乳房链球菌GapC蛋白的表达及其B细胞抗原表位的预测与鉴定

Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes

摘要:

乳房链球菌Streptococcus uberis的GapC蛋白是一种位于该菌表面的具有甘油醛-3-磷酸脱氢酶活性的蛋白，其参与细胞活动，表现出多种生物学活性，此外还具有良好的抗原性。文中旨在对乳房链球菌GapC蛋白可能的B细胞抗原表位进行预测，分析和验证候选表位肽的免疫原性。利用S. uberis分离株RF5-1克隆gapC基因，构建重组表达质粒pET-28a-GapC，诱导表达GapC重组蛋白，并以纯化蛋白免疫家兔，获得抗GapC多抗。利用生物信息学软件预测并分析GapCB细胞抗原表位的三维结构和空间位置及对GapC蛋白及表位的同源性比较。结果表明，表达纯化了44 kDa的GapC蛋白具有良好的反应性。利用表位预测软件筛选并合成针对S. uberis GapC蛋白的6个线性和3个构象优势B细胞表位多肽，三维结构的分析显示，筛选的多肽具有良好的抗原表位形成条件。以纯化的S. uberis GapC蛋白免疫家兔制备多抗，通过间接ELISA对抗原表位进行鉴定。ELISA检测结果显示，9条抗原表位肽均可不同程度地与抗GapC多抗反应，其中表位²⁶⁶AANDSYGYTEDPIVSSD²⁸²与多抗反应水平显著高于其他表位。研究结果为深入了解S. uberis GapC蛋白的免疫学特性和利用其有效抗原表位奠定了基础。

Abstract:

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide ²⁶⁶AANDSYGYTEDPIVSSD²⁸² reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.