Estrada I C, Gutiérrez M C, Esparza J, Quesada-Pascual F, Estrada-Parra S, Possani L D
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México, D.F.
Int J Lepr Other Mycobact Dis. 1992 Mar;60(1):18-27.
In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segments of the amino acid sequence of proteins from Mycobacterium leprae: 65 kDa, 28 kDa, 18 kDa, and 28 kDa superoxide dismutase, recently renamed antigens 2L, 9L, 12L, and 14L, respectively. They were assayed in the guinea pig model for the induction of a delayed-type hypersensitivity response in M. leprae and BCG-sensitized animals. To sensitize the animals two schemes were used: either a single dose of 5 x 10(9) irradiated or autoclaved whole bacilli, or four weekly intramuscular injections each containing 500 micrograms of soluble extract of M. leprae (MLSE) in incomplete Freund's adjuvant. Because the second scheme used far too much antigen, we decided to use the first scheme for the experiments we report here. DTH reactions of sensitized animals were induced after 30 days with intradermal injections of 5 micrograms of MLSE and with each of the 10 peptides at three different concentrations: 250 micrograms, 100 micrograms, and 0.05 micrograms. All M. leprae-sensitized guinea pigs gave indurations of 10 mm or more with MLSE, which indicates that the animals were sensitized. None of them gave DTH indurations with 250 micrograms or 100 micrograms, but some of them had positive DTH reactions with the 0.05 micrograms doses of the synthetic peptides. This is most likely due to the fact that we have used an outbred strain of guinea pigs. The peptides were also tested at 0.05 micrograms in animals sensitized with BCG. P7 and P10 seem to be nonspecific peptides; the remaining peptides only induced DTH in the M. leprae-sensitized guinea pigs. P3 (segments 65-85 of the 65-kDa protein) induced a positive DTH in 58% of the tested animals. In other experiments, guinea pigs were sensitized with a single injection (500 micrograms) of each of the synthetic peptides. All animals, except those sensitized with P4 and P8, had positive DTH responses when the homologous peptide was used. Those sensitized with P2, P4, P5, P7, and P8 were able to produce indurations when MLSE was used for the induction of the DTH reaction.
在本研究中，我们报道了10种肽（P1 - P10）的合成，这些肽对应于麻风分枝杆菌蛋白质氨基酸序列的一个或几个片段：65 kDa、28 kDa、18 kDa和28 kDa超氧化物歧化酶，最近分别重新命名为抗原2L、9L、12L和14L。在豚鼠模型中对它们进行了检测，以诱导麻风分枝杆菌和卡介苗致敏动物的迟发型超敏反应。为使动物致敏，使用了两种方案：要么单次注射5×10⁹个经辐照或高压灭菌的完整杆菌，要么每周进行4次肌肉注射，每次注射含500微克麻风分枝杆菌可溶性提取物（MLSE）的不完全弗氏佐剂。由于第二种方案使用的抗原过多，我们决定在本文报道的实验中使用第一种方案。致敏动物的迟发型超敏反应在30天后通过皮内注射5微克MLSE以及10种肽中的每一种（三种不同浓度：250微克、100微克和0.05微克）来诱导。所有麻风分枝杆菌致敏的豚鼠对MLSE产生了10毫米或更大的硬结，这表明动物已致敏。它们对250微克或100微克剂量均未产生迟发型超敏硬结，但其中一些对0.05微克剂量的合成肽有阳性迟发型超敏反应。这很可能是因为我们使用的是远交系豚鼠。这些肽在0.05微克剂量下也在卡介苗致敏的动物中进行了测试。P7和P10似乎是非特异性肽；其余肽仅在麻风分枝杆菌致敏的豚鼠中诱导迟发型超敏反应。P3（65 kDa蛋白质的65 - 85片段）在58%的受试动物中诱导了阳性迟发型超敏反应。在其他实验中，豚鼠用每种合成肽单次注射（500微克）进行致敏。当使用同源肽时，除用P4和P8致敏的动物外，所有动物均有阳性迟发型超敏反应。用P2、P4、P5、P7和P8致敏的动物在用MLSE诱导迟发型超敏反应时能够产生硬结。
