Pilot-Matias T J, Muerhoff A S, Simons J N, Leary T P, Buijk S L, Chalmers M L, Erker J C, Dawson G J, Desai S M, Mushahwar I K
Diagnostics Division, Abbott Laboratories, North Chicago, IL 60064-4000, USA.
J Med Virol. 1996 Apr;48(4):329-38. doi: 10.1002/(SICI)1096-9071(199604)48:4<329::AID-JMV6>3.0.CO;2-9.
The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently. The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized. Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E. coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins. Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis. Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses. An antigenic region was also identified in the putative core protein of GB virus B. Many of the clones identified originally as encoding antigenic proteins were quite large. To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot. Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened. These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.
最近克隆并测序了与GB病毒型肝炎相关的黄病毒科两个新成员（GB病毒A和B）的基因组。还鉴定并表征了与GB病毒A和B相关但不同的第三种新病毒（GB病毒C）的基因组。包含这三种病毒大开放阅读框的重叠克隆已在大肠杆菌中作为CTP：CMP-3-脱氧-D-甘露糖辛酮酸胞苷转移酶（CKS）融合蛋白表达。使用来自感染GB病毒的绢毛猴血清以及来自各种非甲、非乙、非丙、非丁、非戊型肝炎患者或“有风险”个体的人血清，对细菌裂解物进行蛋白质印迹分析。在所有三种病毒的推定NS3、NS4和NS5蛋白中鉴定出抗原区域。在GB病毒B的推定核心蛋白中也鉴定出一个抗原区域。许多最初鉴定为编码抗原蛋白得克隆相当大。为了更精确地定位这些区域，通过聚合酶链反应（PCR）产生较小的重叠克隆，作为重组CKS融合蛋白表达并通过蛋白质印迹进行检测。此外，从感染性绢毛猴血清构建了一个λgt11表达文库并进行免疫筛选。这些研究已在GB病毒A中鉴定出至少三个表位，在GB病毒B中鉴定出五个表位，在GB病毒C中鉴定出四个表位。
