中国农业科学 ›› 2006, Vol. 39 ›› Issue (12): 2590-2596 .

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

水牛朊蛋白成熟片段基因的克隆与原核表达

袁志栋,王志亮,刘雨田,吴晓东,赵永刚,刘海生   

  1. 湖南科技大学生命科学学院
  • 收稿日期:2005-08-08 修回日期:2005-12-08 出版日期:2006-12-10 发布日期:2006-12-10
  • 通讯作者: 王志亮

Cloning, Prokaryotic Expression of the Buffalo Mature PrP Gene

  1. 湖南科技大学生命科学学院
  • Received:2005-08-08 Revised:2005-12-08 Online:2006-12-10 Published:2006-12-10

摘要: 【目的】克隆、分析水牛朊蛋白(prion protein,PrP)成熟片段基因,并获取纯化的表达产物。【方法】将应用PCR技术扩增的水牛成熟PrP的核酸序列克隆到表达载体pET-32a,并分析其序列;把阳性克隆质粒pET-32a-BPrP转化表达菌BL21(DE3),鉴定纯化的表达产物。【结果】水牛朊蛋白成熟片段核酸序列及其推导的氨基酸序列与已知其他牛科动物的成熟PrP核酸、氨基酸序列间的相似性分别在97.1%和97.2%以上,并且发现水牛成熟PrP有5个重复序列,编码的氨基酸序列其中有2个九肽和3个八肽重复序列。具有较高的表达水平的PrP融合蛋白被纯化后,用Western印迹实验证明该蛋白具有PrP抗原活性。【结论】首次报道了克隆、分析水牛朊蛋白成熟片段基因,发现水牛成熟PrP有5个重复序列,并获得了具PrP抗原活性的水牛成熟PrP融合蛋白。

关键词: 水牛成熟PrP, 克隆, 序列分析, 原核表达

Abstract: There are no reports on buffalo PrP till now. For the sake, with the designed primers, the buffalo mature PrP gene was amplified by PCR method. And the recombinant plasmid was constructed,which expresses buffalo mature PrP fusion protein. Then the vector was sequenced. The PrP sequences of the family Bovidae were analyzed and it shows the buffalo mature PrP gene contain five copies of a short, G-C-rich element which encodes the octapeptide,and there are two nonapeptides and three octapeptides.After transformed the positive recombinant plasmids, which designated as pET-32a- BPrP , to BL21 (DE3), and the product was achieved a high expression level. After ultrasonicated, the sediment was isolated and purified. And the product was confirmed by Western Blot.