建兰花叶病毒TGB1和TGB2基因的原核表达

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

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。

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 TGBI and TGB2 gene, respectively. By reverse transcription-polymerase chain reac- tion （RT-PCR）, the TGB1 and TGB2 genes were amplified from CymMV-infected Phalaenopsis leaves. Both frag- ments 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 a- nalysis 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.