湖北农业科学 ›› 2021, Vol. 60 ›› Issue (10): 142-147.doi: 10.14088/j.cnki.issn0439-8114.2021.10.029

• 生物工程 • 上一篇    下一篇

副猪嗜血杆菌sodA基因的表达及其理化性质

胡基雄1, 王席2, 李国攀1, 荣俊1   

  1. 1. 长江大学生命科学学院,湖北 荆州 434025;
    2. 荆州长新生物有限公司,湖北 荆州 434025
  • 收稿日期:2021-02-09 出版日期:2021-05-25 发布日期:2021-05-28
  • 通讯作者: 荣俊(1959-),男,湖北石首人,教授,博士,主要从事基因工程亚单位疫苗的研究工作,(电子信箱)200459@yangtzeu.edu.cn。
  • 作者简介:胡基雄(1995-),男,湖北武汉人,在读硕士研究生,研究方向为基因工程亚单位疫苗,(电话)13697198511(电子信箱)1320046008@qq.com。
  • 基金资助:
    高校教育综合奖补项目(2018-702090250401)

Expression of the Haemophilus parasuis sodA gene and the study on its physicochemical properties

HU Ji-xiong1, WANG Xi2, LI Guo-pan1, RONG Jun1   

  1. 1. College of Life Science,Yangtze University,Jingzhou 434025,Hubei,China;
    2. Jingzhou Changxin Biotechnology Co. , Ltd. ,Jingzhou 434025,Hubei,China
  • Received:2021-02-09 Online:2021-05-25 Published:2021-05-28

摘要: 为探究副猪嗜血杆菌(Haemophilus parasuissodA基因编码的超氧化物歧化酶(Superoxide Dismutase,SOD)的活性及理化性质,设计特异性引物从HPS中扩增得到目的基因sodA,利用原核表达系统对HPS SOD蛋白进行表达,通过硫酸铵分级沉淀、Sephacryl S-300凝胶过滤层析以及DEAE Sepharose Fast Flow阴离子交换层析等方法对表达蛋白进行了分离纯化,利用邻苯三酚自氧化法对纯化产物进行SOD酶活性检测,通过HPSEC法、火焰原子吸收分光光度法对其理化性质进行了初步探究。结果表明,表达的重组HPS SOD蛋白质为可溶性蛋白质,单亚基分子质量为26 ku,纯化后目的蛋白质纯度为95%,纯化后的重组HPS SOD蛋白质比活性为215.9 U/mg,经HPSEC法检测分析,重组HPS SOD呈多聚体,通过火焰原子吸收分光光度法检测纯化的重组HPS SOD中锰元素含量为0.71 mg/L。

关键词: 副猪嗜血杆菌(Haemophilus parasuis), 超氧化物歧化酶, 邻苯三酚自氧化法

Abstract: In order to study the activity and physiological function of the SOD protein encoded by the sodA gene of Haemophilus parasuis, the sodA gene was amplified from HPS by the specific primers and expressed in Escherichia coli prokaryotic expression system. The expressed protein was purified by the ammonium sulfate fractionation, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose Fast Flow anion exchange chromatography. The activity of the purified protein was determined by the pyrogallol autoxidation method and the physicochemical properties were studied by HPSEC and AAS. The results showed that the recombinant HPS SOD protein was a soluble protein with a size of 26 ku. The purity of the target protein was 95% after purification, and its activity was measured to be 215.9 U/mg by pyrogallol autoxidation assay. HPSEC analysis indicated that the recombinant HPS SOD was a multimer, and the manganese content in the purified HPS SOD was detected to be 0.71 mg/L by Flame Atomic Absorption Spectrophotometry.

Key words: Haemophilus parasuis, superoxide dismutase, pyrogallol autoxidation method

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