食品科学 ›› 2010, Vol. 31 ›› Issue (19): 309-312.doi: 10.7506/spkx1002-6630-201019067

• 生物工程 • 上一篇    下一篇

深海古菌Thermococcus siculi HJ21高温普鲁兰酶基因的克隆及表达

王淑军1,吕明生1,李华钟2,徐金利1 , 2,焦豫良1,房耀维1,刘 姝1   

  1. 1.淮海工学院食品工程学院
    2.江南大学 教育部工业生物技术重点实验室
  • 收稿日期:2010-06-09 修回日期:2010-09-14 出版日期:2010-10-15 发布日期:2010-12-29
  • 通讯作者: 王淑军 E-mail:shujunwang86@hotmail.com
  • 基金资助:

    国家自然科学基金项目(40746030);江苏省高校自然科学研究重大项目(09KJA170001)

Cloning and Expression of a New Thermostable Pullulanase Gene from a Deep-sea Archaeaon Strain Thermococcus siculi HJ21

WANG Shu-jun1,LU Ming-sheng1,LI Hua-zhong2,XU Jin-li1,2,JIAO Yu-liang1,FANG Yao-wei1,LIU Shu1   

  1. 1. College of Food Science and Technology, Huaihai Institute of Technology, Lianyungang 222005, China;
    2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
  • Received:2010-06-09 Revised:2010-09-14 Online:2010-10-15 Published:2010-12-29
  • Contact: WANG Shu-jun1 E-mail:shujunwang86@hotmail.com

摘要:

根据NCBI 上公布的普鲁兰酶基因的保守序列设计简并引物,以Thermococcus siculi HJ21 基因组DNA 为模板进行PCR,得到T. siculi HJ21 普鲁兰酶的基因,测序后通过Blast(NCBI)数据库比对和分析表明扩增基因有一个4056bp、编码1351 个氨基酸的开放阅读框,为一新的普鲁兰酶基因。将该基因插入表达载体pET28a,并转化Escherichia coli BL21(DE3),经IPTG 诱导,测定普鲁兰酶比活力。重组转化子的细胞破碎液有高温普鲁兰酶活性,SDS-PAGE 电泳结果显示出分子质量约为150kD 的特异性条带。

关键词: Thermococcus siculi HJ21, 普鲁兰酶, 基因, PCR

Abstract:

The gene of pullulanase was amplified from Thermococcus siculi HJ21 with degenerate primes designed based on the NCBI published conserved sequence information. The DNA sequencing and BLAST (NCBI) analysis showed that this DNA sequence was a new pullulanase gene with an open reading frame (ORF) of 4056 bp in length encoding 1351 amino acids.The gene was cloned into the expression vector, pET28a, producing a hybrid plasmid pET28a-pull. Subsequently, pET28a-pull was introduced into Escherichia coli BL21(DE3). The lysate of the transformant cells showed thermostable pullulanase activity. The SDS-PAGE analysis showed a band with apparent molecular weight of 150 kD.

Key words: Thermococcus siculi HJ21, pullulanase, gene, PCR

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