热带病与寄生虫学

热带病与寄生虫学 ›› 2014, Vol. 12 ›› Issue (3): 125-128.doi: 10.3969/j.issn.1672-2302.2014.03.001

• 论著 •    下一篇

粉尘螨变应原ProDer f 1 编码基因重组pET28a-YARA-Ihc 体系的构建及表达

刘志明,姜玉新   

  1. ：210048 南京市，东南大学医学院附属南京江北人民医院（刘志明），皖南医学院寄生虫学教研室（刘志明、姜玉新）
  • 出版日期:2014-09-10 发布日期:2014-10-17
  • 基金资助:

    国家自然科学基金资助项目（81172790）

Construction and expression of the recombinant plasmid pET28a- YARA- Ihc- ProDer f 1 of Derma⁃tophagoides farina

Liu Zhiming1,2, Jiang Yuxin2   

  1. 1. Department of clinical laboratory, Affiliated Nanjing Jiangbei Hospital of Medical College, Southeast University Medical College, Nanjing 210048, China. 2. Department of Medical Parasitology, Wannan Medical College.
  • Online:2014-09-10 Published:2014-10-17

摘要： 目的 构建粉尘螨变应原ProDer f 1 原核表达载体pET28a-YARA-IhC-ProDer f 1，为评价其免疫治疗效果奠定基础。方法 用分子克隆技术构建出表达载体pET28a-YARA-IhC-ProDer f 1，在E.coli BL21(DE3)中表达融合蛋白YARA-IhC-ProDer f 1，并进行Ni2+-NTA 树脂柱亲和层析以纯化蛋白。结果 经测序证实成功构建了表达载体pET28a-YARA-IhC-ProDer f 1，YARA-IhC-ProDer f 1融合蛋白在E.coli BL21(DE3)中得到表达，纯化后的蛋白浓度为278 μg/ml。SDS-PAGE 和Western blot 分析表明纯化蛋白为目的蛋白YARA-IhC-ProDer f 1。结论 已成功制备出pET28a-YARA-IhC-ProD⁃er f 1 原核表达载体，融合蛋白得到表达和纯化。

关键词: 细胞穿透肽, 粉尘螨, 恒定链, 变应原

Abstract:

Objective To construct the prokaryotic expression vector pET28a-YARA-Ihc-ProDer f 1 and provide the foundation for exploring the fusion protein effect as vaccine for specific immunotherapy. Methods Two oligonucleotides encoding YARA were synthesized and annealed to generate YARA- encoding DNA. The fused genes, YARA- Ihc- ProDer f 1 was constructed and inserted into the prokaryotic expression vector pET28a(+). The fusion protein YARA-Ihc-ProDer f 1 induced with IPTG in E.coli BL21(DE3) was purified with Ni2 +-resin affinity chromatography and confirmed with SDS-PAGE and Western blot. Results Sequence analysis confirmed the construction of the expression vector pEt28a- YARA-Ihc-ProDer f 1 successfully, the fusion protein YARA-Ihc-ProDer f 1 was expressed and purified with the concentration of 278 μg/ml. SDS-PAGE and Western blot demonstrated the fusion protein was YARA-Ihc-ProDer f 1. Conclusion
The recombinant prokaryotic expression vectors, pET28a(+ )- YARA- Ihc- ProDer f 1 was successfully constructed, and the fusion protein was expressed and purified .

Key words: Cell penetrating peptides, Dermatophagoides farina, invariant chain, allergens