Watelet Bénédicte, Quibriac Martine, Rolland Dominique, Gervasi Gaspard, Gauthier Marie, Jolivet Michel, Letourneur Odile
BioMérieux-Pierre Fabre, Chemin de l'Orme, 69280, Marcy l'Etoile, France.
J Virol Methods. 2002 Jan;99(1-2):99-114. doi: 10.1016/s0166-0934(01)00385-8.
The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexahistidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.
乙肝核心抗原（HBcAg）在大肠杆菌和毕赤酵母中表达。在大肠杆菌表达蛋白的C末端引入了一个六组氨酸标签，使其能够通过镍（2+）螯合亲和层析进行纯化。毕赤酵母表达的HBcAg经过热处理后分离出来。两种重组HBcAg在蔗糖梯度上进一步纯化。质谱分析表明，HBcAg仅在毕赤酵母中发生了N-乙酰化，与埃尔曼试剂反应分别测得每摩尔在大肠杆菌或毕赤酵母中表达的HBcAg单体含有0.37和0.23摩尔游离巯基。电子显微镜显示，大肠杆菌和毕赤酵母的蛋白分别形成了直径为34纳米和28纳米的衣壳样颗粒。在两种颗粒中均发现有核酸成分被困，但只有毕赤酵母来源的颗粒中的核酸成分受到酶处理的保护，这表明两种重组分子之间存在结构差异。这些重组抗原的高纯度使得能够开发一种夹心免疫测定法来检测人血清中的抗-HBc抗体。初步结果表明，在这种诊断测定中，细胞内产生的毕赤酵母HBcAg比复性的大肠杆菌HBcAg更适合用于检测抗-HBc。
