灰飞虱VAP-B蛋白的抗体制备及体内外应用  

Antibody preparation of VAP-B protein from Laodelphax striatellus and application in vivo and vitro

作　　者：赵霞 秦发亮 刘文文[1] 王锡锋[1] ZHAO Xia;QIN Faliang;LIU Wenwen;WANG Xifeng(State Key Laboratory for Biology of Plant Disease and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

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

出　　处：《植物保护》2022年第4期220-225,239,共7页Plant Protection

基　　金：国家自然科学基金(31772134)。

摘　　要：灰飞虱Laodelphax striatellus的囊泡相关膜蛋白相关蛋白B(VAP-B)是内质网膜蛋白家族的蛋白,其主要参与调节囊泡运输、脂类的转运代谢以及未折叠蛋白应答等。本研究克隆了灰飞虱VAP-B基因,构建了pCold-SUMO-VAP-B原核表达载体,再将其转化大肠杆菌感受态细胞Rosetta(DE3),经过IPTG低温诱导表达得到了含有HIS和SUMO标签的VAP-B可溶性蛋白,经过Ni^(2+)-NTA亲和层析柱纯化后的蛋白再利用rTEV酶去除SUMO标签,得到无标签的VAP-B蛋白。通过免疫新西兰雄兔制备了VAP-B的多克隆抗体。Western blot检测结果表明该抗体可以特异性检测到灰飞虱的VAP-B蛋白;同时利用该抗体进行免疫荧光标记,发现其能特异性定位灰飞虱唾液腺内的VAP-B蛋白。研究结果表明制备的VAP-B抗体能够成功用于该蛋白的体内外检测,为阐明VAP-B在灰飞虱体内的关键作用奠定了基础。The vesicle associated membrane protein associated protein B of small brown planthopper(SBPH)(Laodelphax striatellus)belongs to highly conserved endoplasmic reticulum(ER)membrane family protein,which mainly involves in the regulation of vesicle transport,lipid transport and unfolded protein response.In this study,the gene VAP-B of SBPH was cloned and the prokaryotic expression vector pCold-SUMO-VAP-B was constructed.Then the expression vector was transformed into Escherichia coli Rosetta(DE3)to express soluble VAP-B protein by IPTG induction at low temperature.The protein was purified through Ni^(2+)-NTA affinity chromatography prepacked column and then the SUMO tag was cleaved through rTEV protease.Anti-VAP-B polyclonal antibody was produced by immunizing New Zealand male rabbit.Western blot assay revealed that the antibody could detect the VAP-B of SBPH and the recombinant protein.At the same time,this antibody can be used to label the VAP-B in the salivary gland of SBPH specifically by immunofluorescence.These results suggested that the VAP-B antibody can be successfully used for the detection of the protein both in vitro and in vivo,which provide the foundation for clarifying the key role of VAP-B in the body of SPBH.

关 键 词：灰飞虱 囊泡相关膜蛋白相关蛋白B 抗体制备 Western blot 免疫荧光标记 

分 类 号：S433.39[农业科学—农业昆虫与害虫防治]