食品科学 ›› 2017, Vol. 38 ›› Issue (20): 1-5.doi: 10.7506/spkx1002-6630-201720001

• 生物工程 •    下一篇

红鳍东方鲀组织蛋白酶L表达载体的构建及表达结果验证

李妍钰,汤奇龙,张鲁嘉,谢静莉,王文利,刘源   

  1. (1.上海海洋大学食品学院,上海 201306;2.华东理工大学生物工程学院,上海 200237;3.上海交通大学农业与生物学院,上海 200240)
  • 出版日期:2017-10-25 发布日期:2017-09-29
  • 基金资助:
    国家自然科学基金青年科学基金项目(31622042;31371790;31271900); “十三五”国家重点研发计划重点专项(2016YFD040083)

Construction and Verification of Expression Vector for Takifugu rubripes Cathepsin L Gene

LI Yanyu, TANG Qilong, ZHANG Lujia, XIE Jingli, WANG Wenli, LIU Yuan   

  1. (1. College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China; 2. School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China; 3. College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China)
  • Online:2017-10-25 Published:2017-09-29

摘要: 红鳍东方鲀味道鲜美,已从其体内发掘了系列鲜味肽,但其形成机制尚未明确。组织蛋白酶L可通过降解肌原纤维蛋白及肌钙蛋白发挥着重要的滋味形成作用。为了探索其对红鳍东方鲀呈味肽的形成机制,本研究通过查找NCBI数据库得到一条全长为1?336?bp的红鳍东方鲀组织蛋白酶L的序列,从红鳍东方鲀肌肉中提取RNA并转录为cDNA作为模板克隆,选择pET-28a(+)作为表达载体、E. coli BL21作为重组工程菌进行克隆表达,成功得到大小为36?kD的重组组织蛋白酶L,并验证了其可在大肠杆菌表达系统中成功克隆表达。本研究为进一步研究红鳍东方鲀组织蛋白酶L酶学特性及其对滋味形成提供了理论支持。

关键词: 红鳍东方鲀, 组织蛋白酶L, 呈味肽, 大肠杆菌表达系统

Abstract: Takifugu rubripes has a delicious taste. To date, a series of umami peptides have been separated and identified from T. rubripes, but their formation mechanism is not yet clear. Cathepsin L degrades myofibrillar protein and troponin during muscle degradation and thus plays an important role in the flavor formation. This study aimed to elucidate the possible mechanism of flavor peptide formation. We obtained a 1 336 bp sequence of cathepsin L gene from the NCBI database, and extracted RNA from the muscle of T. rubripes, and then reverse-transcribed it into cDNA. PET-28a (+) as the expression vector and E. coli BL21 as engineering bacteria was used to clone and express the cathepsin L gene. Recombinant cathepsin L was obtained with a molecular mass of 36 kD, and the E. coli expression system was also successfully validated. This study can provide useful information for further studies on enzymatic characteristics and of cathepsin L and its role in taste formation in T. rubripes.

Key words: Takifugu rubripes, cathepsin L, flavor peptide, E. coli expression system

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