Production of Mycobacterium bovis Antigens Included in Recombinant Occlusion Bodies of Baculovirus.

IF 1.2 Q2 Biochemistry, Genetics and Molecular Biology
Luciana Villafañe, Marina Andrea Forrellad, María Gabriela López, Sergio Garbaccio, Carlos Garro, Rosana Valeria Rocha, María Emilia Eirin, Mahavir Singh, Oscar A Taboga, Fabiana Bigi
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引用次数: 3

Abstract

Bovine tuberculosis (bTB) is a disease produced by Mycobacterium bovis that affects livestock, wild animals, and humans. The classical diagnostic method to detect bTB is measuring the response induced with the intradermal injection of purified protein derivative of M. bovis (PPDb). Another ancillary bTB test detects IFN-γ produced in whole blood upon stimulation with PPDb, protein/peptide cocktails, or individual antigens. Among the most used M. bovis antigens in IFN-γ assays are the secreted proteins ESAT-6 and CFP-10, which together with antigen Rv3615c improve the sensitivity of the test in comparison to PPDb. Protein reagents for immune stimulation are generally obtained from Escherichia coli, because this bacterium produces a high level of recombinant proteins. However, E. coli recombinant antigens are in general contaminated with lipopolysaccharides and other components that produce non-specific IFN-γ secretion in in vitro assays. In this work, we produced the relevant ESAT-6, CFP-10, and Rv3615c M. bovis antigens as fusions to the polyhedrin protein from the baculovirus AcMNPV. We obtained chimeric proteins effectively incorporated to the occlusion bodies and easily purified the recombinant polyhedra with no reactive contaminants. In an IFN-γ assay, these fusion proteins showed equivalent sensibility but better specificity than the same M. bovis proteins produced in E. coli.

杆状病毒重组闭塞体含牛分枝杆菌抗原的制备。
牛结核病(bTB)是一种由牛分枝杆菌产生的疾病,影响牲畜、野生动物和人类。检测bTB的经典诊断方法是皮内注射纯化的牛分枝杆菌蛋白衍生物(PPDb)引起的反应。另一个辅助的bTB测试检测全血在PPDb、蛋白/肽鸡尾酒或单个抗原刺激下产生的IFN-γ。IFN-γ检测中使用最多的牛支原体抗原是分泌蛋白ESAT-6和CFP-10,它们与抗原Rv3615c一起,与PPDb相比,提高了检测的敏感性。用于免疫刺激的蛋白质试剂通常从大肠杆菌中获得,因为这种细菌产生高水平的重组蛋白。然而,在体外实验中,大肠杆菌重组抗原通常被脂多糖和其他产生非特异性IFN-γ分泌的成分污染。在这项工作中,我们制作了相关的ESAT-6、CFP-10和Rv3615c M. bovis抗原,作为与杆状病毒AcMNPV多面蛋白的融合体。我们得到的嵌合蛋白可以有效地结合到封闭体中,并且很容易纯化重组多面体,没有活性污染物。在IFN-γ实验中,这些融合蛋白与大肠杆菌中产生的相同的牛支原体蛋白具有相同的敏感性,但具有更好的特异性。
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来源期刊
Journal of Molecular Microbiology and Biotechnology
Journal of Molecular Microbiology and Biotechnology 生物-生物工程与应用微生物
CiteScore
3.90
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: We are entering a new and exciting era of microbiological study and application. Recent advances in the now established disciplines of genomics, proteomics and bioinformatics, together with extensive cooperation between academic and industrial concerns have brought about an integration of basic and applied microbiology as never before.
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