猕猴桃种传潜隐病毒外壳蛋白的原核表达及多克隆抗体制备  

Prokaryotic expression and polyclonal antibody preparation of the coat protein of actinidia seed-borne latent virus

作　　者：杨馥韩 余兆耀 尹世欣 龙友华[1] 王勇[1] 陈相儒 YANG Fuhan;YU Zhaoyao;YIN Shixin;LONG Youhua;WANG Yong;CHEN Xiangru(College of Agriculture,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学农学院,贵阳550025

出　　处：《植物保护》2023年第3期187-192,共6页Plant Protection

基　　金：贵州省科技计划(黔科合基础-ZK[2021]一般136);现代农业产业技术体系建设专项;贵州大学引进人才科研项目(贵大人基合字[2019]23号)。

摘　　要：猕猴桃种传潜隐病毒(actinidia seed borne latent virus, ASbLV),属于乙型线状病毒科Betaflexiviridae李属病毒属Prunevirus,是一种在我国猕猴桃上广泛发生的病毒。本研究通过RT-PCR方法克隆ASbLV的外壳蛋白基因,并连接到原核表达载体pET28a(+)上,转化大肠杆菌Rosetta (DE3),经IPTG诱导后可表达分子量约为28 kD的融合蛋白。经过Ni^(2+)-NTA树脂纯化融合蛋白,然后以其为抗原制备多克隆抗体。Western blot结果表明,多克隆抗体的效价为1∶4 000。该多克隆抗体只与ASbLV发生特异性反应,而不与猕猴桃病毒1、猕猴桃病毒A、苹果茎沟病毒、柑橘叶斑驳病毒、黄瓜花叶病毒和马铃薯X病毒发生反应,说明该多克隆抗体特异性良好。利用间接ELISA对36份猕猴桃田间样品进行了ASbLV检测,检测结果表明其中20份样品被ASbLV侵染,且间接ELISA检测结果与RT-PCR检测结果一致。本研究建立的间接ELISA方法能够有效地应用于猕猴桃田间样品中的ASbLV检测。Actinidia seed-borne latent virus(ASbLV),which belongs to the genus Prunevirus in the family Betaflexiviridae,is a common virus on kiwifruit in China.In this study,the coat protein(CP)gene of ASbLV was amplified from ASbLV-infected leaves of kiwifruit by RT-PCR,fused to the prokaryotic vector pET28a(+)and transformed into Escherichia coli Rosetta(DE3).After induced with IPTG,the fusion protein with a molecular weight of 28 kD was expressed.The fusion protein purified by Ni^(2+)-NTA resin was used for preparation of polyclonal antibody.Western blot result showed that the polyclonal antibody titer was 1∶4000.This polyclonal antibody reacted specifically with ASbLV,but did not react with the other six viruses infecting kiwifruit,including actinidia virus 1,actinidia virus A,apple stem grooving virus,citrus leaf blotch virus,cucumber mosaic virus and potato virus X.Indirect ELISA(ID-ELISA)result showed that 20 of the 36 samples were positive.The ID-ELISA result was consistent with those of RT-PCR.In this study,we successfully established an ID-ELISA method based on the specific antibody of ASbLV.This method can be efficiently applied to detecting ASbLV from field samples.

关 键 词：猕猴桃种传潜隐病毒 外壳蛋白 原核表达 多克隆抗体 

分 类 号：S436.634.1[农业科学—农业昆虫与害虫防治]