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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (19): 115-120.

所属专题： 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

齿兰环斑病毒CP基因的原核表达及其产物抗血清制备

罗金水 吴祖建 谢联辉   

  • 收稿日期:2011-02-09 修回日期:2011-03-09 出版日期:2011-08-05 发布日期:2011-08-05
  • 基金资助:

    福建省重大专项前期研究项目

Prokaryotic Expression and Antiserum Preparation of the CP gene of Odontoglossum Ringspot Virus

  • Received:2011-02-09 Revised:2011-03-09 Online:2011-08-05 Published:2011-08-05

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摘要：

本研究在于探讨侵染兰花的重要病毒之一齿兰环斑病毒（Odontoglossum ringspot virus，ORSV）的检测技术。从福建省漳州市采集感染齿兰环斑病毒的建兰病样，设计一对特异性引物扩增并克隆得到该病毒的外壳蛋白基因，该基因开放阅读框长477 bp，编码158 aa约合18.0 kDa的蛋白质，随后将目的基因插入pET-29a（+）中构建相应的原核表达载体进行诱导表达，目的蛋白经纯化后免疫家兔获得了特异性抗血清。Western blot检测结果表明，抗血清与诱导表达的ORSV外壳蛋白发生特异性反应。间接酶联免疫吸附检测结果表明，抗血清可检测病叶的最低稀释度达1:25600（v/v），最佳工作浓度为1:12800（v/v），病汁液灵敏度为0.195 mg/mL（w/v），而与TMV等11种同源或异源病毒均无明显的血清学交叉反应。

关键词: 蔬菜, 蔬菜, 日光温室, 施肥, 土壤养分, 研究

Abstract:

The aim was to study the technology which detected Odontoglossum ringspot virus (ORSV), which was one of the most important and worldwide viruses attacking orchids. Chinese Cymbidium plants which showed disease symptoms were collected from Zhangzhou, Fujian. One pair of specific primers was designed for amplification of the coat protein (CP) gene from the samples infected with ORSV. The open reading frame encoding CP of ORSV isolate obtained from Zhangzhou, Fujian is 477 bp, encoding a 18.0 kD protein with 158 aa. The expected CP gene was then inserted into the pET-29a (+) vector for the prokaryotic expression. And the aimed protein was purified and used to immune the rabbit for antiserum preparation. According to the result of ID-ELISA analysis, specific rabbit anti-ORSV serum was prepared with a high titre of 1:25600, a working concentration of 1:12800, and a sap sensitivity of 0.195 mg/mL. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of ORSV. There were no obvious cross reactions between the antiserum and 11 kinds of homologous or heterologous viruses such as Tobacco Mosaic Virus (TMV).

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