苹果坏死花叶病毒CP基因原核表达及其抗血清制备  

Prokaryotic Expression and Preparation of Antiserum of Coat Protein Gene of Apple Necrotic Mosaic Virus

作　　者：邢飞[1] 王红清 李世访[1] XING Fei;WANG Hongqing;LI Shifang(Institute of Plant Protection,Chinese Academy of Agricultural Sciences/State Key Laboratory for Biology of Plant Diseases and Insect Pests,Beijing 100193,China;College of Horticulture,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100193 [2]中国农业大学园艺学院,北京100193

出　　处：《西南大学学报（自然科学版）》2024年第4期63-69,共7页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金面上项目(31872922).

摘　　要：苹果坏死花叶病毒(Apple necrotic mosaic virus,ApNMV)是近年新发现的病原物,并且是与我国苹果花叶病症状高度相关的重要病毒,进行ApNMV外壳蛋白(coat protein,CP)基因原核表达、制备多克隆抗血清,以建立快速、灵敏、准确的ApNMV常规检测方法尤为必要.通过设计特异性引物,以RT-PCR方法成功获得ApNMV CP基因序列,插入到原核表达载体pET-28a(+)中构建重组质粒,转化至大肠杆菌BL21(DE3),利用IPTG进行重组蛋白(含His-tag)诱导表达.SDS-PAGE和Western blot分析结果表明,CP基因在大肠杆菌中获得了高效表达,用Ni Sepharose 6 Fast Flow进行蛋白纯化,回收共得到重组蛋白2.4 mg.在屏障环境下免疫2只SPF级新西兰兔,制备出多克隆抗血清,稀释400倍后仍能与ApNMV阳性苹果叶片样品发生免疫反应.由此证明,本研究建立的ApNMV间接ELISA检测方法具有较好的灵敏性,检测效率高,能够用于田间大量样品ApNMV的诊断.In recent years,apple necrotic mosaic virus(ApNMV),a novel ilarvirus,was identified as the main causal agent of apple mosaic disease in China.The objective of this study is to conduct prokaryotic expression and preparation of antiserum of coat protein gene of apple necrotic mosaic virus to establish a rapid,sensitive and accurate diagnostic method for ApNMV.In the present study,ApNMV coat protein(CP)gene sequence was successfully cloned by RT-PCR using specific primers and inserted into prokaryotic expression vector pET-28a(+)to construct recombinant plasmid.And then the recombinant plasmid was transformed into E.coli BL21(DE3)strains.The target recombinant protein with His-tag was induced by IPTG.SDS-PAGE and Western blot analysis showed that CP gene was highly expressed in E.coli.The expected fusion protein with His-tag was purified using Ni Sepharose 6 Fast Flow.Total 2.4 mg recombinant protein was obtained,and used to immunize two specific pathogen-free New Zealand rabbits to produce polyclonal antibody under the barrier environment.After 400 times dilution of the antiserum,the immune reaction could be still efficient to detect ApNMV in apple leaf samples by enzyme-linked immunosorbent assay(ELISA).Taken together,a fast,simple,sensitive and specific indirect ELISA method was established for the detection of ApNMV.

关 键 词：苹果坏死花叶病毒 外壳蛋白基因 原核表达 抗血清 酶联免疫吸附分析方法 

分 类 号：S436.611.1[农业科学—农业昆虫与害虫防治]