牛分枝杆菌分子标识物的筛选及在豚鼠体内的评价

Screening of molecular markers of Mycobacterium bovis and preliminary evaluation in guinea pigs

摘要:

【背景】牛结核病(bovine tuberculosis, BTB)是一种由结核分枝杆菌(Mycobacterium tuberculosis)复合体中的牛分枝杆菌(Mycobacterium bovis)引起的慢性传染性疾病，对牛群健康、畜牧业经济和公共卫生构成了重大威胁。【目的】筛选牛分枝杆菌新型诊断分子标识物，以期建立牛结核病新型诊断方法。【方法】利用同位素标记的相对与绝对定量技术并结合文献报道筛选出Mpb70、GroEL2、HspX、DnaK、Mpb83、EsxW、BfrB、Hrp1、EsxL、AtpD和EsxN这11种蛋白质进行制备，并通过豚鼠模型评价其致敏效果。【结果】当重组蛋白量为100 μg时，Mpb70、HspX、Mpb83和EsxN可引起明显的迟发型过敏反应，并且出现破溃。当使用量为50 μg时，Mpb70、HspX、Mpb83、EsxW、EsxL和EsxN可引起明显的迟发型过敏反应。当使用量低至12.5 μg时，则无法引起豚鼠的迟发型过敏反应。对不同蛋白进行组合使用，EsxW/EsxL/EsxN组合的活性最好，可刺激豚鼠产生明显的迟发型过敏反应；EsxW/EsxL、EsxW/EsxN、EsxL/EsxN组合也可引起迟发型过敏反应，但其反应程度不如EsxW/EsxL/EsxN组合。【结论】重组蛋白Mpb70、HspX、Mpb83、EsxW、EsxL和EsxN可引起较好的迟发型过敏反应，具有可替代结核菌素的应用前景。

关键词:豚鼠模型;迟发型过敏反应;重组蛋白;结核菌素

Abstract:

[Background] Bovine tuberculosis (BTB) is a chronic infectious disease caused by Mycobacterium bovis, a part of the Mycobacterium tuberculosis complex, posing threats to cattle health, livestock economies, and public health. [Objective] In order to screen new diagnostic molecular markers of Mycobacterium bovis and establish new diagnostic method for bovine tuberculosis. [Methods] Using isotopic labeling-based relative and absolute quantification techniques and article screening, we identified 11 proteins (Mpb70, GroEL2, HspX, DnaK, Mpb83, EsxW, BfrB, Hrp1, EsxL, AtpD, and EsxN) for further evaluation. These recombinant proteins were then assessed for sensitization effects in a guinea pig model. [Results] At a dose of 100 μg, Mpb70, HspX, Mpb83, and EsxN induced marked delayed-type hypersensitivity (DTH) and rupture. At a dose of 50 μg, Mpb70, HspX, Mpb83, EsxW, EsxL, and EsxN provoked significant DTH. At a dose of 12.5 μg, the proteins did not induce DTH. Among the protein combinations, EsxW/EsxL/EsxN showed the highest activity, triggering robust DTH in guinea pigs. Other combinations such as EsxW/EsxL, EsxW/EsxN, and EsxL/EsxN also induced DTH, though to a lesser extent than EsxW/EsxL/EsxN. [Conclusion] Recombinant proteins Mpb70, HspX, Mpb83, EsxW, EsxL, and EsxN elicited notable DTH, demonstrating the potential as tuberculin substitutes.

Key words:guinea pig model;delayed-type hypersensitivity;recombinant protein;tuberculin