大豆症青相关病毒CP蛋白的抗血清的制备及检测应用  

Preparation and application of antiserum to soybean stay-green associated virus coat protein

作　　者：张玉阳 李庆伦 于连伟 谢莉娜 姜兴林 王贺 李洪连[1] 施艳[1] 杨雪 袁虹霞[1] ZHANG Yuyang;LI Qinglun;YU Lianwei;XIE Lina;JIANG Xingling;WANG He;LI Honglian;SHI Yan;YANG Xue;YUAN Hongxia(Department of Plant Pathology,College of Plant Protection,Henan Agricultural University,Zhengzhou 450002,China;Crop Science Postdoctoral Programme of Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学植物保护学院植物病理学系,郑州450002 [2]河南农业大学作物学博士后流动站,郑州450002

出　　处：《植物病理学报》2024年第3期555-560,共6页Acta Phytopathologica Sinica

基　　金：河南省重大科技专项(221100110300);河南省青年人才托举工程项目(2022HYTP037);河南农业大学科技创新项目(KJCX2021A13)。

摘　　要：大豆症青相关病毒(soybean stay-green associated virus,SoSGV)是新发现的一种可以引起大豆症青的植物病毒。大豆症青在中国大豆产区广泛发生流行,严重威胁大豆产业的健康发展,因此建立快速有效的SoSGV检测方法对于大豆症青的监测和防控具有重要意义。本研究依据SoSGV外壳蛋白(coat protein,CP)基因的核苷酸序列设计引物,从发病大豆中扩增得到786 bp的目的基因,将其克隆到原核表达载体pET-28a上,获得重组载体pET-28a-CP。在大肠杆菌Rosseta菌株中经过IPTG诱导表达,获得30 kDa的蛋白,与预期SoSGV CP大小一致。以纯化后SoSGV CP蛋白为抗原,免疫小鼠,制备了特异性SoSGV CP抗血清。In-ELISA检测结果表明,血清效价≥3.2×10~4。Western blot分析表明所制备的SoSGV CP抗体可以在SoSGV侵染的烟草叶片和大豆症青发病的大豆叶片中检测到30 kDa的特异性条带。本研究为SoSGV引起的大豆症青的快速检测提供了重要的技术支撑。Soybean stay-green associated virus(SoSGV)is a newly discovered plant virus that can cause soybean stay-green.Soybean stay-green is widespread in the soybean producing area of China and seriously threatens the development of soybean industry.Therefore,it is of great importance to establish a rapid and effective SoSGV detection method for the monitoring and control of soybean stay-green.In this study,SoSGV CP primers were designed according to the nucleotide sequence of the SoSGV coat protein(CP)gene,CP gene of 786 bp length was amplified from infected soybean,and cloned into prokaryotic expression vector pET-28a to obtain the recombinant plasmid pET-28a-CP.The recombinant vector pET-28a-CP was transformed into Escherichia coli strain Rosseta.After induction with IPTG,a 30 kDa protein was obtained,which was consistent with the molecular weight of SoSGV CP.SoSGV CP antiserum was prepared by immunizing mice with recombinant SoSGV CP protein.The In-ELISA test results showed that the serum titer of the SoSGV CP antiserum was≥3.2×104.Western blot analysis showed that the SoSGV CP antiserum could specifically recognize the SoSGV CP protein,which was 30 kDa,in the SoSGV-infected tobacco and soybean plants.These results indicate that the SoSGV CP antiserum provides favorable conditions for rapid detection of soybean stay-green in soybeans-infected by SoSGV.

关 键 词：大豆症青相关病毒 大豆症青 外壳蛋白 原核表达 抗血清 

分 类 号：S432.44[农业科学—植物病理学]