Bitter G A, Egan K M, Burnette W N, Samal B, Fieschko J C, Peterson D L, Downing M R, Wypych J, Langley K E
Amgen, Thousand Oaks, California 91320.
J Med Virol. 1988 Jun;25(2):123-40. doi: 10.1002/jmv.1890250202.
A gene encoding the 226 amino acid hepatitis B surface antigen (HBsAg), subtype adw, was cloned into a generalized vector for the expression of heterologous genes in Saccharomyces cerevisiae. The 5' end of the genomic HBsAg gene was replaced with a chemically synthesized DNA segment that conserved the amino acid sequence of the protein but utilized DNA sequences that optimize translation initiation in yeast. High-cell-density fermentations of laboratory strains of Saccharomyces cerevisiae have been developed in which HBsAg production increases linearly with respect to cell mass. The HBsAg is present as a lipoprotein particle in cell lysates and has been purified to homogeneity. The evidence presented indicates that the HBsAg particles may be formed during lysis of the yeast cells. The purified HBsAg particles have a morphology similar to that of the 22 nm particles present in the serum of human chronic carriers of hepatitis B. The reactivity of the yeast-derived HBsAg particles with a series of monoclonal antibodies is essentially identical to that of human plasma HBsAg. By this analysis, therefore, the structure of the HBsAg protein is similar in yeast and in human particles. The purified yeast HBsAg particles were formulated with alum adjuvant and subsequently were shown to confer immunity in chimpanzees to challenge with two heterologous serotypes (adr, ayw) of hepatitis B virus.
编码226个氨基酸的乙型肝炎表面抗原（HBsAg）adw亚型的基因被克隆到一个用于在酿酒酵母中表达异源基因的通用载体中。基因组HBsAg基因的5'端被一个化学合成的DNA片段取代，该片段保留了蛋白质的氨基酸序列，但使用了优化酵母中翻译起始的DNA序列。已开发出酿酒酵母实验室菌株的高细胞密度发酵方法，其中HBsAg的产量随细胞量呈线性增加。HBsAg以脂蛋白颗粒的形式存在于细胞裂解物中，并已纯化至同质。所提供的证据表明，HBsAg颗粒可能在酵母细胞裂解过程中形成。纯化的HBsAg颗粒的形态与慢性乙型肝炎携带者血清中存在的22 nm颗粒相似。酵母来源的HBsAg颗粒与一系列单克隆抗体的反应性与人类血浆HBsAg基本相同。因此，通过这种分析，HBsAg蛋白在酵母颗粒和人类颗粒中的结构相似。纯化的酵母HBsAg颗粒与明矾佐剂一起配制，随后在黑猩猩中显示出对两种异源血清型（adr、ayw）乙型肝炎病毒攻击的免疫保护作用。
