EPITOPE MAPPING OF THE EBV MEMBRANE ANTIGEN

EBV膜抗原(EBV-MA)gp350含有病毒中和成分 表位，目前正在作为可能的EBV候选疫苗进行测试 在英国。一系列可识别的单抗(MAbs) 从Louis Qualtiere获得EBV-MA的不同表位。几个 从加里·皮尔逊那里获得了更多的单抗。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。EB病毒Bam L片段的克隆 将gp350的编码序列用于细菌表达载体， PWS50，生成10个表达不同部分的重叠克隆 去糖基化的gp350。其中八个克隆产生的抗原 经纯化后与单抗进行斑点杂交免疫分析 EBV-MA上的表位。在测试的15株单抗中，有4株与 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(见L)。剩下的 单抗可能只识别糖基化形式的gp350。蛋白质 其中4个克隆表达的抗体被用来制备兔抗血清。 当在组织培养中检测EBV时，兔子 抗血清以及识别这三个表位的单抗都不能 中和，表明这些表位没有参与 中和。描述这些数据的手稿已被接受为 发表在J Gen Virol杂志上。整个gp350编码序列也被 在杆状病毒表达系统中表达，糖基化 蛋白。杆状病毒表达的蛋白的性质包括 它的糖基化和与单抗的反应性正在评估中。亚克隆 它们以糖基化形式表达gp350的不同部分。 杆状病毒表达系统目前正在为其他 表位图谱研究。

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.