凤仙花坏死斑病毒NP基因原核表达及抗血清制备  

Prokaryoticexpression and antiserum preparation of NP gene of impatiens necrotic spot virus

作　　者：蒋润州 纪开燕 赵海婷 丁诗文 秦朗 张坤[1] 赵景奎 贺振[1] JIANG Runzhou;JI Kaiyan;ZHAO Haiting;DING Shiwen;QIN Lang;ZHANG Kun;ZHAO Jingkui;HE Zhen(College of Plant Protection,Yangzhou University,Yangzhou 225009,China;Hengyang Technician College,Hengyang 421101,China;Yangzhou Forestry Management Station,Yangzhou 225009,China)

机构地区：[1]扬州大学植物保护学院,江苏扬州225009 [2]衡阳技师学院,湖南衡阳421101 [3]扬州市林业管理站,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2024年第5期45-51,共7页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(32272485);泰州市“凤城英才计划”双创引进专项(2022-12);江苏省自然科学基金资助项目(BK20211323);国家重点研发计划政府间国际科技创新合作重点专项(2022YFE0130900);江苏省“科技副总”项目(FZ20221535);扬州大学学科交叉高层次青年人才培育项目(2022-11);扬州大学高端人才支持计划项目(2022-12)。

摘　　要：凤仙花坏死斑病毒(impatiens necrotic spot virus,INSV)可引起多种农业和园艺观赏作物的严重病害,该病害在世界范围内普遍发生,建立快速有效的检测方法对INSV的防控有着重要意义。以INSV Nuclear Protein基因序列引物进行扩增,获得789 bp的目的基因,将其与原核表达载体pET28a连接,获得重组质粒pET28a-NPINSV,将其转化大肠埃希菌Rosetta菌株。经IPTG诱导后,SDS-PAGE电泳检测显示在分子量约28.7 ku处有目的蛋白质条带,与预期的INSV NP大小一致。利用镍柱亲和纯化INSV NP蛋白,并免疫健康新西兰大白兔,制备兔多克隆抗血清。通过Dot blot和ELISA方法对INSV NP多克隆抗血清进行检测,结果显示其能特异性识别INSV的NP蛋白。应用此抗血清在受INSV侵染的桃叶样品中能检测到NP蛋白的表达,而在健康植株叶片中未检测到。对制备的抗血清用纯水稀释至1:20000时,仍能特异地检测到目的蛋白质条带,说明该抗血清特异性较强,效价较高,可为INSV的快速检测提供有效的方法参考。Impatiens necrotic spot virus(INSV)is capable of causing serious diseases in a wide range of agricultural and horticultural ornamental crops,and the disease is widespread worldwide.The establishment of a rapid and effective detection method is important for the prevention and control of INSV.In this study,we used a pair of primers based on the sequence of INSV Nuclear Protein to clone the 789 bp gene.Then ligated the target gene to the prokaryotic expression vector pET28a to get the recombinant plasmid pET28a-NPINSV,which was transformed into Escherichia coli Rosetta strain,and then induced by IPTG.The SDS-PAGE showed that the molecular weight of the target gene was about 1000 mg.After IPTG induction,SDS-PAGE showed a target protein band at a molecular weight of about 28.7 kDa,which was consistent with the expected INSV NP.The INSV NP protein was purified using column affinity and immunised against healthy New Zealand Large White rabbits to prepare rabbit polyclonal antisera.In this study,the INSV NP polyclonal an-tiserum was tested by Dot blot and ELASA,and the results showed that it was able to specifically recognise the NP protein of INSV.It could be applied to the detection of different environmental samples:the expression of NP protein could be detected in peach leaf samples infested with INSV,while it failed to be detected in the leaves of healthy plants.When the prepared antiserum was diluted to 1:20000 with ultrapure water,INSV NP could still be specifically detected.It indicates that the INSV antiserum prepared by E.coli expressing NP is more specific and more potent,and it can provide an effective method reference for the rapid detection of INSV.

关 键 词：凤仙花坏死斑病毒 NP蛋白 原核表达 抗血清 

分 类 号：S436.45[农业科学—农业昆虫与害虫防治]