中华按蚊气味结合蛋白AsinOBP2的基因克隆、表达谱及与人体气味物质的结合特性分析

doi:10.16380/j.kcxb.2021.02.003

摘要/Abstract

摘要：

【目的】克隆中华按蚊Anopheles sinensis气味结合蛋白2(odorant binding protein 2, OBP2)基因AsinOBP2，分析该基因的表达及其重组蛋白与人体气味物质的结合能力。【方法】采用RT-PCR和RACE技术克隆AsinOBP2的全长cDNA序列，通过qPCR分析AsinOBP2基因在中华按蚊不同发育时期(雌雄蛹及羽化后第0, 1, 2, 3, 6和9天的成虫)、3日龄成虫不同嗅觉组织(触角、喙和下颚须)和吸血前后3日龄雌成虫中的表达水平；通过原核表达和亲和层析，表达并纯化AsinOBP2重组蛋白，运用荧光竞争结合实验测定重组蛋白AsinOBP2与39种人体气味物质的结合能力。【结果】克隆并测序了AsinOBP2的全长cDNA序列(GenBank登录号: MT700441)，其开放阅读框长492 bp，编码163个氨基酸，N末端30个氨基酸为信号肽序列，具有气味结合蛋白典型的6个保守半胱氨酸结合位点，属于Classic亚家族成员。qPCR结果显示，AsinOBP2在中华按蚊羽化后的表达水平逐渐增强，羽化后第6天表达水平最高，随后开始下降；AsinOBP2主要在雌成蚊触角中表达；吸食血液后3日龄雌成虫中AsinOBP2的表达水平显著下降。荧光竞争结合实验显示，在测定的39种人体气味物质中，重组蛋白AsinOBP2与12种化合物具有结合活性，其中与吲哚结合能力较强，解离常数Ki=27.15 μmol /L，其次是与正丙胺、2-甲基丁醛和葵醛，解离常数Ki分别为42.49, 48.33和47.90 μmol /L。【结论】根据AsinOBP2基因的表达特点及其重组蛋白与人体气味物质的结合能力，后者表明AsinOBP2对人体气味物质具有明显的选择结合特性，我们推断AsinOBP2在雌蚊追寻宿主的行为中起着重要的作用。

关键词: 中华按蚊, 气味结合蛋白, 基因克隆, 原核表达, 人体气味物质, 结合能力

Abstract:

【Aim】 This study aims to clone the odorant binding protein 2 gene AsinOBP2 of Anopheles sinensis, and to analyze the expression profiles of AsinOBP2 and the binding affinities of its recombinant protein with human odorants. 【Methods】 The full-length cDNA sequence of AsinOBP2 was cloned by RT-PCR and RACE techniques. The expression levels of AsinOBP2 in different developmental stages (female and male pupa, and 0-, 1-, 2, 3-, 6- and 9-day-old adults), different chemosensory tissues (antenna, proboscis and maxillary palp) of the 3-day-old adult, and the 3-day-old female adults before and post blood meal were measured by qPCR. The recombinant protein AsinOBP2 was expressed and purified by prokaryotic expression and affinity chromatography, respectively. The binding affinities of the recombinant AsinOBP2 with 39 human odorants were analyzed using fluorescence competitive binding assay. 【Results】 The full-length cDNA of AsinOBP2 (GenBank accession no.: MT700441) was successfully cloned and sequenced. Its ORF is 492 bp in length, encoding 163 amino acids with the signal peptide of 30 amino acids at the N-terminus. The mature protein possesses six conserved cysteines and belongs to the Classic OBP subfamily. The qPCR results showed that the expression level of AsinOBP2 in adults gradually increased with the eclosion time, peaked on the 6th day after eclosion, and then went down. AsinOBP2 was mainly expressed in the antenna of female adult mosquito. The expression level of AsinOBP2 in the 3-day-old female adult decreased significantly after blood meal. The results of fluorescence competitive binding assay showed that among the 39 human odorants tested, the recombinant AsinOBP2 had binding activities with 12 compounds. AsinOBP2 had a higher binding affinity to indole, with the dissociation constant Ki value of 27.15 μmol/L, followed by n-propylamine, 2methylbutanal and decanal with the Ki values of 42.49, 48.33 and 47.90 μmol/L, respectively. 【Conclusion】 Based on the expression profiles of AsinOBP2 and the binding affinities of its recombinant protein to human odorants, which indicated that AsinOBP2 has obviously selective binding properties to human odorants, we inferred that AsinOBP2 plays an important role in host-seeking behavior of female mosquito.

Key words: Anopheles sinensis, odorant-binding protein, gene cloning, prokaryotic expression, human odorant, binding affinity

张嘉俊, 何兴菲, 张婷婷, 司风玲, 陈斌, 何正波. 中华按蚊气味结合蛋白AsinOBP2的基因克隆、表达谱及与人体气味物质的结合特性分析[J]. 昆虫学报, 2021, 64( 2): 158-169.

ZHANG Jia-Jun, HE Xing-Fei, ZHANG Ting-Ting, SI Feng-Ling, CHEN Bin, HE Zheng-Bo. cDNA cloning and expression profiling of the odorant binding protein AsinOBP2 in Anopheles sinensis (Diptera: Culicidae) and analysis of its binding characteristics with human odorants [J]. Acta Entomologica Sinica, 2021, 64( 2): 158-169.

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中华按蚊气味结合蛋白AsinOBP2的基因克隆、表达谱及与人体气味物质的结合特性分析

cDNA cloning and expression profiling of the odorant binding protein AsinOBP2 in Anopheles sinensis (Diptera: Culicidae) and analysis of its binding characteristics with human odorants

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