6与1-十一醇和（+）-柠檬烯具有很强的结合能力，K_i值分别为6.89和9.50μmol·L^-1。当pH降至5.0时，BminOBP6却丧失了对1-十一醇的结合能力，同时与（+）-柠檬烯的结合能力也大幅减弱，K_i值从9.50μmol·L^-1升至31.26μmol·L^-1；而不论在何种pH条件下，TOBP6均丧失了对这两种气味配体的结合能力。【结论】pH在柑橘大实蝇BminOBP6与其配体结合和释放过程中发挥了重要作用，并且BminOBP6的C末端在配体的结合中起着关键作用。更多还原

【Abstract】 【Objective】To explore the recognition mechanisms of Bmin OBP6 and its ligands,a C terminus-truncated Bmin OBP6（TOBP6） was constructed in this study.Subsequently,the binding affinities of TOBP6 and the wild type protein Bmin OBP6 to1-undecanol and （+）-limonene under different pH conditions were determined.【Method】3D structure of BminOBP6 was built by homology modeling through the online software SWISS MODEL.Based on the established BminOBP6 3D model,the C terminus sequence after sixth α-helix of BminOBP6 was determined.This C terminus sequence is the sequence that will be removed from Bmin OBP6 in the subsequent study.Specific primers were designed to amplify the encoding sequence of BminOBP6 C terminustruncated mutant TOBP6.Then the recombinant expression vector pET-32a/TOBP6 was constructed.Recombinant expression vectors pET-32a/TOBP6 and pET32a/BminOBP6 that has been made before in our lab were transformed into Escherichia coli cells BL21 （DE3） to express the TOBP6 and BminOBP6 recombinant proteins,respectively.Fluorescence competitive binding assays were conducted to determine the binding affinities of TOBP6 and BminOBP6 to 1-undecanol and （+）-limonene under different pH（7.4 and 5.0） conditions.【Result】Amino acid sequence alignments revealed that BminOBP6 shared 62.6%identity with Culex quinquefasciatus Cqui OBP1,and thus the 3D structure of Cqui OBP1 was used as the template in the homology modeling of BminOBP6.The 3D structure of BminOBP6 indicated that the C terminus sequence after α6 of BminOBP6 is a sequence of 8 amino acid residues （D117-P124）.Based on this result,the encoding sequence of TOBP6 was cloned and its recombinant expression vector pET-32a/TOBP6 was built.Recombinant expression vectors pET-32a/TOBP6 and pET32a/BminOBP6 were then transformed into E.coli cells BL21 （DE3） to express the TOBP6 and BminOBP6 recombinant proteins,respectively.The fluorescence competitive binding results showed that the binding affinity of BminOBP6 to 1-undecanol and （+）-limonene was quite strong at pH 7.4,the K_i values were 6.89 and 9.50μmol·L^-1,respectively.However,when the pH decreased to 5.0,BminOBP6 completely lost its binding capacity to 1-undecanol and exhibited an obvious decrease in binding to （+）-limonene (the K_i value increased from 9.50μmol·L^-1 to31.26μmol·L^-1).The binding affinity of TOBP6 to 1-undecanol and （+）-limonene was completely abolished regardless of p H conditions.【Conclusion】Changes in pH have a significant effect on the ligand binding and release of BminOBP6,and the C terminus of BminOBP6 is crucial for BminOBP6 to bind its ligands.更多还原