Bannantine John P, Hansen Janis K, Paustian Michael L, Amonsin Alongkorn, Li Ling-Ling, Stabel Judith R, Kapur Vivek
National Animal Disease Center, USDA Agricultural Research Service, Ames, Iowa 50010, USA.
J Clin Microbiol. 2004 Jan;42(1):106-14. doi: 10.1128/JCM.42.1.106-114.2004.
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.
开发用于诊断牛副结核病的特异性免疫测定法需要针对副结核分枝杆菌的特异性抗原。然而，由于与构成鸟分枝杆菌复合群的其他分枝杆菌存在基因相似性，尚未发现此类抗原。通过比较基因组学方法，先前已鉴定出副结核分枝杆菌的21个潜在编码序列，在所测试的任何其他分枝杆菌物种（n = 9）中均未出现（J. P. Bannantine、E. Baechler、Q. Zhang、L. Li和V. Kapur，《临床微生物学杂志》40:1303 - 1310，2002年）。在此，我们描述了这些副结核分枝杆菌特异性序列在大肠杆菌中的克隆、异源表达和抗原分析。代表每个独特预测编码区域的核苷酸序列被扩增并克隆到两个不同的大肠杆菌表达载体中，这两个载体编码多组氨酸或麦芽糖结合蛋白（MBP）亲和纯化标签。所有21种MBP融合蛋白均在变性条件下成功纯化，并在用副结核分枝杆菌免疫的兔和小鼠血清进行的免疫印迹研究中进行了评估。这些研究表明，21种基因产物中的5种由副结核分枝杆菌产生且具有抗原性。用来自9头健康牛和10头患有临床疾病的牛的一组血清进行的免疫印迹分析表明，在感染情况下也能检测到相同的5种副结核分枝杆菌蛋白。总体而言，这些研究采用基因组方法鉴定出了在任何其他分枝杆菌中均不存在的新型副结核分枝杆菌抗原。这些发现可能对改进副结核病的诊断产生重大影响。
