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园艺学报 ›› 2004, Vol. 31 ›› Issue (4): 517-519.

• 研究报告 • 上一篇    下一篇

马铃薯S病毒外壳蛋白基因的克隆及其在大肠杆菌中的表达

李广存 杨 煜 王秀丽 杨元军 毕玉平
  

  1. ( 山东省农业科学院生物中心,济南250100; 山东省农业科学院蔬菜研究所,济南250100)
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2004-08-25 发布日期:2004-08-25

Cloning of Potato Virus S Coat Protein Gene and Its Expression in E.coliJM 109

  1. ( Bio—tech.Center,Shandong Academy of Agricultural Sciences,Ji'nan 250100,China; Vegetable Institute,ShandongAcademy ofAgricultural Sciences,Ji'nan 250100,China)
  • Received:1900-01-01 Revised:1900-01-01 Online:2004-08-25 Published:2004-08-25

摘要: 以田间采集的马铃薯病叶中提取的马铃薯病毒s(PVS)总RNA为模板,通过RT—PCR获取
长度为890 bp的P 一cp的cDNA,克隆至pGEM—T载体上。酶切回收该基因片段,并构建了该基因的原核
表达质粒pBV—pvs。SDS—PAGE凝胶电泳和Western印迹分析表明:P 一印基因在大肠杆菌JM109中可特异
地高效表达分子量约33kD的蛋白,且表达蛋白具有良好的抗原活性。利用该表达产物免疫动物家兔,获
得的抗血清可用于大田马铃薯s病毒的快速检测。

关键词: 马铃薯, 马铃薯s病毒, 外壳蛋白基因, RT—PCR, 序列分析, Western印迹, 抗血清制备

Abstract: Total RNA of potato virus S (PVS)was isolated from diseased potato leaves collected in
field,The cDNA encoding PVS coat protein(PVS-cp)was amplified by RT-PCR and cloned into the plasmid
pGEM—T vector. The expression vector of P 一cp gene was constructed through inserting the cDNA fragment
into the expression plasmid pBV220. The results of SDS-PAGE and W estern blot analysis showed that
the P -cp gene highly expressed in E.coli JM109 and the molecular size of the expression product was about
33 kD. The product was used as antigen to immunize rabbits and the specific anti-serum can be used to detect
PVS rapidly.

Key words: Potato, Potato virus S, Coat protein gene, RT-PCR, Sequence analysis, Western blot, Anti-serum preparation