›› 2012, Vol. 24 ›› Issue (6): 0-1049.

• 论文 •    

建兰花叶病毒TGB1和TGB2基因的原核表达

邓衍福,陈芝娟,程晓非,章鹏程,胡凤,施农农*   

  1. 杭州师范大学 生命与环境科学学院植物RNA信号传导研究中心,浙江 杭州 310036
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-11-25 发布日期:2012-11-25

Cloning and prokaryotic expression of Cymbidium mosaic virus TGB1 and TGB2 gene

DENGYanfu;CHEN Zhijuan;CHENG Xiaofei;ZHANG Pengcheng;HU Feng;SHI Nongnong*   

  1. Research Centor for Plant RNA Signaling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2012-11-25 Published:2012-11-25

摘要: 建兰花叶病毒(Cymbidium mosaic virus,CymMV)是严重危害兰科植物的主要病毒之一。本研究根据NCBI登录序列号(AB197937)的CymMV-TGB1,TGB2基因分别设计特异性引物,采用逆转录聚合酶链式反应(RTPCR)方法从感染CymMV蝴蝶兰病叶中扩增TGB1,TGB2基因,并将目的基因插入pGEX-4T3中,构建相应的原核表达载体。将表达载体转入大肠杆菌BL21,经IPTG诱导后成功表达出目的蛋白;SDS-PAGE及Western blot检测证实目的蛋白分别是融合GST的CymMV TGB1和TGB2蛋白。两个蛋白的表达为下一步抗体制备以深入开展CymMV TGB1和TGB2功能研究奠定了基础。

关键词: 蝴蝶兰, 建兰花叶病毒, 原核表达, Western blot

Abstract: Cymbidium mosaic virus (CymMV) is an important virus infecting Orchidaceous plants severely In this study, according to the genomic sequence of CymMV in GenBank (Accession No AB197937), the specific primers were designed for amplifying its TGB1 and TGB2 gene, respectively By reverse transcriptionpolymerase chain reaction (RTPCR), the TGB1 and TGB2 genes were amplified from CymMVinfected Phalaenopsis leaves Both fragments were then inserted into pGEX4T3 vector for prokaryotic expression The constructed expression vectors were transformed into E coli BL21 that was induced by IPTG for expressing the proteins SDSPAGE and Western blot analysis confirmed that the expressed proteins were GSTfused CymMV TGB1 and TGB2 proteins Expression of TGB1 and TGB2 will help to prepare the antibodies for their further functional analysis.

Key words: Phalaenopsis, CymMV, prokaryotic expression, Western blot