| 张潇婉, 张 璐, 刘文文, 李 莉*, 王锡锋.白背飞虱VAMP7和Vti1a蛋白的原核表达､抗体制备及应用[J].植物保护,2021,47(1):55-60. |
| 白背飞虱VAMP7和Vti1a蛋白的原核表达､抗体制备及应用 |
| Preparation and application of the antibodies of Sogatella furcifera VAMP7 and Vti1a proteins expressed in Escherichia coli |
| 投稿时间：2020-11-14 修订日期：2020-01-21 |
| DOI：DOI:10.16688/j.zwbh.2019622 |
| 中文关键词: 白背飞虱 囊泡相关膜蛋白7 囊泡转运蛋白 原核表达 Western blot 免疫荧光标记 |
| 英文关键词:Sogatella furcifera VAMP7 Vti1a prokaryotic expression Western blot immunofluorescence |
| 基金项目:国家自然科学基金(31772134); 国家重点研发计划(2017YFD0200907) |
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| 中文摘要: |
| 白背飞虱Sogatella furcifera的囊泡相关膜蛋白7(VAMP7)和囊泡转运蛋白(Vti1a)隶属于SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor)家族, 该家族蛋白主要参与生物体中关键的膜转运过程?前期研究发现这两种蛋白分别与南方水稻黑条矮缩病毒Southern rice black-streaked dwarf virus (SRBSDV)的主要外层衣壳蛋白P10存在显著互作, 推测可能协助病毒粒体在介体白背飞虱内的转运和扩散?为了进一步利用血清学技术研究VAMP7和Vti1a在传毒过程中的功能, 本研究克隆了白背飞虱编码这两种蛋白的基因, 成功构建了VAMP7和Vti1a基因的原核表达载体, 并将载体分别转入大肠杆菌中进行诱导表达, 得到了相应的原核表达蛋白?在蛋白纯化后, 将纯化蛋白注射于新西兰大白兔体内进行免疫, 分别制备得到VAMP7和Vti1a的抗体?两种抗体经Western blot检测发现, 均可分别与白背飞虱体内的VAMP7和Vti1a特异性结合?利用制备的抗体对白背飞虱的肠道进行免疫标记, 激光共聚焦显微镜观察发现所制备抗体能够在白背飞虱中肠上皮细胞的胞质中特异性标记到VAMP7和Vti1a, 表明制备的抗体能够成功用于这两种蛋白的体内外检测, 为阐明这两种蛋白参与传播SRBSDV的机制研究奠定了基础? |
| 英文摘要: |
| Vesicle-associated membrane protein 7 (VAMP7) and vesicle transport through interaction with t-SNAREs homolog 1a (Vti1a) of Sogatella furcifera belong to the SNARE family, participating in the vesicular transport. In our previous study, they interacted with the P10 of Southern rice black-streaked dwarf virus (SRBSDV) respectively, and might facilitate virion transport in insects. In order to use the serological techniques to further validate their functions, the prokaryotic expression vectors of VAMP7 and Vti1a genes were constructed and then transformed into strain BL21 (DE3) of Escherichia coli and expressed by IPTG induction. Purified proteins were used to immunize rabbits for preparation of polyclonal antibodies. Western blot showed that the prepared antibodies could specifically bind to the recombinant proteins and VAMP7 and Vti1a in S. furcifera, respectively. Immunofluorescent labeling assay revealed that VAMP7 and Vti1a were located in the cytoplasm of the midgut epithelia in S. furcifera, which laid the foundation for the functional study of VAMP7 and Vti1a in SRBSDV transmission. |
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