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

Abstract

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 ...

Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative‐stress regulatory gene:implications for sensitivity to isoniazid

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ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex

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Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis

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