两种方法测定绿盲蝽气味结合蛋白AlucOBP21配体结合特性的结果比较  

Comparison of Two Methods in Analysis of Binding Characteristics of Odorant Binding Proteins AlucOBP21 of Apolygus lucorum

作　　者：刘航玮 张强[1] 耿亭[2] 董昆 安兴奎 王琪 张永军[1] 郭予元[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193 [2]中国农业科学院廊坊科研中试基地,河北廊坊065000

出　　处：《中国农业科学》2017年第23期4585-4592,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31471778;31672038;31621064;31772176)

摘　　要：【目的】比较荧光竞争结合试验(fluorescence competitive binding assay)和微量热涌动(microscale thermophoresis,MST)技术在研究绿盲蝽(Apolygus lucorum)气味结合蛋白(odorant binding proteins,OBPs)Aluc OBP21配体结合特性上的差异,探索一种新的测定昆虫OBPs结合功能的方法。【方法】提取绿盲蝽雌、雄成虫触角总RNA并进行反转录,获得绿盲蝽成虫触角c DNA。采用特异性克隆引物,以绿盲蝽成虫触角c DNA为模板进行PCR扩增,构建p ET32a/Aluc OBP21重组质粒。将重组质粒转化至BL21(DE3)感受态细胞进行原核表达,获得含表达标签的重组Aluc OBP21蛋白。通过高亲和Ni-NTA纯化介质对重组粗蛋白进行纯化,采用重组肠激酶切掉His-tag标签,最终获得无表达标签的重组Aluc OBP21蛋白。利用荧光竞争结合试验,以1-N-phenylnaphthylamine(1-NPN)为荧光探针研究重组Aluc OBP21蛋白与候选配体的结合特性,其中1-NPN和气味标样均溶解在质谱纯级的甲醇中。同时,利用MST技术解析重组Aluc OBP21蛋白与候选配体的结合特性,候选配体标样溶解在二甲基亚砜(dimethyl sulfoxide,DMSO)溶液中。候选配体化合物包括8种潜在的盲蝽性信息素及性信息素类似物、12种植物绿叶挥发物和绿盲蝽驱避剂主要组分二甲基二硫醚等。【结果】重组Aluc OBP21蛋白在上清液和包涵体均能够表达,最终选择上清组分进行目标蛋白纯化,利用重组肠激酶在22℃切除His-tag标签得到无标签的重组Aluc OBP21蛋白。荧光竞争结合试验结果显示,重组Aluc OBP21与荧光探针1-NPN的解离常数为(6.88±0.31)μmol·L^(-1),Aluc OBP21能够与β-紫罗兰酮和β-石竹烯结合,解离常数分别为(13.74±1.93)和(13.24±2.12)μmol·L^(-1),而其他候选配体均不能与重组Aluc OBP21有效结合。MST测试结果显示,Aluc OBP21可以与β-石竹烯、β-紫罗兰酮、β-蒎烯和柠檬烯有效结合,解离常数分别为(0.20±0.02)、(0.05±0.01)、(0.70±0.04)和(0【Objective】The objective of this study is to compare two methods of fluorescence competitive binding assay and microscale thermophoresis（MST） in analysis of binding characteristics of odorant binding proteins Aluc OBP21 of Apolygus lucorum, and to explore a new method for determination of binding function of insect OBPs. 【Method】 Total RNA was extracted from antennae of both female and male A. lucorum adults by using Trizol reagent. The c DNAs were synthesized using the Superscript III Reverse Transcriptase system. Aluc OBP21 was PCR-amplified using gene-specific primers. The sample c DNA of the antennae was used as the template. The PCR product was cloned into an expression vector PET-32 a（＋） for expression in prokaryotic BL21（DE3） cells. The transformation of the strain with p ET32 a/Aluc OBP21 was incubated in cultures and the crude protein with His-tag was obtained. The supernatant was obtained by sonication, purified by Ni ion affinity chromatography. Soon after the His-tag was removed with recombinant enterokinase, then the purified Aluc OBP21 without His-tag was harvested. To investigate the binding abilities of Aluc OBP21 to candidate ligand chemicals, fluorescence binding test was performed using 1-N-phenylnaphthylamine（1-NPN） as a fluorescence probe. 1-NPN and odor standard samples were dissolved in methanol（mass spectrometry grade）. And also, the binding characteristics of recombinant Aluc OBP21 to candidate ligands were explored by MST technique. In this assay, the candidate ligand samples were dissolved in dimethyl sulfoxide（DMSO） solution. Candidate ligands included 8 potential sex pheromones and sex pheromone analogues of mirids, 12 green leaf volatiles and a repellent（dimethyl disulfide） of A. lucorum.【Result】Recombinant Aluc OBP21 was expressed in both supernatant and inclusion bodies. Finally, the supernatant fraction was selected to purify the target protein. Recombinant Aluc OBP21 without His-tag was obtained by using recombinant entherokinase at 22℃

关 键 词：绿盲蝽 气味结合蛋白 AlucOBP21 竞争结合试验 微量热涌动测定 

分 类 号：S433[农业科学—农业昆虫与害虫防治]