The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes
The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes
复制标题
DOI：
10.1016/s0378-1119(96)00348-4
复制
发表时间：
1996-11-14
期刊：
影响因子：
3.5
通讯作者：
Martin, C
中科院分区：
文献类型：
--
作者：
Mechin, MC;DerVartanian, M;Martin, C
以前，来自肠致病性传染性胃肠炎病毒（TGEV）的两个B细胞表位，即刺突S蛋白（TGEV-S）的C表位（TGEV-C）氨基酸（aa）363-371和A表位（TGEV-A）aa 522-531，在插入ClpG的相同区域后分别在大肠杆菌表面的CS31 A纤丝上表达。然而，所得嵌合体在小鼠中诱导边际TGEV中和抗体（Ab）应答。在这里，为了改善这种反应，我们在ClpG的12个暴露于嘌呤的区域中单独引入TGEV-C或与TGEV-A（A：：C或C：：A）不同的串联结合。在所得到的28个工程蛋白中，只有15个携带多达51个额外的氨基酸，基本上没有干扰正确的CS31 A原纤维形成过程。在整个ClpG中鉴定了仅接受TGEV-C的六个部分允许位点和耐受A：：C或C：：A串联肽的三个高度允许位点。完整的细菌或提取的CS31 A杂合原纤维在任何允许位点表达TGEV表位，被针对外源亲本蛋白的Ab识别，为在细胞表面暴露相应的ClpG区域和在多聚CS31 A原纤维背景下表位的抗原性提供了直接的论据。CS31 A原纤维作为TGEV肽载体的潜力表明，ClpG中可能存在三个位置（N末端，aa 202-204和202-218），这可能是重要的融合位点，因此与TGEV疫苗的最终设计相关。出乎意料的是，TGEV-A，无论其在ClpG中的位置如何，都介导了杂合蛋白的部分蛋白水解降解，这表明它作为细胞蛋白酶的底物起作用，因此其作为疫苗抗原候选物的适合性是值得怀疑的。
Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding ClpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202-204 and 202-218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.