Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes.
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 beta-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 B- and 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.
Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan.
R
R
Yamaguchi
K
K
Matsuo
A
A
Yamazaki
M
M
Takahashi
Y
Y
Fukasawa
M
M
Wada
C
C
Abe
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006968:Hypersensitivity, Delayed; D008969:Molecular Sequence Data; D009162:Mycobacterium avium / Q000235:genetics / Q000276:immunology*; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology
D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D000939:Epitopes