LIANG Wan-qi and ZHANG Da-Bing. Purification and Observation of the Recombinant Hepatitis B Virus Core Antigen (rHBcAg) Particles Produced in the Yeast[J]. Virologica Sinica, 2004, 19(2): 101-104.
Citation: LIANG Wan-qi, ZHANG Da-Bing. Purification and Observation of the Recombinant Hepatitis B Virus Core Antigen (rHBcAg) Particles Produced in the Yeast .VIROLOGICA SINICA, 2004, 19(2) : 101-104.

酿酒酵母表达的rHBcAg颗粒的纯化和鉴定及其透射电镜观察

  • 通讯作者: 张大兵, 
  • 乙肝核心抗原(HBcAg)蛋白基因(C基因)在酿酒酵母中表达,表达产物经过分离和Sepharose CL-4B柱子的初步纯化。产物经SDS-PAGE和Western blotting鉴定为一分子量约21.5kDa的多肽。再经蔗糖密度梯度超离心和CsCl等密度梯度超离心等过程而被纯化。分管收集的超离心纯化产物经ELISA抗原活性检测和密度分析,可知ELISA反应强度较高的收集管中的颗粒密度主要分布在1.27g/mL和1.40 g/mL两个峰值处。将rHBcAg抗原活性最高的收集管合并,再经TEM观察,发现酵母表达的rHBcAg蛋白(核心蛋白)能自主装配成大小不同的两种核心颗粒,大颗粒直径约为30.1±2.4 nm,小颗粒直径约为21.5±3.3 nm。这表明,酿酒酵母表达的rHBcAg颗粒具有大小不同的二态性,其生物学意义还未明了,需进一步研究和探讨。

Purification and Observation of the Recombinant Hepatitis B Virus Core Antigen (rHBcAg) Particles Produced in the Yeast

  • Corresponding author: ZHANG Da-Bing, 
  • Hepatitis B virus core antigen protein gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg particles) were purified from crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation, and CsCl-isopycnic ultracentrifugation. rHBcAg was synthesized in yeast cells as a particle consisting of polypeptides which have a molecular weight of 21.5 kDa (p21.5). ELISA and CsCl- isopycnic ultracentrifugation purification products (rHBcAg particles) in each fraction indicated that the particle densities of the fraction with higher rHBcAg antigenicity mainly distributed at the densities of 1.27g•mL-1 and 1.40 g•mL-1, respectively. Observation of the purified products (rHBcAg particles) by TEM indicated that rHBcAg peptides could self-assemble into two kinds of different sizes of rHBcAg particles (core particles); the large particle was about 30.1±2.4 nm in diameter and the small particle about 21.5±3.3 nm. This indicated that the rHBcAg particles displayed dimorphism, but the biological significance of this dimorphism is not clear and needs to be studied further.

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    Purification and Observation of the Recombinant Hepatitis B Virus Core Antigen (rHBcAg) Particles Produced in the Yeast

      Corresponding author: ZHANG Da-Bing,
    • 1. 1.School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030,China
    • 2. School of Life Science, Shanghai University, Shanghai 200436,China
    • 3. Biological Technology Center of Shanghai Agriculture Science Institute, Shanghai 201106,China

    Abstract: Hepatitis B virus core antigen protein gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg particles) were purified from crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation, and CsCl-isopycnic ultracentrifugation. rHBcAg was synthesized in yeast cells as a particle consisting of polypeptides which have a molecular weight of 21.5 kDa (p21.5). ELISA and CsCl- isopycnic ultracentrifugation purification products (rHBcAg particles) in each fraction indicated that the particle densities of the fraction with higher rHBcAg antigenicity mainly distributed at the densities of 1.27g•mL-1 and 1.40 g•mL-1, respectively. Observation of the purified products (rHBcAg particles) by TEM indicated that rHBcAg peptides could self-assemble into two kinds of different sizes of rHBcAg particles (core particles); the large particle was about 30.1±2.4 nm in diameter and the small particle about 21.5±3.3 nm. This indicated that the rHBcAg particles displayed dimorphism, but the biological significance of this dimorphism is not clear and needs to be studied further.

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