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  

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

作　　者：裴业春[1] 安晓荣[2] 侯健[2] 陈永福[2] 闫凤祥 关宏[2] 韦双双[1] 王大勇[1] PEI Ye-chun AN Xiao-rong HOU Jian CHEN Yong-fu YAN Feng-xiang GUAN Hongz WEI Shuang-shuang WANG Da-yong(College of Agriculture, Hainan University, Haikou 570228, China College of Biological Science, China Agricultural University, Beijing 100193, China)

机构地区：[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

出　　处：《中国畜牧兽医》2016年第11期2826-2833,共8页China Animal Husbandry & Veterinary Medicine

基　　金：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)

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chain1和chain2拼接在一起形成重组Fel d 1(rFel d 1),然后插入到HBcAg的c/e1loop区,取代HBcAg c/e1loop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pET28aHBcAg-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-r Fel 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HBcAgrFel 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。To expose the cat recombinant allergen Fel d 1protein 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 1and chain 2that composed the Fel d 1protein.Then the rFel d 1sequence was inserted into the HBcAg c/e1 loop area,replacing the amino acids between D78 and E83in 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 1was 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 1was expressed successfully in E.coli expression system and the pure fusion protein HBcAg-rFel d 1was purified by Ni-NTA affinity chromatography.Further,TEM confirmed the fusion protein HBcAg-rFel d1 could assemble into VLPs.The fusion protein HBcAg-rFel d 1could assemble into VLPs spon-taneously,which laid a solid foundation for the research of the preventive and therapeutic vaccines for cat allergy.

关 键 词：猫变应原 乙肝病毒核心抗原 FEL d 1 病毒样颗粒 

分 类 号：R392.8[医药卫生—免疫学]