<FONT face="Verdana">水稻黑条矮缩病病毒外壳蛋白基因S10的原核表达、多克隆抗体制备及应用</FONT>

欧阳元龙,吴建祥,熊如意,周益军,周雪平

doi:10.3969/j.issn.1001-7216.2010.01.05

中国水稻科学 >

2010 , Vol. 24 >Issue 1: 25 - 30

DOI: https://doi.org/10.3969/j.issn.1001-7216.2010.01.05

研究报告

水稻黑条矮缩病病毒外壳蛋白基因S10的原核表达、多克隆抗体制备及应用

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1南京师范大学生命科学学院，江苏南京 210046； 2浙江大学农业与生物技术学院生物技术研究所，浙江杭州 310029； 3江苏省农业科学院植物保护研究所，江苏南京 210014； *通讯联系人， E-mail:wujx@zju.edu.cn

收稿日期: 1900-01-01

修回日期: 1900-01-01

网络出版日期: 2010-01-10

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Prokaryotic Expression of Coat Protein Gene S10 of Rice BlackStreaked Dwarf Virus, and Preparation and Application of Its Polyclonal Antibody

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1College of Life Science, Nanjing Normal University, Nanjing 210046, China； 2Institute of Biotechnology， College of Agriculture & Biotechnology, Zhejiang University， Hangzhou 310029, China； 3Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; *Corresponding author, E-mail: wujx@zju.edu.cn

Received date: 1900-01-01

Revised date: 1900-01-01

Online published: 2010-01-10

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摘要

用RT-PCR方法从感染水稻黑条矮缩病毒（rice blackstreaked dwarf virus, RBSDV）水稻中克隆该病毒的外壳蛋白基因S10,然后将此外壳蛋白基因再亚克隆到原核表达载体PET32a中构建成重组原核表达载体pET32aCP。将重组表达载体转化大肠杆菌BL21（DE3）,经IPTG诱导, Ni+ NTA亲和柱纯化获得分子量约为76 kD含硫氧还蛋白的融合蛋白。以纯化的重组蛋白为抗原免疫兔子制备RBSDV外壳蛋白的多克隆抗体，并用制备的多克隆抗体建立了可靠、灵敏、特异的检测RBSDV的免疫捕获RTPCR及Dotblot ELISA方法，为该水稻病毒病的诊断提供技术支持。

关键词： 水稻黑条矮缩病病毒; 原核表达; 多克隆抗体; 免疫捕获反转录聚合酶链式反应; 斑点酶联免疫吸附测定

本文引用格式

欧阳元龙,吴建祥,熊如意,周益军,周雪平 . 水稻黑条矮缩病病毒外壳蛋白基因S10的原核表达、多克隆抗体制备及应用[J]. 中国水稻科学, 2010 , 24(1) : 25 -30 . DOI: 10.3969/j.issn.1001-7216.2010.01.05

Abstract

The full length cDNA of rice blackstreaked dwarf virus (RBSDV) segment 10 (S10) which encoded coat protein was cloned from the virus infected rice samples by RTPCR，and subcloned into a prokaryotic expression vector pET32a. The recombinant prokaryotic expression vector (pET32aCP) was used to transform Escherichia coli BL21 (DE3). A 76 kD TrxA fusion protein was obtained with induction of IPTG and purification of Ni+ NTA affinity column. The purified recombinant protein was used to immunize rabbits for production of polyclonal antibodies against the coat protein of RBSDV. Using polyclonal antibodies, immunocapture RTPCR and Dotblot ELISA were established for reliable, sensitive and specific detection of RBSDV. The two detection methods utilized polyclonal antibodies provide technical support for the diagnosis of RBSDV disease.

Key words： rice blackstreaked dwarf virus; prokaryotic expression; polyclonal antibody; immunocapture RTPCR; dotblot ELISA

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