食品与发酵工业 >

2021 , Vol. 47 >Issue 16: 15 - 21

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.026534

研究报告

牡蛎过敏原肌浆钙结合蛋白的分离纯化和鉴定

  • 程青丽 ,
  • 李国明 ,
  • 赵晓涵 ,
  • 冯晓文 ,
  • 卢知浩 ,
  • 谷瑞增 ,
  • 鲁军 ,
  • 刘文颖
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  • (中国食品发酵工业研究院,北京市蛋白功能肽工程技术研究中心,北京,100015)
程青丽(硕士研究生)和李国明(高级工程师)为共同第一作者(刘文颖高级工程师为通讯作者,E-mail:wenyingliu888@126.com)

收稿日期: 2020-12-24

  修回日期: 2021-02-07

  网络出版日期: 2021-09-10

基金资助

中国食品发酵工业研究院强院工程培育专项(院强院20-功能肽505);国家自然科学基金项目(31671963)

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Isolation, purification and identification of sarcoplasmic calcium-binding protein from oyster (Crassostrea gigas) allergen

  • CHENG Qingli ,
  • LI Guoming ,
  • ZHAO Xiaohan ,
  • FENG Xiaowen ,
  • LU Zhihao ,
  • GU Ruizeng ,
  • LU Jun ,
  • LIU Wenying
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  • (Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries, Beijing 100015, China)

Received date: 2020-12-24

  Revised date: 2021-02-07

  Online published: 2021-09-10

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摘要

利用质谱鉴定从太平洋牡蛎肌浆里提取的单一蛋白,通过免疫印迹分析诱导表达的重组蛋白和之前已鉴定的天然蛋白的分子质量一致性,利用圆二色光谱分析两蛋白在二级结构上的一致性,鉴定牡蛎肌浆钙结合蛋白(sarcoplasmic calcium-binding protein, SCP)糖蛋白类型并测定其多糖含量。结果表明,从牡蛎粗提物中纯化20 kDa蛋白,经LC-MS/MS证实为牡蛎肌浆钙结合蛋白SCP;电泳得到天然SCP和重组SCP分别为20 kDa和22 kDa的条带;测定二者α螺旋和β折叠含量分别为13.5%、18.9%以及14.5%、18.2%;SCP蛋白含O-糖苷键型糖多糖,含量为7.89%。重组SCP与天然SCP除分子质量稍有差异外,二者空间结构近乎相同,是太平洋牡蛎中新型潜在致敏原。该研究为今后用重组SCP替代天然SCP进行致敏性和致敏机制研究奠定了基础。

关键词： 太平洋牡蛎; 肌浆钙结合蛋白; 重组表达; 分离纯化; 糖蛋白

本文引用格式

程青丽 , 李国明 , 赵晓涵 , 冯晓文 , 卢知浩 , 谷瑞增 , 鲁军 , 刘文颖 . 牡蛎过敏原肌浆钙结合蛋白的分离纯化和鉴定[J]. 食品与发酵工业, 2021 , 47(16) : 15 -21 . DOI: 10.13995/j.cnki.11-1802/ts.026534

Abstract

The aim of this study was to identify the single protein extracted from Crassostrea gigas muscle sarcoplasm by mass spectrometry and analyze the molecular weight consistency of the induced recombinant protein and the natural protein identified by western blot(WB). The consistency of the two protein on the two level structure was analyzed by cireular dichroism(CD) spectrum, and the sarcoplasmic calcium-binding protein (SCP) glycoprotein type of oyster was identified. The content of polysaccharide was also determined. The results revealed that 20 kDa protein was purified from oyster crude extract and confirmed as SCP by LC-MS / MS,the bands of natural SCP and recombinant SCP were 20 kDa and 22 kDa respectively,which including 13.5%, 18.9% and 14.5%, 18.2% Helix and Beta-Turn , respectively. SCP protein contained 7.89% O-glycosidic glycopolysaccharide. The molecular weight of recombinant SCP was slightly different from that of natural SCP, but their spatial structures were almost the same. It is a new potential allergen in Crassostrea gigas, which lays an important foundation for the study of allergenicity and sensitization mechanism of recombinant SCP instead of natural SCP in the future.

Key words： Crassostrea gigas; sarcoplasmic calcium-binding protein; recombinant expression; isolation and purification; glycoprotein

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