Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries
Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries
复制标题
DOI：
10.1099/0022-1317-81-4-993
复制
发表时间：
2000-04-01
影响因子：
3.8
通讯作者：
du Plessis, DH
中科院分区：
文献类型：
--
作者：
Bentley, L;Fehrsen, J;du Plessis, DH
VP2是非洲马瘟病毒（AHSV）的外壳蛋白，可被分型中和抗体识别。为了定位其抗原区域，构建了丝状噬菌体文库，该文库展示源自编码VP2的基因的cDNA拷贝的片段化的肽。将大小在约30至100个氨基酸范围内的肽与展示载体fUSE 2的附着蛋白pill融合。为了确保最大的多样性，最终的图书馆由三个子图书馆组成。第一种方法利用仅编码VP2基因的酶促片段化DNA，第二种方法包括质粒序列，而第三种方法包括PCR步骤，该PCR步骤被设计为允许不同的肽编码序列在连接到载体中之前重组。用AHSV特异性多克隆鸡IgY、多克隆马免疫球蛋白和已知中和AHSV的单克隆抗体（MAb）对所得复合文库进行免疫亲和选择，用该方法定位的抗原决定簇大多位于VP2的N端，通过确定所选抗原决定簇的最小重叠区域，以高分辨率定位了重要的结合区域，并利用该单克隆抗体筛选了随机17肽表位文库。鉴定了可能是不连续中和表位的一部分的序列。将血清型3的VP2上的抗原区域的氨基酸序列与其他三种血清型上的相应区域进行比较，揭示了具有在血清学上区分AHSV血清型的潜力的区域。
VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2, Peptides ranging in size from approximately 30 to 100 amino acids were fused with pill, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector, The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV, Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies, Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2, Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides, The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified, The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.