C端截短的乙型肝炎病毒核心抗原的原核表达及免疫原性

Prokaryotic expression and immunogenicity of C-terminal truncated hepatitis B virus core antigen

目的 原核表达C端截短的乙型肝炎病毒核心抗原（hepatitis B virus core antigen,HBcAg）[HBcAg（aa 1-149）],并研究其在小鼠中的免疫原性.方法 用聚合酶链反应扩增C端截去34个氨基酸的HBcAg基因片段,构建含HBcAg（aa 1-149）基因的克隆质粒及表达质粒,在大肠杆菌Rossatta2中以异丙基-β-D-硫代半乳糖苷诱导重组蛋白的表达.表达产物经Ni2＋亲和层析柱纯化后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质印迹法鉴定.将纯化的重组蛋白免疫BALB/c小鼠,用ELISA法检测血清抗-HBc水平.结果 重组原核表达质粒pET15bHBcAg（aa 1-149）经双酶切和SDS-PAGE鉴定证明构建正确.重组HBcAg（aa 1-149）在大肠杆菌中获得高效表达,表达形式主要为可溶性蛋白.蛋白质印迹法显示在预期位置（相对分子质量约17 000位置）出现特异性条带.纯化后的重组蛋白纯度较高（＞90％）,并可在小鼠中诱生较高水平的抗-HBc.结论 重组HBcAg（aa 1-149）在原核系统中获得成功表达,并可在小鼠中诱导抗体应答.

Objective To express the C-terminal truncated HBcAg（aa 1-149） in E.coli using genetic engineering methods,and study the immunogenicity of truncated HBcAg in BALB/c mice.Methods The C-terminal 34 aa truncated HBcAg gene fragment was amplified by PCR,and the cloning and expression plasmids containing HBcAg（aa 1-149） gene were constructed.The recombinant plasmid was transformed into E.coli Rossatta2 host cell The HBcAg（aa 1-149） fusion protein was expressed under the induction of IPTG,and identified by SDS-PAGE and Western blotting after Ni2＋ affinity chromatography purification.BALB/c mice were immunized with HBcAg（aa 1-149）,and the levels of antibody in serum was detected by ELISA.Results The restriction and SDS-PAGE analyses proved that recombinant plasmid pET15bHBcAg （aa 1-149） was constructed correctly.HBcAg （aa 1-149） was expressed in E.coli.The specific band of recombinant protein was showed at expected position （Mr 17 000 position） by Western blotting.The purity of purified HBcAg（aa 1-149） was more than 90%,and high levels of anti-HBc were induced in mice.Conclusion C-terminal truncated HBcAg is successfully expressed in prokaryotic cells and can induce humoral immune response in mice.