Bannantine John P, Paulson Avery L, Chacon Ofelia, Fenton Robert J, Zinniel Denise K, McVey David S, Smith David R, Czuprynski Charles J, Barletta Raúl G
Bacterial Diseases of Livestock Research Unit, USDA ARS National Animal Disease Center, Ames, Iowa 50010, USA.
Clin Vaccine Immunol. 2011 Jan;18(1):105-12. doi: 10.1128/CVI.00297-10. Epub 2010 Nov 17.
Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5' and 3' regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.
副结核分枝杆菌鸟亚种可引起反刍动物的副结核病（JD）。遗传工具的开发以及副结核分枝杆菌鸟亚种基因组测序项目的完成，扩大了抗原发现的机会。在本研究中，我们测定了MAP1152 - MAP1156基因簇5'和3'区域编码的两种蛋白质的血清反应性。MAP1152编码一种PPE蛋白，MAP1156编码一种参与甘油三酯代谢的二酰基甘油酰基转移酶，属于未分类的UPF0089蛋白家族。重组MAP蛋白作为麦芽糖结合蛋白（MBP）融合蛋白在大肠杆菌中过量表达并纯化。免疫印迹分析表明，MAP1152和MAP1156对用活的副结核分枝杆菌鸟亚种细胞免疫的小鼠和兔血清以及来自自然感染牛的样本均有反应性。在免疫印迹试验中，与患有JD的牛的血清相比，MAP1156产生的阳性信号比MAP1152更强。开发了针对重组蛋白的酶联免疫吸附试验，并用于检测来自自然感染和未感染牛的预先分类的阳性和阴性血清样本。除一个样本外，所有样本对MBP - LacZ融合蛋白（阴性对照抗原）均无血清反应性（P > 0.05）。MAP1152对所有阳性血清有血清反应性，但对阴性血清无反应性（P < 0.01）。MAP1156比MAP1152表现出更强且更具变异性的反应性，但在未感染和感染牛之间观察到显著差异（P < 0.05）。此外，反应程度与阳性参考抗原遵循相同趋势。总之，这两种蛋白质在小鼠和兔中具有免疫原性，并且副结核分枝杆菌鸟亚种感染的牛对MAP1152和MAP1156的交叉反应表位产生体液反应。这些发现对诊断、疫苗生产以及阐明JD的免疫发病机制具有潜在应用价值。
