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

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.