羊朊毒体单抗结合表位分析

Analysis of monoclonal antibody binding sites in ovine prion protein

摘要:

通过分段表达PrP核心片段和人工合成多肽, 分析5株羊朊毒体单抗结合表位。分段表达PrP核心片段, 通过PCR方法扩增目的片段, 经酶切、连接后, 将目的片段插入质粒pET32a, 在大肠杆菌BL21中表达。将表达的系列融合蛋白与单抗进行免疫转印试验, 根据反应情况确定单抗结合的大致部位, 在此基础上设计合成多条针对性多肽, 用ELISA方法进一步确定3株单抗的结合部位; 通过与6段融合蛋白反应证明5株单抗的结合部位分别为: 2H3在199 aa~ 213 aa之间, 4C6、5F11和7F11在139 aa~168 aa之间, 7F1在214 aa~227 aa之间, 与3段人工合成多肽进行ELISA反应进一步得到4C6、5F11和7F11抗原结合表位在149 aa~158 aa之间; 本研究确定了5株单抗在PrP分子上的结合部位,为羊痒病和牛海绵状脑病的检测、发病机制的研究奠定了基础。

Abstract:

Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa~158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.