| 赵 霞, 秦发亮, 刘文文*, 王锡锋.灰飞虱VAP-B蛋白的抗体制备及体内外应用[J].植物保护,2022,48(4):220-225. |
| 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 |
| 投稿时间：2021-04-21 修订日期：2021-05-16 |
| DOI：10.16688/j.zwbh.2021246 |
| 中文关键词: 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 |
| 英文关键词:Laodelphax striatellus vesicle associated membrane protein associated protein B antibody preparation Western blot immunofluorescence |
| 基金项目:国家自然科学基金（31772134） |
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| 中文摘要: |
| 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Ni2+-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。 |
| 英文摘要: |
| 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 Ni2+-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. |
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