摘要
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.
出处
《国际生物制品学杂志》
CAS
2015年第1期1-5,共5页
International Journal of Biologicals
关键词
肝炎核心抗原
乙型
原核细胞
基因表达
蛋白纯化
免疫原性
Hepatitis B core antigen
Prokaryotic cells
Gene expression
Protein purification
Immunogenicity