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

doi:10.3969/j.issn.1001-7216.2010.01.05

中国水稻科学

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

欧阳元龙1,2,3;吴建祥2,*;熊如意3;周益军3;周雪平2

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 出版日期:2010-01-10 发布日期:2010-01-10

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

OUYANG Yuan-long1,2,3, WU Jian-xiang2,*, XIONG Ru-yi3, ZHOU Yi-jun3, ZHOU Xue-ping2

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:1900-01-01 Revised:1900-01-01 Online:2010-01-10 Published:2010-01-10

摘要/Abstract

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

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

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 .

OUYANG Yuan-long,WU Jian-xiang, XIONG Ru-yi,ZHOU Yi-jun,ZHOU Xue-ping. Prokaryotic Expression of Coat Protein Gene S10 of Rice BlackStreaked Dwarf Virus, and Preparation and Application of Its Polyclonal Antibody [J]. Chinese Journal of Rice Science 2010,24(1): 25-30 ., DOI: 10.3969/j.issn.1001-7216.2010.01.05 .

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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

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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