《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (11): 2826-2833.doi: 10.16431/j.cnki.1671-7236.2016.11.004

• 生物技术 • 上一篇    下一篇

猫重组变应原Fel d 1与乙肝病毒核心抗原融合基因的原核表达

裴业春1, 安晓荣2, 侯健2, 陈永福2, 闫凤祥2, 关宏2, 韦双双1, 王大勇1   

  1. 1. 海南大学农学院, 生物技术与分子药理学实验室, 海口 570228;
    2. 中国农业大学生物学院, 农业生物技术国家重点实验室, 北京 100193
  • 修回日期:2016-07-26 出版日期:2016-11-20 发布日期:2016-11-18
  • 通讯作者: 王大勇 E-mail:wangdy@hainu.edu.cn
  • 作者简介:裴业春(1985-),男,海南万宁人,博士,讲师,研究方向:生物技术与分子药理学,E-mail:ycpei@hainu.edu.cn
  • 基金资助:

    国家自然科学基金青年项目(31402257);海南省自然科学基金项目(20153084)

Prokaryotic Expression of Fusion Gene of Cat Recombinant Allergen Fel d 1 with Hepatitis B Core Antigen

PEI Ye-chun1, AN Xiao-rong2, HOU Jian2, CHEN Yong-fu2, YAN Feng-xiang2, GUAN Hong2, WEI Shuang-shuang1, WANG Da-yong1   

  1. 1. College of Agriculture, Hainan University, Haikou 570228, China;
    2. College of Biological Science, China Agricultural University, Beijing 100193, China
  • Revised:2016-07-26 Online:2016-11-20 Published:2016-11-18

摘要:

为将猫重组变应原Fel d 1蛋白展示在乙肝病毒核心抗原(HBcAg)病毒样颗粒的表面,本试验将编码Fel d 1蛋白的两个基因chain 1和chain 2拼接在一起形成重组Fel d 1(rFel d 1),然后插入到HBcAg的c/e1 loop区,取代HBcAg c/e1 loop区的D78与E83之间的氨基酸。经密码子优化后进行全基因合成,成功构建了pET28a-HBcAg-rFel d 1原核表达载体,将其转化入大肠杆菌BL21(DE3)中,进行原核诱导表达与Ni-NTA亲和层析纯化,并进行SDS-PAGE电泳、Western blotting和透射电镜检测。结果显示,本试验成功表达了HBcAg-rFel d 1融合蛋白,并利用镍柱纯化得到了较纯的HBcAg-rFel d 1融合蛋白,进一步利用负染法透射电子显微镜检测到HBcAg-rFel d 1融合蛋白呈现病毒样颗粒结构。HBcAg-rFel d 1融合蛋白能自发形成病毒样颗粒结构,为猫过敏症的预防与治疗性疫苗的开发奠定基础。

关键词: 猫变应原; 乙肝病毒核心抗原; Fel d 1; 病毒样颗粒

Abstract:

To expose the cat recombinant allergen Fel d 1 protein on the outer surface of hepatitis B core antigen(HBcAg)virus-like particles(VLPs), the recombinant Fel d 1(rFel d 1)was created by linking the two genes chain 1 and chain 2 that composed the Fel d 1 protein. Then the rFel d 1 sequence was inserted into the HBcAg c/e1 loop area, replacing the amino acids between D78 and E83 in HBcAg c/e1 loop area. We successfully constructed the prokaryotic expression vector pET28a-HBcAg-rFel d 1 via gene codon optimization and synthesis. The recombinant plasmid pET28a-HBcAg-rFel d 1 was transformed into E.coli BL21(DE3)cells, then induced by IPTG, purified by Ni-NTA affinity chromatography and tested by SDS-PAGE, Western blotting and transmission electron microscopy(TEM). The fusion protein HBcAg-rFel d 1 was expressed successfully in E. coli expression system and the pure fusion protein HBcAg-rFel d 1 was purified by Ni-NTA affinity chromatography. Further, TEM confirmed the fusion protein HBcAg-rFel d 1 could assemble into VLPs. The fusion protein HBcAg-rFel d 1 could assemble into VLPs spontaneously, which laid a solid foundation for the research of the preventive and therapeutic vaccines for cat allergy.

Key words: cat allergen; hepatitis B virus core antigen; Fel d 1; virus-like particles

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