白蜡窄吉丁气味结合蛋白AplaOBP3的免疫定位及配体结合特性

【目的】本研究旨在解析白蜡窄吉丁Agrilus planipennis气味结合蛋白AplaOBP3在触角中的定位及配体结合特性，同时比较AplaOBP3与已报道的AplaOBP1和AplaOBP2的功能异同。【方法】原核表达白蜡窄吉丁气味结合蛋白AplaOBP3，制备多克隆抗体并进行Western blot检测；采用免疫组织化学技术在白蜡窄吉丁触角感器中对AplaOBP3的表达进行定位；通过荧光竞争结合实验分析AplaOBP3重组蛋白与58种化合物的结合特性，并与前期报道的白蜡窄吉丁AplaOBP1和AplaOBP2蛋白的配体结合特性进行比较。【结果】原核表达系统中成功表达AplaOBP3重组蛋白；免疫定位结果显示AplaOBP3在白蜡窄吉丁触角的锥形感器类型I中表达。荧光竞争结合实验结果表明，重组AplaOBP3蛋白与反-2-己烯醛、苯甲醛、4′-乙基苯乙酮、β-紫罗兰酮和罗勒烯具有强的结合能力，其解离常数KD值分别为6.20，4.03，5.24，1.72和4.83 μmol/L。AplaOBP3与AplaOBP2具有相似的表达及配体结合特性。【结论】白蜡窄吉丁气味结合蛋白AplaOBP3与AplaOBP2具有相似的表达及配体结合特性，它们可能共同参与白蜡窄吉丁的嗅觉感受，在白蜡窄吉丁寄主定位过程中发挥作用。

Immunolocalization and ligand binding characteristics of the odorant binding proteinAplaOBP3 ofAgrilus planipennis(Coleoptera: Buprestidae)

【Aim】 The aim of this study is to explore the localization of the odorantbinding protein AplaOBP3 in antennae of Agrilus planipennis and its ligand bindingcharacteristics, and to compare the function of AplaOBP3 with the reported AplaOBP1 andAplaOBP2. 【Methods】 The recombinant AplaOBP3 of A. planipennis was expressed inprokaryotic expression system. The polyclonal antibody against AplaOBP3 was prepared anddetected by Western blot assay. Immunocytochemical technique was used to localize theexpression of AplaOBP3 in antennal sensilla of A. planipennis. Its binding characteristicsto 58 compounds were determined by fluorescence competitive binding assay, and comparedwith those of AplaOBP1 and AplaOBP2 previously reported. 【Results】 The recombinantprotein AplaOBP3 was successfully expressed in prokaryotic expression system. Theimmunolocalization results showed that AplaOBP3 was expressed in the antennal sensillabasiconica type I. The results of fluorescence competitive binding assay showed thatAplaOBP3 had strong binding capabilities with trans-2-hexenal, benzaldehyde,4′-ethylacetophenone, β-ionone and ocimene, with the dissociation constant KD values of6.20, 4.03, 5.24, 1.72 and 4.83 μmol/L, respectively. AplaOBP3 and AplaOBP2 had similarexpression and ligand binding properties. 【Conclusion】 AplaOBP3 and AplaOBP2 have similarexpression and ligand binding properties, and both may be involved in olfactory perceptiontogether and play a role in host localization in A. planipennis.