A major immunogenic 36,000-molecular-weight antigen from Mycobacterium leprae contains an immunoreactive region of proline-rich repeats

Thole, Jelle; Stabel, L F; Suykerbuyk, M.E.G.; Wit, Maartje; Klatser, P. R.; Kolk, A. H. J.; Hartskeerl, Rudy A.

doi:10.1128/iai.58.1.80-87.1990

Cited by 45 publications

(24 citation statements)

References 35 publications

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“…The ORF-14 protein has several short repetitive motifs containing proline residues at the C-terminal end, a feature shared among several antigenic proteins from M. tuberculosis and other mycobacteria [11,12,31,32]. It has been reported that three other proline-rich antigenic proteins from M. tuberculosis, MTC 28, 36 kDa and 45/47 kDa antigens [12,31,32], migrate slowly in SDS-PAGE gels compared to their expected molecular sizes. The discrepancies between the calculated and apparent molecular sizes of these antigenic proteins are attributed to the rigidity and reduced mobility of the protein backbone due to the abundance of proline residues.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene

et al. 1999

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A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guérin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF‐10, ORF‐14 and ORF‐15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX‐4T vector system and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti‐GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST‐ORF‐14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF‐14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST‐ORF‐14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF‐14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG‐vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long‐term contacts of TB patients also had antibodies reactive to the ORF‐14 protein. These results suggest that the ORF‐14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.

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Section: Discussionmentioning

confidence: 99%

Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene

et al. 1999

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“…Previously, we described a polymerase chain reaction (PCR) using heat-stable Taq polymerase for the specific detection of M. leprae (3). A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the proline-rich antigen of M. leprae (3,9). With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium when using suspensions of bacilli.…”

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Application of a polymerase chain reaction for the detection of Mycobacterium leprae in skin tissues

et al. 1991

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The polymerase chain reaction (PCR) based on the selective amplification of a 530-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied on sections of fixed or frozen biopsy samples from leprosy patients. A simple procedure for the extraction of DNA from M. leprae in clinical specimens that provided suitable template DNA for amplification was developed. When PCR was applied on frozen sections, positive amplification in samples from all untreated acid-fast bacillus (AFB)-positive patients and in samples from 56% of the untreated AFB-negative patients could be detected, while biopsy samples from patients with skin diseases other than leprosy were all PCR negative. With neutral Formalin-fixed biopsy samples, positive amplification in 92% of the samples from untreated AFB-positive patients and in 61% of the samples from untreated AFB-negative patients could be detected by PCR. Biopsy samples exposed to mercuric chloride or nonbuffered formaldehyde containing fixatives were not suitable for application of PCR. This PCR holds promise as a tool for studies on M. leprae infection.

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“…The nucleotide sequence of Avi-3 shows a 194-amino-acid polypeptide with a calculated molecular mass of 21.5 kDa, which is a little lower than the apparent molecular mass of approximately 27 kDa estimated by SDS-PAGE (1). Such discrepancy has often been observed for other mycobacterial proteins (43,48), especially those which are proline rich (44). The conclusion that the cloned DNA contained a gene for Avi-3 was based on the following evidence: (i) the N-terminal amino acid sequence coincided with that deduced from the nucleotide sequence except for three amino acids, including two ambiguous residues; and (ii) expression of the gene in E. coli resulted in the production of a protein that was recognized by the polyclonal and monoclonal antibodies raised against the Avi-3 antigen, and the recombinant protein had a molecular mass of approximately 23 kDa (Fig.…”

Section: Discussionmentioning

confidence: 95%

Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes

et al. 1992

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The Avi-3 antigen, which is found only in Mycobacterium avium culture sonic extracts, is species specific and results in strong skin test activity in guinea pigs sensitized with heat-killed M. avium. Its gene was cloned by using a previously developed single-probe method and was sequenced. The gene encoded a 194-amino-acid polypeptide with a molecular weight of 21,500. A recombinant Avi-3 antigen expressed in Escherichia coli reacted with monoclonal and polyclonal antibodies raised against the native Avi-3 antigen. To identify epitopes on this protein for immunodiagnostic purposes, various parts of the Avi-3 antigen were expressed as 13-galactosidase fusion proteins, using pUR and pURS expression vectors. The clones screened by both antibody reactivity and T-cell proliferative activity defined fragments with coexisting Band T-cell epitopes. A B-cell epitope (Asn-176 to Ala-186) and two T-cell epitopes (Glu-75 to Ile-86 and Arg-155 to Leu-164) were thus defined. The synthetic polymerized peptides of the T-cell epitopes were proven to elicit a delayed cutaneous hypersensitivity reaction in guinea pigs. This mapping method would be useful in the development of a subunit vaccine consisting of an immunodominant B-cell epitope linked to a T-cell epitope in the vicinity. * Corresponding author. MATERIALS AND METHODS Reagents. Restriction endonucleases, vectors, E. coli JM109 competent cells, and DNA ligation and 7-deaza sequencing kits were purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). [-y-32P]ATP and [a_-32P]dCTP were from Amersham International plc (Amersham, United Kingdom). Peroxidase-conjugated goat anti-mouse immunoglobulin G was purchased from Bio-Rad Laboratories (Richmond, Calif.). The E. coli expression vector pKK233-2 (3) was from Pharmacia (Uppsala, Sweden). Bacterial strains. M. avium ATCC 15769 (B92, serotype 1) was cultivated in Sauton medium (42), and E. coli JM109 and EQ192 (49) were grown in YT broth (0.8% tryptone, 0.5% NaCl, 0.5% yeast extract). Design ofoligonucleotide probes. A purified Avi-3 antigen was analyzed by a 470A gas-phase protein sequencer (Applied Biosystems Inc.) to determine the sequence of the N-terminal 22 amino acids. Two oligonucleotide probes were synthesized automatically with a DNA synthesizer (model 380A; Applied Biosystems Inc.) and purified by two cycles of reverse-phase liquid chromatography. The sequences of single probes 25 and 35 were 5'-TGCAGATCGGCGACCAGTTCCCGGC-3' (corresponding to Leu-3 to Ala-11) and 5'-GACCAGTTCCCG GCCTACGAGCTGACCGCCCITGAT-3' (Asp-7 to Ile-18), respectively. About 100 pmol of purified oligonucleotide probes was labeled with 2P by T4 polynucleotide kinase. The reaction mixture was extracted once with phenol, and labeled probes were purified on NENSORB (Du Pont Co.). These purified probes were used for the hybridization test. Genomic Southern hybridization. M. avium DNA was prepared by the method of Suzuki et al. (42). About 5 ,ug of purified M. avium DNA was completely digested with BamHI, KpnI, and PstI and then fractionated by 0.8% agarose ...

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A major immunogenic 36,000-molecular-weight antigen from Mycobacterium leprae contains an immunoreactive region of proline-rich repeats

Cited by 45 publications

References 35 publications

Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene

Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene

Application of a polymerase chain reaction for the detection of Mycobacterium leprae in skin tissues

Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes

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