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首页> 外文期刊>Journal of Clinical Microbiology >Expression and Immunogenicity of Proteins Encoded by Sequences Specific to Mycobacterium avium subsp. paratuberculosis
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Expression and Immunogenicity of Proteins Encoded by Sequences Specific to Mycobacterium avium subsp. paratuberculosis

机译：鸟分枝杆菌亚种特异序列编码的蛋白质的表达和免疫原性副结核病

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The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium avium subsp. paratuberculosis. However, because of genetic similarity to other mycobacteria comprising the M. avium complex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. avium subsp. paratuberculosis that are not represented in any other mycobacterial species tested (n = 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol. >40:1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of these M. avium subsp. paratuberculosis-specific sequences in Escherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coli expression vectors encoding polyhistidine or maltose binding protein (MBP) affinity purification tags. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. avium subsp. paratuberculosis. These studies showed that 5 of the 21 gene products are produced by M. avium subsp. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same five M. avium subsp. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. avium subsp. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.
机译：牛约翰尼氏病诊断的特异性免疫测定方法的发展需要禽分枝杆菌亚种的特异性抗原。 肺结核。但是，由于与构成 M的其他分枝杆菌的遗传相似性。鸟复合体，未发现此类抗原。通过比较基因组学方法，21个潜在的M编码序列。 avium 子空间先前已鉴定出在任何其他分枝杆菌种（ n = 9）中均不存在的副结核病（JP Bannantine，E.Baechler，Q.Zhang，L.Li和V.Kapur，J.Clin.Microbiol。> 40： 1303-1310，2002）。在这里，我们描述了这些 M的克隆，异源表达和抗原分析。 avium 子空间大肠杆菌中的副结核特定序列。代表每个独特的预测编码区的核苷酸序列被扩增并克隆到两个不同的 E中。编码聚组氨酸或麦芽糖结合蛋白（MBP）亲和纯化标签的大肠杆菌表达载体。所有的21种MBP融合蛋白均在变性条件下成功纯化，并在免疫印迹研究中用来自兔和经 M免疫的小鼠的血清进行了免疫印迹研究。 avium 子空间肺结核。这些研究表明21种基因产物中有5种是 M产生的。 avium 子空间肺结核并且具有抗原性。用9名健康牛和10名患有临床疾病的牛的血清进行的免疫印迹分析表明，相同的5个 M。 avium 子空间在感染的背景下还可以检测到肺结核蛋白。总体而言，这些研究已使用基因组学方法来鉴定新型 M。 avium 子空间在其他分枝杆菌中不存在的肺结核抗原。这些发现可能会对改善约翰尼氏病的诊断产生重大影响。

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