https://www.ahs.ac.cn/images/0513-353X/images/top-banner1.jpg|#|苹果
https://www.ahs.ac.cn/images/0513-353X/images/top-banner2.jpg|#|甘蓝
https://www.ahs.ac.cn/images/0513-353X/images/top-banner3.jpg|#|菊花
https://www.ahs.ac.cn/images/0513-353X/images/top-banner4.jpg|#|灵芝
https://www.ahs.ac.cn/images/0513-353X/images/top-banner5.jpg|#|桃
https://www.ahs.ac.cn/images/0513-353X/images/top-banner6.jpg|#|黄瓜
https://www.ahs.ac.cn/images/0513-353X/images/top-banner7.jpg|#|蝴蝶兰
https://www.ahs.ac.cn/images/0513-353X/images/top-banner8.jpg|#|樱桃
https://www.ahs.ac.cn/images/0513-353X/images/top-banner9.jpg|#|观赏荷花
https://www.ahs.ac.cn/images/0513-353X/images/top-banner10.jpg|#|菊花
https://www.ahs.ac.cn/images/0513-353X/images/top-banner11.jpg|#|月季
https://www.ahs.ac.cn/images/0513-353X/images/top-banner12.jpg|#|菊花

园艺学报 ›› 2016, Vol. 43 ›› Issue (11): 2233-2242.doi: 10.16420/j.issn.0513-353x.2016-0208

• 研究报告 • 上一篇    下一篇

葡萄病毒B 外壳蛋白原核表达及抗血清制备

任 芳,董雅凤*,张尊平,范旭东,胡国君,李正男,周 俊   

  1. 中国农业科学院果树研究所,国家落叶果树脱毒中心,辽宁兴城 125100
  • 出版日期:2016-11-25 发布日期:2016-11-25
  • 基金资助:

    国家现代农业产业技术体系建设专项资金项目(CARS-30-bc-3)

Prokaryotic Expression of Grapevine virus B Coat Protein and Antiserum#br# Preparation

REN Fang,DONG Ya-feng*,ZHANG Zun-ping,FAN Xu-dong,HU Guo-jun,LI Zheng-nan,and ZHOU#br# Jun   

  1. National Center for Eliminating Viruses from Deciduous Fruit Trees,Research Institute of Pomology,Chinese Academy of
    Agriculture Sciences,Xingcheng 125100,China
  • Online:2016-11-25 Published:2016-11-25

摘要:

根据已报道的葡萄病毒B(Grapevine virus B,GVB)核苷酸序列设计引物,采用RT-PCR
方法从葡萄中扩增获得GVB 外壳蛋白基因(cp),大小为594 bp。通过序列测定和分析,选取优势分离
物GVB-JFL cp 基因克隆到原核表达载体pET-28a,转化大肠杆菌BL21(DE3),IPTG 诱导表达并纯化融
合蛋白pET-GVB CP。SDS-PAGE 电泳结果显示,融合蛋白获得过量表达,分子量约26 kD。以纯化蛋白
为抗原免疫新西兰大耳白兔制备抗血清。采用间接ELISA 和dot-ELISA 对抗血清特异性进行检测,结果
表明,抗血清可与感染GVB 的西方烟和葡萄样品发生阳性反应,与健康植株及感染同属另一种病毒葡萄
病毒A(Grapevine virus A,GVA)的样品不反应,阳性样品检测效价达1︰4 000,16 个田间样品摩擦接
种至烟草后有9 个样品ELISA 检测为阳性,表明制备的抗血清可用于GVB 的特异性检测。

关键词: 葡萄病毒B, 外壳蛋白, 原核表达, 抗血清

Abstract:

In this study,the coat protein gene(cp,594 bp)of Grapevine virus B(GVB)was amplified
from grapevine by RT-PCR using primers designed according to previous reported GVB sequences. After
sequence determination and analysis,the cp gene of preponderant isolate GVB-JFL was inserted into
expression vector pET-28a and constructed recombinant plasmid pET-GVB cp,pET-GVB cp was then
transformed into E. coli BL21(DE3)and induced by IPTG. SDS-PAGE analysis showed that the coat
protein(approximately 26 kD)was induced at a high level. The purified coat protein was used as antigen
for raising antiserum in rabbits,and the specificity of the antiserum was tested by ELISA and dot-ELISA.
The results showed that the antiserum could be successfully used to detect GVB in the infected Nicotiana
occidentalis and grapevine,but had no reaction with the healthy plants and Grapevine virus A(GVA,
another member of vitiruses)infected plants,the antibody titer for positive samples could be up to 1︰4 000,
and 9 of the 16 field samples detected after inoculated in tobacco were positive,these results indicated that the antiserum obtained in the study could be used for efficient and specific detection of GVB.

Key words: Grapevine virus B, coat protein, prokaryotic expression, antiserum

中图分类号: