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玉米黄花叶病毒运动蛋白的原核表达纯化与抗血清制备
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引用本文:马秋萌,刘玉姿,吴迪,李洪蕊,王颖,韩成贵.玉米黄花叶病毒运动蛋白的原核表达纯化与抗血清制备.植物保护学报,2023,50(2):343-349
DOI:10.13802/j.cnki.zwbhxb.2023.2021138
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马秋萌 中国农业大学植物病理学系, 农业农村部作物有害生物监测与绿色防控重点实验室, 北京 100193  
刘玉姿 中国农业大学植物病理学系, 农业农村部作物有害生物监测与绿色防控重点实验室, 北京 100193  
吴迪 中国农业大学植物病理学系, 农业农村部作物有害生物监测与绿色防控重点实验室, 北京 100193  
李洪蕊 中国农业大学植物病理学系, 农业农村部作物有害生物监测与绿色防控重点实验室, 北京 100193  
王颖 中国农业大学植物病理学系, 农业农村部作物有害生物监测与绿色防控重点实验室, 北京 100193  
韩成贵 中国农业大学植物病理学系, 农业农村部作物有害生物监测与绿色防控重点实验室, 北京 100193 hanchenggui@cau.edu.cn 
中文摘要:为实现对玉米黄花叶病毒(maize yellow mosaic virus,MaYMV)的血清学检测,丰富该病毒的检测方法,将编码MaYMV运动蛋白(movement protein,MP)的基因连接到原核表达载体pDBHis-MBP上,将构建成功的原核表达质粒转化到大肠杆菌Escherichia coli中诱导表达融合蛋白,将纯化后的融合蛋白对新西兰大白兔Oryctolagus cuniculus进行免疫并制备MaYMV MP多克隆抗血清,并采用Western blot对抗血清的效价、灵敏度和特异性进行检测。结果显示,利用成功构建的原核表达载体经诱导表达获得分子量大小约为62 kD的融合蛋白,纯化后对新西兰大白兔进行免疫获得MaYMV MP抗血清,该抗血清的效价为1:128 000,灵敏度为1:32,且该抗血清能够特异性地检测到本氏烟Nicotiana benthamiana中瞬时表达的MaYMV MP,而不与马铃薯卷叶病毒属Polero-virus及黄症病毒属Luteovirus的其他病毒发生血清学交叉反应,证明该抗血清具有良好的特异性。表明本研究制备的MaYMV MP抗血清能特异性地检测到MaYMV MP,可用于对田间样品的MaYMV血清学检测。
中文关键词:玉米黄花叶病毒  血清制备  原核表达  瞬时表达  运动蛋白
 
Prokaryotic expression and purification of maize yellow mosaic virus movement protein and antiserum preparation
Author NameAffiliationE-mail
Ma Qiumeng Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, Beijing 100193, China  
Liu Yuzi Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, Beijing 100193, China  
Wu Di Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, Beijing 100193, China  
Li Hongrui Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, Beijing 100193, China  
Wang Ying Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, Beijing 100193, China  
Han Chenggui Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, Beijing 100193, China hanchenggui@cau.edu.cn 
Abstract:In order to achieve serological test of maize yellow mosaic virus (MaYMV) and provide more detection methods for the virus, the movement protein (MP) gene of MaYMV was cloned and ligated into the prokaryotic expression vector pDB-His-MBP. The ligated plasmid was transformed into Escherichia coli to express fusion protein. The fusion protein (62 kD) was purified and used to immunize Oryctolagus cuniculus to prepare MaYMV MP polyclonal antiserum, and the titer, sensitivity and specificity of the antiserum were determined by Western blot. The results showed that the titer of the antiserum was 1:128 000 and its sensitivity was 1:32. The antiserum could react specifically with the MaYMV MP transiently expressed in Nicotiana benthamiana and did not have cross-reactions with MPs of other members in the genera Polerovirus and Luteovirus. This antiserum developed in this study will be useful in detection of MaYMV MP in the transient expression system and in detection of MaYMVin field samples.
keywords:maize yellow mosaic virus (MaYMV)  antiserum preparation  prokaryotic expression  transient expression  movement protein (MP)
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