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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
引用本文:张鹏艳,叶琳. 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[J]. 国际生物制品学杂志, 2015, 38(1): 1-5. DOI: 10.3760/cma.j.issn.1673-4211.2015.01.001
作者姓名:张鹏艳  叶琳
作者单位:610023 成都生物制品研究所有限责任公司生物技术研究室
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。

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  

Prokaryotic expression and immunogenicity of C-terminal truncated hepatitis B virus core antigen
Zhang Pengyan,Ye Lin. Prokaryotic expression and immunogenicity of C-terminal truncated hepatitis B virus core antigen[J]. International Journal of Biologicals, 2015, 38(1): 1-5. DOI: 10.3760/cma.j.issn.1673-4211.2015.01.001
Authors:Zhang Pengyan  Ye Lin
Affiliation:Bio-Technology Research Laboratory, Chengdu Institute of Biological Products Co., Ltd., Chengdu 610023, China
Abstract: 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 (Mr 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.
Keywords:Hepatitis B core antigen  Prokaryotic cells  Gene expression  Protein purification  Immunogenicity
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