EPITOPE MAPPING OF THE EBV MEMBRANE ANTIGEN

EBV 膜抗原的表位作图

• 批准号: 3804788
• 负责人: C J MARCUS-SEKURA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804788
• 关键词: Epstein Barr virus; antibody neutralization test; epitope mapping; gene expression; insect virus; laboratory rabbit; molecular cloning; monoclonal antibody; nucleic acid sequence; protein purification; surface antigens; tissue /cell culture; virus antigen; western blottings

The EBV membrane antigen (EBV-MA) gp350 contains virus neutralizing epitopes and is currently being tested as a possible EBV vaccine candidate in England. A series of monoclonal antibodies (mAbs) to identifiably different epitopes on EBV-MA was obtained from Louis Qualtiere. Several additional mAbs were obtained from Gary Pearson. These have been used to screen antigen expressing clones for reactivity to different epitopes in order to identify the nucleic acid sequences important for virus neutralization. A clone of the EBV Bam L fragment which contains the coding sequence for the gp350 was used with a bacterial expression vector, pWS50, to generate ten overlapping clones which express different portions of unglycosylated gp350. The antigens produced by eight of these clones were purified and used in a dot blot immunoassay with the mAbs to map the epitopes on EBV-MA. Four of fifteen mAbs tested reacted with the recombinant E.coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3186-3528 and 3528-3576 in Bam HI L) identified. The remaining mAbs may only recognize the glycosylated form of gp350. Proteins expressed by four of the clones were used to produce antisera in rabbits. When tested for neutralization of EBV in tissue culture, the rabbit antisera as well as the mAbs which recognized the three epitopes failed to neutralize, suggesting that these epitopes are not involved in neutralization. A manuscripts describing these data has been accepted for publication in J Gen Virol. The entire gp350 coding sequence has also been expressed in a Baculovirus expression system which glycosylates the protein. The properties of the Baculovirus-expressed protein, including its glycosylation and reactivity with mAbs, are being evaluated. Subclones which express different portions of gp350 in a glycosylated form in the Baculovirus expression system are currently being prepared for additional epitope mapping studies.

EBV膜抗原（EBV-MA）gp 350含有病毒中和性抗原， 表位，目前正在测试作为一个可能的EBV疫苗候选人 在英国 一系列单克隆抗体（mAb），可识别 EBV-MA上的不同表位从Louis Qualtiere获得。 几 从加里·皮尔森（Gary Pearson）处获得了额外的mAb。 这些已经被用来 筛选抗原表达克隆对不同表位的反应性， 为了鉴定对病毒重要的核酸序列， 中和一种EBV Bam L片段的克隆，其含有 将gp 350的编码序列与细菌表达载体一起使用， pWS 50，以产生表达不同部分的10个重叠克隆 非糖基化的GP 350。这些克隆中的八个产生的抗原 纯化并用于与mAb的斑点印迹免疫测定，以绘制 EBV-MA上的表位。检测的15种mAb中有4种与 重组大肠杆菌抗原和三个抗原表位（核苷酸 1980-2307、3186-3528和3528-3576）。其余 mAb可能仅识别糖基化形式的gp 350。 蛋白 用其中四个克隆表达的抗体在兔中产生抗血清。 当在组织培养中测试EB病毒中和时，兔子 抗血清以及识别这三个表位的单克隆抗体未能 中和，表明这些表位不参与 中和描述这些数据的手稿已被接受， 发表于J Gen Virol。整个gp 350编码序列也已被 在杆状病毒表达系统中表达，所述杆状病毒表达系统使所述糖基化， 蛋白杆状病毒表达蛋白的性质，包括 其糖基化和与mAb的反应性正在评估中。亚克隆 其在细胞中以糖基化形式表达gp 350的不同部分， 杆状病毒表达系统目前正在准备用于其他 表位作图研究。