Yamaguchi R, Matsuo K, Yamazaki A, Takahashi M, Fukasawa Y, Wada M, Abe C
Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan.
Infect Immun. 1992 Mar;60(3):1210-6. doi: 10.1128/iai.60.3.1210-1216.1992.
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.
Avi-3抗原仅存在于鸟分枝杆菌培养物的超声提取物中，具有种属特异性，在用热灭活的鸟分枝杆菌致敏的豚鼠中可引起强烈的皮肤试验反应。其基因通过先前开发的单探针方法进行克隆并测序。该基因编码一种分子量为21,500的194个氨基酸的多肽。在大肠杆菌中表达的重组Avi-3抗原与针对天然Avi-3抗原产生的单克隆和多克隆抗体发生反应。为了鉴定该蛋白上用于免疫诊断的表位，使用pUR和pURS表达载体将Avi-3抗原的各个部分表达为β-半乳糖苷酶融合蛋白。通过抗体反应性和T细胞增殖活性筛选的克隆确定了同时存在B细胞和T细胞表位的片段。由此确定了一个B细胞表位（Asn-176至Ala-186）和两个T细胞表位（Glu-75至Ile-86和Arg-155至Leu-164）。T细胞表位的合成聚合肽被证明可在豚鼠中引发迟发型皮肤超敏反应。这种定位方法将有助于开发一种亚单位疫苗，该疫苗由一个免疫显性B细胞表位与附近的一个T细胞表位相连组成。
