Ross B C, Anderson D A
Department of Clinical Pathology, Fairfield Hospital, Victoria, Australia.
J Virol Methods. 1991 May;32(2-3):213-20. doi: 10.1016/0166-0934(91)90052-2.
The capsid proteins of hepatitis A virus (HAV) were expressed as fusion proteins of beta-galactosidase in E. coli using the expression vector lambda gt11. Four fusion proteins were stably expressed and used to immunize rabbits to obtain mono-specific antisera. The antisera were unable to neutralize viral infectivity or react with HAV by radioimmunoassay. Three of the antisera were able to recognize HAV antigens in infected BS-C-1 cells by immunofluorescence and denatured capsid proteins by immunoblot analysis. The antisera were used to investigate the migration of the capsid proteins in gels by immunoblot analysis using standard SDS-PAGE conditions and in gels containing urea. The migration of VP1 and VP3 correlated with their molecular weights predicted from the nucleotide sequence and was consistent in either the presence or absence of urea. However, VP2 migrated with an apparent molecular weight significantly higher than the predicted value and, in gels containing urea, migrated as a doublet. It is proposed that the upper band of this doublet represents VP0, the proteolytic precursor of VP2 and VP4. The relative molecular mass (Mr) of VP4 was estimated to be less than 1 kDa, which is substantially lower than the 2.5 kDa predicted from the nucleotide sequence.
利用表达载体λgt11在大肠杆菌中将甲型肝炎病毒（HAV）的衣壳蛋白表达为β-半乳糖苷酶的融合蛋白。四种融合蛋白稳定表达，并用于免疫兔子以获得单特异性抗血清。这些抗血清不能中和病毒感染性，也不能通过放射免疫测定与HAV发生反应。其中三种抗血清能够通过免疫荧光识别感染的BS-C-1细胞中的HAV抗原，并通过免疫印迹分析识别变性的衣壳蛋白。这些抗血清用于通过使用标准SDS-PAGE条件的免疫印迹分析以及在含有尿素的凝胶中研究衣壳蛋白在凝胶中的迁移情况。VP1和VP3的迁移与其从核苷酸序列预测的分子量相关，并且在有或没有尿素的情况下都是一致的。然而，VP2迁移时的表观分子量明显高于预测值，并且在含有尿素的凝胶中迁移时呈现为双峰。有人提出，这个双峰的上带代表VP0，即VP2和VP4的蛋白水解前体。VP4的相对分子质量（Mr）估计小于1 kDa，这大大低于从核苷酸序列预测的2.5 kDa。
