食品科学 ›› 2019, Vol. 40 ›› Issue (20): 114-121.doi: 10.7506/spkx1002-6630-20190308-104

• 生物工程 • 上一篇    下一篇

融合型原肌球蛋白MBP-CTB-TM构建及其口服致敏性评价

傅玲琳,黄健健,谢梦华,王翀,王彦波   

  1. (浙江工商大学食品与生物工程学院,浙江食品质量安全工程研究院,浙江 杭州 310018)
  • 出版日期:2019-10-25 发布日期:2019-10-25
  • 基金资助:
    国家自然科学基金面上项目(31871735)

FU Linglin, HUANG Jianjian, XIE Menghua, WANG Chong, WANG Yanbo

FU Linglin, HUANG Jianjian, XIE Menghua, WANG Chong, WANG Yanbo   

  1. (Zhejiang Engineering Institute of Food Quality and Safety, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China)
  • Online:2019-10-25 Published:2019-10-25

摘要: 将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP-CTB-EGFP穿透细胞膜的能力,从而开发新型黏膜佐剂。进一步地,利用原肌球蛋白(tropomyosin,TM)重组融合蛋白MBP-CTB-TM,将其应用于Balb/c小鼠致敏实验,探究融合蛋白的致敏性及其对小鼠食物过敏相关免疫反应的影响,以达到降低过敏原剂量提高建模效率的效果。结果表明:MBP-CTB-EGFP具有作为黏膜佐剂的能力,从而促进外源蛋白进入HEK293T细胞内,且与转染方式相比,携带外源蛋白进入细胞的效率更高。另一方面,利用融合蛋白MBP-CTB-TM免疫小鼠,TM特异性IgE的OD450 nm达到0.4,而天然TM致敏组仅为0.05,此外融合蛋白致敏还导致高水平的TM特异性IgG1、IgG2a产生。本研究表明,融合蛋白MBP-CTB-TM致敏效果更好,有可能达到降低过敏原用量的效果,为后续食物过敏研究提供了有力工具。

关键词: 霍乱毒素B亚基, 原肌球蛋白, 融合蛋白, 食物过敏, 小鼠模型

Abstract: In the present study, maltose-binding protein/cholera toxin B subunit/enhanced green fluorescent protein (MBP-CTB-EGFP) complex was constructed by the recombination of MBP, CTB and EGFP, expressed by E. coli and purified. To assess the viability of the recombinant protein as a novel mucosal adjuvant, a cell model was used to explore the ability of MBP-CTB-EGFP to penetrate the cell membrane. Further, the shrimp allergen tropomyosin (TM) was used to produce the recombinant fusion protein MBP-CTB-TM, and we investigated whether it could reduce allergen usage and improve modeling efficiency in food allergy studies. Therefore, MBP-CTB-TM was applied to sensitize Balb/c mice to investigate the allergenicity of the fusion protein, and to evaluate its influence on the food allergy-related immune reactions. The results showed that MBP-CTB-EGFP was able to act as a mucosal adjuvant in promoting the transportation of foreign proteins into cells, and the efficiency was higher than that of transfection. Furthermore, sensitization of mice with the fusion protein MBP-CTB-TM resulted in an OD450 nm of 0.4 for TM specific IgE in ELISA, while the OD450 nm for natural TM sensitization group was only 0.05. In addition, fusion protein sensitization also promoted TM specific IgG1 and IgG2a production. This study shows that the fusion protein MBP-CTB-TM has a better sensitization effect and thus may reduce the requirement for allergens, therefore providing a powerful tool for further allergy studies.

Key words: cholera toxin B subunit, tropomyosin, fusion protein, food allergy, mouse model

中图分类号: