Lamb F I, Kingston A E, Estrada I, Colston M J
Laboratory for Leprosy and Mycobacterial Research, National Institute for Medical Research, London, United Kingdom.
Infect Immun. 1988 May;56(5):1237-41. doi: 10.1128/iai.56.5.1237-1241.1988.
The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gt11 clone into the high-copy-number plasmid pUC8. Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea. The ability of E. coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd. A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans. Mice immunized with killed M. leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.
编码麻风分枝杆菌免疫显性65千道尔顿抗原的基因从λgt11克隆亚克隆到高拷贝数质粒pUC8中。含有这些重组体的大肠杆菌产生大量抗原，该抗原在尿素存在下通过聚丙烯酰胺凝胶电泳纯化。通过构建另外的克隆证实了大肠杆菌识别分枝杆菌启动子的能力，在这些克隆中该基因两侧是来自噬菌体fd的转录终止子。采用类似方法证明了该基因在变铅青链霉菌中的表达。用灭活的麻风分枝杆菌免疫的小鼠对纯化的65千道尔顿蛋白表现出细胞介导的免疫反应性，该蛋白刺激体外淋巴细胞增殖和体内迟发型超敏反应。
