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1、GENE ELSEVIER Gene 179 (1996) 211-218 The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes Marie-Claire M6chin, Maurice Der Vartanian *, Christine Martin Laboratoire de Microbiologie, lnstitut National de la Re
2、cherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genbs-Champanelle, France Received 16 October 1995; revised 25 March 1996; accepted 2 April 1996 Abstract Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the
3、 C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-53l 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 margi
4、nal 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 t
5、o 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 fibrilla
6、e 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. T
7、he 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
8、 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. Keywords: Recombinant DNA; Genetic fusion; Hybrid protein; Tan
9、dem insertion; Transmissible gastroenteritis virus; Peptide presentation; Surface exposure I. Introduction Epitope-based recombinant vaccine technologies offer the potential for oral or mucosal delivery, especially * Corresponding author. Tel.: + 33 4 73624243; Fax: + 33 4 73624581; e-mail: dvartanc
10、lermont.inra.fr Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); BSA, bovine serum albumin; CIpG, major CS31A fibrillar subunit; clpG, gene encoding ClpG; kb, kilobase(s) or 1000 bp; LB, Luria- Bertani (medium); mAb monoclonal Ab; nt, nucleotide(s); oligo, oligo- deoxyribonucle
11、otide; pAb, polyclonal Ab; PAGE, polyacrylamide-gel electrophoresis; PBS, phosphate-buffered saline (0.14 M NaC1/2.7 mM KCI/1.47mM KH2PO4/20mM Na2HPO4, pH 7.4); SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline (0.15 M NaCI/10 mM Tris- HC1, pH 7.4); TGEV, transmissible gastroenteritis virus; TG
12、EV-A, peptide/site/epitope A of TGEV-S; TGEV-C, peptide/site/epitope C of TGEV-S; TGEV-S, spike S glycoprotein of TGEV; wt, wild type; :, novel junction (fusion or insertion); , denotes plasmid-carrier state. 0378-1119/96/$15.00 1996 Elsevier Science B.V. All rights reserved PH S0378-1119(96)00348-4
13、 when the relevant epitope genetically fused to a carrier protein, is displayed as a heterologous peptide on the surface of a bacterial strain (Rabinovich et al., 1994). However, coupling foreign peptides to carriers can result in a poor immunogenicity of the chimeric antigens due to the local confo
14、rmational restrictions imposed on epitopes by the embedding structure (Benito et al., 1995). Therefore, approaches to enhancing immunogenicity are critical. Since the conformation of a foreign sequence within a carrier varies widely depending on the flanking sequences (Tishminetzky et al., 1994), th
15、e insertion of an antigen into different exposed regions of a delivery protein, thus changing antigen conformation, could permit a strategy for finding appropriate environments for peptide presentation among the complex assortment of molecular contexts offered by the carrier. A second strategy to im
16、proving immunogenicity is the fusion of 212 M.-C. Mkchin et al./Gene 179 (1996) 211-218 A 5- ATG- -TAA-3 B clpG CIpG C cc CIpG a b C d e l 66 ATG . GCGCATGCG M . AHA -21 607 717 834 bp 12 TGG Spe I Bgl II W I I -I 1 pPSX10S ACTAGT GGAGATCTA CIpG420 T* S G D L* processing site 1 1 / 2 34 5 678910 L pPSX6S ACT AGT Mun I , CIpG202 T* S I C AA TTG CTG GGC CAG cTr CAA GCA GTA AAC CCT AAC GCG GGC AAT AGAGG A CAA GTA AAT AAA AAT AGT GCG GT
