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首页> 外文期刊>Infection and immunity >Identification of Mycobacterium leprae antigens from a cosmid library: characterization of a 15-kilodalton antigen that is recognized by both the humoral and cellular immune systems in leprosy patients.
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Identification of Mycobacterium leprae antigens from a cosmid library: characterization of a 15-kilodalton antigen that is recognized by both the humoral and cellular immune systems in leprosy patients.

机译：从粘粒文库中鉴定麻风分枝杆菌抗原：麻风病人的体液和细胞免疫系统均识别的15-千达尔顿抗原的特征。

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Screening of the Mycobacterium leprae cosmid library with pooled sera from lepromatous leprosy (LL) patients by a colony immunoblot technique resulted in the identification of about 100 colonies that produced immunologically reactive proteins. Twenty-four of these clones were purified, analyzed, and found to comprise two groups according to the reactivity of the recombinant proteins with LL sera and to the DNA restriction patterns of the recombinant plasmids and cosmids. Proteins specified by clones from group I reacted strongly with LL patients' sera on a Western blot (immunoblot), demonstrating a 15-kDa protein band designated A15. The A15 antigen also reacted with pooled sera from patients with tuberculoid leprosy from the United States and Brazil. Clones from group II did not show any reactive protein band on a Western blot, when reacted with patients' sera. DNAs from cosmids of group II all contain a 10-kb PstI fragment that hybridized to the unique repetitive M. leprae DNA. Sequence analysis of a 1.2-kb fragment containing the entire coding sequence of A15 revealed three open reading frames (ORFs), only one of which (ORF II) contains sufficient genetic information to encode for A15. Part of the A15 gene was found to exist also in a group of lambda gt11:M. leprae clones previously isolated in our laboratory by immunological screening with LL patients' sera. One of the lambda gt11 clones (L8) expresses a beta-galactosidase fusion protein with 89 amino acids from the C terminus of A15. An important result was that the fusion protein was clearly recognized by T cells from leprosy patients. Interestingly, Mycobacterium tuberculosis-stimulated T cells from M. leprae nonresponder (LL as well as borderline tuberculoid) patients were able to respond to the isolated recombinant M. leprae antigen, indicating that nonresponsiveness to M. leprae antigens can be reversible. The sequence of the M. leprae DNA fused to the beta-galactosidase gene of lambda gt11 clone L8 was identical to that of a lambda gt11:M. leprae clone isolated recently that expresses an immunologically reactive fusion protein (S. Laal, Y. D. Sharma, H. K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. Misra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991). Besides the complete sequence of the A15 gene, sequencing data of two flanking ORFs are presented. Downstream from ORF II (A15), ORF III has a high degree of similarity to the genes for tomato ATP-dependent proteases that are members of a larger class of highly conserved proteases ubiquitous among prokaryotes and eukaryotes.(ABSTRACT TRUNCATED AT 400 WORDS)
机译：通过菌落免疫印迹技术用麻风麻风病患者的合并血清筛选麻风分枝杆菌粘粒文库，鉴定出约100个产生免疫反应蛋白的菌落。根据重组蛋白与LL血清的反应性以及重组质粒和粘粒的DNA限制性图谱，对其中的24个克隆进行了纯化，分析和发现，包括两组。 I组克隆指定的蛋白质与LL患者的血清在Western印迹（免疫印迹）上发生强烈反应，显示出一条15 kDa的蛋白带，称为A15。 A15抗原还与来自美国和巴西的结核性麻风患者的合并血清反应。当与患者血清反应时，第二组的克隆在蛋白质印迹上未显示任何反应性蛋白条带。来自第II组粘粒的DNA均包含一个10 kb的PstI片段，该片段与独特的重复性麻风杆菌DNA杂交。包含A15整个编码序列的1.2 kb片段的序列分析显示了三个开放阅读框（ORF），其中只有一个（ORF II）包含足以编码A15的遗传信息。发现一部分A15基因也存在于一组λgt11：M中。以前在我们实验室中通过对LL患者的血清进行免疫筛选而分离出的麻风病克隆。 λgt11克隆之一（L8）表达具有A15 C末端89个氨基酸的β-半乳糖苷酶融合蛋白。一个重要的结果是，融合蛋白被麻风病人的T细胞清楚地识别。有趣的是，来自麻风分枝杆菌无反应者（LL以及交界性结核）患者的结核分枝杆菌刺激的T细胞能够对分离的重组麻风分枝杆菌抗原产生反应，表明对麻风分枝杆菌抗原的无反应性是可逆的。与λgt11克隆L8的β-半乳糖苷酶基因融合的麻风杆菌DNA的序列与λgt11：M的序列相同。最近分离出表达免疫反应性融合蛋白的麻风克隆（S. Laal，YD Sharma，HK Prasad，A.Murtaza，S.Singh，S.Tangri，R.Misra，and I.Nath，Proc.Natl.Acad.Sci （USA 88：1054-1058，1991）。除了A15基因的完整序列，还提供了两个侧翼ORF的测序数据。在ORF II（A15）的下游，ORF III与番茄ATP依赖型蛋白酶的基因高度相似，后者是原核生物和真核生物中普遍存在的一类更高级别的高度保守的蛋白酶的成员（抽象截短于400字）

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