Méchin M C, Der Vartanian M, Martin C
Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, Saint-Genès-Champanelle, France.
Gene. 1996 Nov 14;179(2):211-8. doi: 10.1016/s0378-1119(96)00348-4.
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 CIpG 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.
此前，肠道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的同一区域后，已分别在大肠杆菌表面的CS31A菌毛上表达。然而，所得嵌合体在小鼠中诱导出微弱的TGEV中和抗体（Ab）反应。在此，为了改善这种反应，我们将TGEV-C单独或与TGEV-A以不同的串联组合（A::C或C::A）引入ClpG的12个假定暴露区域。在所得的28种工程蛋白中，只有15种携带多达51个额外氨基酸，基本未干扰正确的CS31A菌毛形成过程。在整个ClpG中鉴定出6个仅接受TGEV-C的部分允许位点和3个耐受A::C或C::A串联肽的高度允许位点。在任何允许位点表达TGEV表位的完整细菌或提取的CS31A杂交菌毛，都被针对外源亲本蛋白的抗体识别，这为相应的ClpG区域在细胞表面的暴露以及表位在聚合CS31A菌毛环境中的抗原性提供了直接证据。CS31A菌毛作为TGEV肽载体的潜力表明，ClpG中可能有三个位置（N端、aa 202 - 204和202 - 218）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，无论TGEV-A在ClpG中的位置如何，它都会介导杂交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发挥作用，因此其作为疫苗抗原候选物的适用性值得怀疑。
