Composition and Immunological Properties of the Protein Fraction of A36, a Major Antigen Complex of <i>Mycobacterium paratuberculosis</i>

Kesel, Myriam De; Gilot, Philippe; Coene, M.; Cocito, Carlo

doi:10.1111/j.1365-3083.1992.tb03092.x

Cited by 41 publications

(58 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Mycobacterium paratuberculosis, the etiological agent of Johne's disease, contains a major antigen complex called A36 which is highly recognized by sera from cattle infected with M. paratuberculosis (6,9). We have recently shown that a 34-kDa protein of the A36 complex is immunodominant and contains B epitopes specific for all tested mycobacteria, including Mycobacterium bovis and Mycobacterium avium (6).…”

mentioning

confidence: 99%

“…The 897-bp open reading frame coded for a novel protein containing specific B epitopes. The occurrence of well-defined hydrophobic and hydrophilic portions suggests the wall location of the protein.Mycobacterium paratuberculosis, the etiological agent of Johne's disease, contains a major antigen complex called A36 which is highly recognized by sera from cattle infected with M. paratuberculosis (6,9). We (18), and the insert (boxed in Fig.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…We have recently shown that a 34-kDa protein of the A36 complex is immunodominant and contains B epitopes specific for all tested mycobacteria, including Mycobacterium bovis and Mycobacterium avium (6). A 500-bp DNA fragment coding for a portion of the 34-kDa protein has been isolated from a Xgtll genomic library ofM.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis

Gilot¹,

Kesel²,

Machtelinckx³

et al. 1993

J Bacteriol

View full text Add to dashboard Cite

The gene coding for an antigenic 34-kDa protein of Mycobacterium paratuberculosis was isolated and sequenced. The 897-bp open reading frame coded for a novel protein containing specific B epitopes. The occurrence of well-defined hydrophobic and hydrophilic portions suggests the wall location of the protein.Mycobacterium paratuberculosis, the etiological agent of Johne's disease, contains a major antigen complex called A36 which is highly recognized by sera from cattle infected with M. paratuberculosis (6,9). We (18), and the insert (boxed in Fig. 1) was sequenced with the universal primers SK and KS (Stratagene) and the T7 sequencing kit used with Deaza G/A T7 sequencing mix (Pharmacia, Uppsala, Sweden).The gene sequence revealed two open reading frames, possibly in phase with the ,-galactosidase gene of Xgtll, at each extremity of the inserted mycobacterial DNA. In one direction, a stretch of 299 bp (nucleotides 1340 to 1638 in Fig. 1) corresponding to a 13.6-kDa polypeptide was found, whereas in the opposite direction there was a 179-bp frame (nucleotides 1832 to 1654 in Fig. 1) coding for a 7.2-kDa polypeptide. The original orientation of the inserted mycobacterial DNA fragment in phage Xgtll-a362 (unidentifiable after its transfer into the sequencing vector) was established as follows. The mycobacterial DNA insert was cloned in both orientations into the expression plasmid pmTNF.MPH, and both reading frames were translated (Fig. 2). This vector allows the expression of cloned genes as fusion proteins with a polypeptide comprising the first 25 amino acids of mouse tumor necrosis factor and a hexahistidine peptide (4). The resulting recombinant plasmids were introduced into E. coli K-12 A H1 (ATCC 33767) by transformation (18). Single * Corresponding author. transformed colonies were grown at 28°C in the presence of tetracycline to an A6. of 0.2 and then heat shocked (4 h, 42°C). Cell lysates were tested, in Western blot (immunoblot) experiments, with rabbit antiserum directed against the fusion protein expressed by phage Xgtll-a362. Restriction analysis of positive clones (pmTNF.MPHa362) indicated that the correct orientation of the mycobacterial DNA fragment in phage Xgtll-a362 involved the transcription of the 299-bp open reading frame, which encoded a 13.6-kDa polypeptide.Confirmation of this result, and of the corresponding nucleotide sequence, was obtained by sequencing the purified recombinant polypeptide. A sonic extract of the induced recombinant bacteria containing pmTNF.MPHa362 was centrifuged (25,000 x g, 15 min, 4°C), and supernatant proteins were precipitated [30% (wt/vol) (NH4)2SO4], dissolved (0.5 M NaCl-15 mM potassium phosphate buffer, pH 5.2), and dialyzed overnight at 4°C (against the same buffer). Guanidine chloride was added (final concentration, 4 M in 50 mM phosphate buffer, pH 7.4), and the recombinant polypeptide carrying six adjacent histidines (Fig. 2) was purified from E. coli proteins by selective interaction between the polyhistidine peptide and a metal chelate adsorbent (Ni...

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis

Gilot¹,

Kesel²,

Machtelinckx³

et al. 1993

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…paratuberculosis possesses a complex cell-wall containing several immunogenic proteins, glycopeptides and glycolipids such as lipoarabinomannan, shared with other mycobacterial species [11,12]. Amongst the protein antigens, protein p34 was identified as immunodominant for bovine B cells [13]. The hydrophilicity profile of p34 identified two distinct regions: a strongly hydrophobic aminoterminal region composed of aminoacids 40-160 and a highly hydrophilic domain corresponding to residues 161-298.…”

Section: Introductionmentioning

confidence: 99%

B‐Cell Epitopes in the Immunodominant p34 Antigen ofMycobacterium aviumssp.paratuberculosisRecognized by Antibodies from Infected Cattle

et al. 2003

View full text Add to dashboard Cite

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) causes Johne's disease, a chronic and fatal enteritis in ruminants. In the last stage of the disease, antibody titres rise and levels of interferon-g decrease, suggesting that the hostimmune response is switching from a T helper 1 (Th1) to a Th2 profile. In infected cattle, the membrane protein p34 elicits the predominant humoral response against M. paratuberculosis. To map the B-cell epitopes of this antigen, affinity-purified bovine antibodies against the carboxy-terminal region of p34 were used to screen a 12-mer phage display library. Several phage clones carrying peptides resembling fragments of p34 were affinity selected. Based on the predicted amino acid sequence, peptides were chemically synthesized, which demonstrated reactivity with serum from naturally infected and p34-vaccinated cattle. Immunization of mice with these peptides elicited an anti-p34 antibody response. Two B-cell epitopes were identified and characterized. Based on the reactivity and the type of immune response elicited, epitope A was determined to be conformational, whereas epitope B was demonstrated to be sequential. Both epitopes were shown to be present in p34 proteins from M. avium ssp. avium or M. paratuberculosis but absent from M. intracellulare, the other member of the M. avium complex. Furthermore, both epitopes were mapped to regions of p34 that display high variability when compared to homologous proteins from other mycobacterial species of public and animal health importance. We hypothesize that these variable regions of p34 may play a role in the immunobiology of M. paratuberculosis infections.

show abstract

Composition and immunogenicity of the polysaccharide components of the thermostable macromolecular antigen group of mycobacterial antigens

Bruneteau

Perret

Vanlinden³

et al. 1992

Med Microbiol Immunol

Self Cite

View full text Add to dashboard Cite

The thermostable macromolecular antigen (TMA) group includes major components of the mycobacterial cell envelope and cytoplasm, which elicit humoral and cellular immune reactions, and seems to play important roles in infectious diseases. The best known member of this group, antigen A60 of Mycobacterium bovis BCG, was previously shown to contain three moieties of polysaccharides, free lipids, and polypeptides. In this work, the TMA polysaccharides of three pathogenic mycobacteria (M. avium, M. bovis and M. paratuberculosis) have been analyzed by coupled gas chromatography-mass spectrometry. In all cases the cores of the TMA complexes were represented by branched glucans of high molecular mass (about 10(6) daltons), for which structural models have been proposed. The immunogenicity of the polysaccharide components from the three TMA was verified with several immunological procedures (immunodiffusion and immunoelectrophoresis of the antigen, and immunoblotting of the corresponding electrofocused immunoglobulins). All tests tallied in showing a negligible immunogenicity of the glucans examined (inability to produce, upon injection, the synthesis of specific immunoglobulins), thus pointing to the protein moiety of TMA as the one responsible for the high immunoreactivity of the complexes.

show abstract

Composition and Immunological Properties of the Protein Fraction of A36, a Major Antigen Complex of Mycobacterium paratuberculosis

Cited by 41 publications

References 28 publications

Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis

Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis

B‐Cell Epitopes in the Immunodominant p34 Antigen ofMycobacterium aviumssp.paratuberculosisRecognized by Antibodies from Infected Cattle

Composition and immunogenicity of the polysaccharide components of the thermostable macromolecular antigen group of mycobacterial antigens

Contact Info

Product

Resources

About