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首页> 外文期刊>Infection and immunity >Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.
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Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

机译：鉴定包含B细胞和T细胞表位的35千株结核分枝杆菌蛋白。

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Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.
机译：通过使用结核病患者的血清和识别仅存在于结核分枝杆菌复合体上的分枝杆菌35-千蛋白的鼠单克隆抗体H61.3作为探针，在λgt11表达载体中筛选结核分枝杆菌基因组DNA文库。存在于粗裂解物中的重组β-半乳糖苷酶融合蛋白诱导结核性胸膜炎患者的T淋巴细胞增殖。由于重组插入片段包含一个内部EcoRI限制性酶切位点，因此可以鉴定两个片段，一个片段位于lacZ基因的近端，大小为1.7 kb（kb），另一个片段位于lacZ基因的远端，大小为2.2 kb。 Southern印迹分析表明，它们都与结核分枝杆菌和牛分枝杆菌的基因组DNA杂交，但不与其他分枝杆菌种的DNA杂交。为了进行广泛的免疫学研究，β-半乳糖苷酶融合蛋白的量非常低，我们将1.7kb的片段融合到了表达载体pEx34中编码噬菌体MS2 DNA聚合酶的基因的N端部分。融合蛋白已部分纯化，随后的蛋白质印迹（免疫印迹）和T细胞增殖实验证实了B细胞和T细胞分枝杆菌表位的存在。此外，为了分离包含35-千达尔顿基因的染色体区域，我们通过克隆15至20 kb的外源DNA在lambda 2001载体中构建了另一个分枝杆菌基因组文库。该文库的筛选是通过使用1.7和2.2kb重组片段作为探针进行的。确定了一些克隆的限制性图谱。

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