Gal-Gal pyelonephritis Escherichia coli pili linear immunogenic and antigenic epitopes.

Abstract

The linear immunogenic and antigenic structure of E. coli Gal-Gal pili from the recombinant strain HU 849 was investigated with nine synthetic peptides corresponding to regions of the pilus sequence predicted to contain hydrophilic beta-turns. Five peptides, as bovine serum albumin conjugates, were found by anti-HU 849 pilus serum and were thus designated "immunogenic epitopes." Peptides corresponding to R 25-38, R 38-50, and R 48-61 (which jointly comprise the single intramolecular disulfide loop), and R 103-116, were bound in low titer. A prominent immunogenic epitope was specified by a peptide corresponding to R 65-75. Four peptides, as thyroglobulin conjugates, elicited antisera in rabbits that bound intact HU 849 pili. These were designated "antigenic epitopes." Two prominent antigenic epitopes were localized to peptides corresponding to R 5-12 and R 93-104, whereas peptides corresponding to R 65-75 and R 119-131 represented two minor antigenic epitopes. None of the peptide antisera bound Gal-Gal pili from heterologous strains except anti-R 93-104 and anti-R 5-12. In 8 of the 10 Gal-Gal-binding pyelonephritis isolates tested, anti-R 5-12 detected a protein with an apparent molecular weight of 18,000 co-migrating with several Gal-Gal pili. Anti-R 93-104 detected a corresponding protein in 4 of 8 fecal and 7 of 12 pyelonephritis Gal-Gal-binding isolates; however, it also bound apparently unrelated proteins of higher molecular weight.

Characterization of in vivo expression of the human papillomavirus type 16 E4 protein in cervical biopsy tissues.

“…In a separate experiment, polystyrene microtiter wells were incubated overnight with 5 Mg/ml ofHPV16 E4 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] for the full-length protein described above was studied by RIA (as described above), and by Western blot analysis. 2 synthetic full-length E4 protein were subjected to SDS-PAGE using 18% polyacrylamide in the resolving gel (29), and the protein was transferred to a nitrocellulose membrane (30).…”

“…To elicit antibodies to synthetic peptide HPV 16 E4 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34], the peptide-thyroglobulin conjugate was emulsified with CFA and injected into New Zealand White rabbits; 6 wk later, the conjugate was administered with IFA, and the animals were bled 10 d thereafter. The IgG fraction was precipitated from the corresponding pre-immune sera and from the hyperimmune E4 antisera with 2 M ammonium sulfate, followed by anion-exchange chromatography with DEAE-cellulose (Sigma Chemical Co.).…”

“…Preparation of the HPV 16 and HPV 6 E4 synthetic peptides. A peptide corresponding to amino acids 20-34 (Gly-Ser-Thr-Trp-ProThr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro) of the E4 ORF of HPV 16 (HPV 16 E4 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]) was synthesized by solid-phase techniques using commercially available amino acids and tert-butoxy carbonyl-protecting groups (Peninsula Laboratories, Belmont, CA) (25). A peptide sequence found in both HPV 6 and 11, corresponding to amino acids 33-46 (Asn-Leu-Leu-His-Thr-Pro-Pro-His-Arg-Pro-Pro-Pro-Leu-Cys) of the E4 ORF of HPV 6 was also synthesized.…”

“…Bound (E4 peptide-specific) IgG was eluted in 20 mM glycine buffer, pH 2.5, and neutralized with 30 mM Tris HC1 buffer, pH 8.0. The E4 peptide-specific IgG, the pre-immune IgG, and the IgG fraction of the same immune sera that did not bind the column were assessed for reactivity to the HPV 16 E4 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34], at an initial concentration of 1.4 ug/ml, were then added in serial 1:2 dilutions in blocking buffer containing 0.17% BRIJ 35 (Pierce Chemical Co.), beginning with a dilution ofthe initial antibody preparation of 1:50. Each dilution was tested in duplicate.…”

“…After synthesis, the amino acid compositions and sequences of the peptides were confirmed by amino acid analysis and automated Edman degradation, respectively. For the purpose of covalent coupling of the HPV 16 E4 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] peptide to thyroglobulin, a cysteine residue was added to the peptide's carboxyl terminus during synthesis; conjugation of the peptide to thyroglobulin was then performed using the heterobifunctional cross-linker m-maleimidobenzoyl-n-hydroxysuccinimide ester (MBS) (Pierce Chemical Co., Rockford, IL) (26).…”