Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes.
Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes.
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DOI：
10.4049/jimmunol.151.8.3971
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发表时间：
1993-10
影响因子：
4.4
通讯作者：
Mark Gavin;Mark J. Gilbert;Stanley R. Riddell;Philip D. Greenberg;Michael J. Bevan
中科院分区：
文献类型：
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作者：
Mark Gavin;Mark J. Gilbert;Stanley R. Riddell;Philip D. Greenberg;Michael J. Bevan
CTL 识别的表位的表征提供了对保护性免疫反应本质的深入了解，并有助于开发增强对人类病原体免疫力的方法。然而，尚未开发出易于应用的 CTL 表位鉴定方法。我们提出了一种快速有效的方法来定位蛋白质内的 CTL 表位。将编码目的蛋白的基因插入诱导型原核表达载体中。然后通过碱消化表达蛋白质的完整或裂解的大肠杆菌来产生随机肽，并通过包被靶细胞进行标准 CTL 靶向测定来测定表位的存在。然后通过核酸外切酶 III 消化产生一大组含有该基因连续 3'-缺失的克隆，并类似地分析表达的截短蛋白是否存在抗原肽。通过确定表达连续截短且Ag表达不同的克隆的缺失点来定位表位。该技术用于鉴定大肠杆菌 β-半乳糖苷酶中的 H-2Ld 限制性九聚体，其中残基 876-884 代表自然加工的表位。为了测试该方法对其他蛋白质的适用性，对来自人类 CMV（免疫功能低下个体中通常致命的病原体）的两个基因进行了 HLA I 类限制性表位的筛选。发现来自 CMV 主要立即早期蛋白的 HLA-B18 限制性表位位于残基 378 和 389 之间，来自 CMV pp65 基质蛋白的 HLA-B35 限制性表位被表征为残基 123 至 131。结果表明，该技术可用于快速识别所选蛋白质内的 CTL 表位，并且应可用于分析病毒分离株或肿瘤的 CTL 表位。突变和宿主免疫反应选择后表位丢失。
The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However, no easily applicable approach for CTL epitope identification has been developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector. Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide. The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2Ld-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131. The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.