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

 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pET15b HBcAg(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。

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

 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 comtaining 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 pET15b HBcAg(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 (M_r 17 000 position) by Western blot. The purity of purified HBcAg(aa 1-149) was more then 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.