乙肝病毒核心抗原展示载体的原核表达与电镜检测  

Prokaryotic expression and electron microscopic examination of Hepatitis B virus core antigen display vector

作　　者：开丽霞 刘安康 朱贵坤 肖正泮 韦双双 王大勇 裴业春 KAI Li-xia LIU An-kang ZHU Gui-kun XIAO Zheng-pan WEI Shuang-shuang WANG Da-yong PEI Ye-chun(Hainan Key Lab of Tropical Animal Reproduction ＆ Breeding and Epidemic Disease Research, College of Animal Science and Technology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou , Hainan 570228 College of Biological Science, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou , Hainan 570228, China)

机构地区：[1]海南大学热带农林学院动物科技学院海南省热带动物繁育与疫病研究重点实验室,海南海口570228 [2]海南大学热带农林学院生物学院,海南海口570228

出　　处：《热带医学杂志》2017年第10期1297-1300,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金青年项目(31402257);海南省科技项目(ZDYF2017110)

摘　　要：目的将多克隆酶切位点MCS插入乙肝病毒核心抗原(HBcAg)的loop区,构建乙肝病毒核心抗原展示载体。方法将多克隆酶切位点MCS插入到HBcAg的c/e1 loop区,取代了HBcAg c/e1 loop区的Pro79与Ala80氨基酸残基,成功构建了pET28a-HBcAg-MCS原核表达载体,将其转化入大肠杆菌BL21(DE3)中,利用不同IPTG浓度梯度进行原核诱导表达与NI-NTA亲和层析纯化,利用SDS-PAGE电泳和透射电镜检测。结果成功表达了HBcAg-MCS融合蛋白,且最佳的IPTG诱导浓度为0.1 mmol/L,利用镍柱纯化得到了较纯的HBcAg-MCS融合蛋白,进一步利用负染法透射电子显微镜检测到HBcAg-MCS融合蛋白呈现病毒样颗粒结构。结论 HBcAg-MCS融合蛋白能够自发形成病毒样颗粒结构,为乙肝核心抗原HBcAg作为展示载体的广泛应用打下基础。Objective To construct a hepatitis B virus core antigen display vector by inserting multiple cloning site（MCS）into the loop region of the hepatitis B virus core antigen（HBcAg）.MethodsThe multiple cloning site（MCS）was inserted into HBcAg c/e1 loop area,replaced the Pro79 and Ala80 amino acid residues of HBcAg c/e1 loop.The prokaryotic expression vector pET28a-HBcAg-MCS was successfully constructed,transformed into E.coli BL21（DE3）and expressed by different concentration of IPTG.The recombinant protein HBcAg was purified by Ni-NTA affinity chromatography and tested by SDS-PAGE electrophoresis and transmission electron microscopy（TEM）.ResultsThe fusion protein HBcAg-HBcAg was expressed successfully in E.coli expression system,and the best induced concentration of IPTG was 0.1 mmol/L.The pure fusion protein HBcAg-MCS was purified by Ni-NTA affinity chromatography.TEM confirmed the fusion protein HBcAgMCS could be assembled into virus-like particles.ConclusionThe fusion protein HBcAg-MCS spontaneously assembled into virus-like particles,laying a solid foundation for the widespread use of hepatitis B core antigen HBcAg as a display vector.

关 键 词：乙肝病毒核心抗原 病毒样颗粒 多克隆酶切位点 

分 类 号：R373.21[医药卫生—病原生物学]