Mehra V, Sweetser D, Young R A
Proc Natl Acad Sci U S A. 1986 Sep;83(18):7013-7. doi: 10.1073/pnas.83.18.7013.
A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a lambda gt11 gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic determinants were isolated by screening with monoclonal antibodies, and the sequences of their insert DNAs were determined with a rapid primer-extension method. The amino acid sequence of each determinant was deduced from the minimum overlap of insert DNAs from multiple antibody-positive DNA clones. Amino acid sequences for six different epitopes were elucidated. A peptide containing sequences for one of these epitopes was synthesized and shown to bind the appropriate monoclonal antibody; this antigenic determinant is unique to M. leprae. The approach described here can be used to rapidly elucidate protein epitopes that are recognized by antibodies or T cells.
一种重组DNA表达策略已被用于推导麻风分枝杆菌（麻风病的病原体）单一蛋白质中六个不同抗原决定簇的氨基酸序列。对编码麻风分枝杆菌65 kDa抗原的基因进行了测序，并用该基因的片段构建了一个λgt11基因亚文库。通过用单克隆抗体筛选分离出产生特定抗原决定簇的重组DNA克隆，并采用快速引物延伸法测定其插入DNA的序列。从多个抗体阳性DNA克隆的插入DNA的最小重叠中推导出每个决定簇的氨基酸序列。阐明了六个不同表位的氨基酸序列。合成了一个含有其中一个表位序列的肽，并证明它能结合相应的单克隆抗体；这种抗原决定簇是麻风分枝杆菌所特有的。本文所述方法可用于快速阐明被抗体或T细胞识别的蛋白质表位。
