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中国农学通报 ›› 2014, Vol. 30 ›› Issue (15): 286-291.doi: 10.11924/j.issn.1000-6850.2013-1817

所属专题: 生物技术

• 生物技术科学 • 上一篇    下一篇

芽孢杆菌蛋白酶基因apr的克隆、 表达及序列分析

李曹龙 柴秀娟 杨晶 王爱英 祝建波   

  • 收稿日期:2013-07-03 修回日期:2013-08-01 出版日期:2014-05-25 发布日期:2014-05-25
  • 基金资助:
    校企合作项目横向课题 “微生物资源筛选” (0257-5001601)。

Cloning, Expression and Sequence Analysis of Protease Gene apr by Bacillus tequilensis

  • Received:2013-07-03 Revised:2013-08-01 Online:2014-05-25 Published:2014-05-25

摘要: 克隆从新疆吐鲁番地区土样分离获得产蛋白酶菌株Bacillus tequilensis C9蛋白酶基因apr,建立蛋白酶基因异源表达体系。采用PCR技术克隆获得目的基因,利用生物信息学软件进行序列分析,构建pET-28a原核表达重组质粒,转化BL21大肠杆菌,37℃下IPTG诱导表达融合蛋白,测定酶活。蛋白酶基因apr全长1098 bp,与Bacillus subtilis aprE (AB734697)有98%的同源性,编码含有299个氨基酸残基的成熟蛋白,是易溶、亲水性较强的蛋白,属于Peptidases_S8_S53 superfamily蛋白家族,融合蛋白分子量约为29 kDa,蛋白酶酶活为28.4 u/mL。试验为构建蛋白酶基因高效表达体系奠定理论基础。

关键词: 西瓜, 西瓜

Abstract: Cloning of protease gene apr from protease production strain Bacillus tequilensis C9 which was collected from the soil of turpan in Xinjiang Province, establish the expression system of protease gene. Protease gene was amplified by PCR and bioinformatics software for sequence analysis, construction of pET- 28a prokaryotic expression recombinant plasmid, and the recombinant protein was expressed in E. coli BL21 though IPTG in 37℃and enzyme activity was determined. The gene fragment of protease gene apr was 1098 bp, the nucleotides sequence homology was 98%, compared with Bacillus subtilis aprE (AB734697), encoded a mature protein containing 299 amino acids, which was easy to protein solution, strong hydrophilicity, the protein belonged to the peptidases_S8_S53 superfamily protein family, the molecular mass of fusion protein was 29 kDa, protease activity was 28.4 u/mL. The experiments can lay the theoretical foundation for establish the efficient expression system of protease gene.