EPITOPE MAPPING OF THE EBV MEMBRANE ANTIGEN

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LouisQualtiere获得。几 从加里·皮尔森（Gary Pearson）处获得了额外的mAb。这些已经被用于 筛选抗原表达克隆对不同表位的反应性， ELISA鉴定病毒重要核酸序列 中和一种EBV BamL片段的克隆，其含有编码 gp 350的序列与核酸序列数据结合 通过计算机从Genebank中获得，用于推导适当的 用于在E.杆菌一种细菌表达载体， pWS 50能与E.大肠杆菌β-半乳糖苷酶和a 高水平蛋白表达，用于产生10个重叠的 表达未糖基化的GP 350的不同部分的克隆。的 由这些克隆中的八个产生的抗原已被纯化并用于 用mAb进行ELISA、斑点印迹免疫分析和Western印迹分析， EBV-MA上的表位。5株mAb与重组E.杆菌 抗原和三个抗原表位（核苷酸1980-2307，3185-3527 和35273576在BamHIL中）。蛋白质表达的四个 克隆用于在兔中产生抗血清。当测试 中和组织培养中的EBV，兔抗血清以及 识别这三个表位的mAb不能中和，这表明 这些表位不参与中和作用。整个GP 350 编码序列也已在杆状病毒表达系统中表达 使蛋白质糖基化。表达的杆状病毒的性质 蛋白质，包括其糖基化和与mAb的反应性， 评估。

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 the 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 by ELISA 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 in conjunction with nucleic acid sequence data obtained by computer from Genebank was used to derive appropriate constructs for gene expression in E. coli. A bacterial expression vector, pWS50, which produces fusion proteins with E. coli beta-galactosidase and a high level of protein expression, was used to generate ten overlapping clones which express different portions of unglycosylated gp350. The antigens produced by eight of these clones have been purified and used in ELISA, dot blot immunoassay and Western blot analysis with the mAbs to map the epitopes on EBV-MA. Five mAbs reacted with the recombinant E. coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3185-3527 and 35273576 in Bam HI L) identified. Protein expressed by four of the clones was 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. 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.