乙酰丙酸-十二烷基硫酸钠对结核分枝杆菌的杀灭作用及其杀菌机制的蛋白组学研究

目的 研究消毒剂乙酰丙酸-十二烷基硫酸钠（LVA-SDS）对结核分枝杆菌标准株H37Rv的杀灭作用，并对其在蛋白质水平的杀菌机制进行研究。方法 按照2002年版卫生部《消毒技术规范》要求，首先采用悬液定量杀菌试验，观察LVA-SDS对结核分枝杆菌H37Rv是否存在杀灭作用；然后采用Label-free定量蛋白质组学技术筛选3次独立重复的实验组（LVA-SDS与结核分枝杆菌H37Rv的菌悬液接触30 min）和对照组的差异表达蛋白，通过生物信息学分析差异蛋白之间的相互作用，利用Western blot验证差异蛋白。结果 20% LVA+2% SDS消毒剂对结核分枝杆菌H37Rv有较好的杀灭作用；筛选差异表达蛋白414种（P<0.05），其中44种蛋白表达上调，370种蛋白表达下调。蛋白相互作用发现异柠檬酸裂解酶（ICL）等14种蛋白处于相互作用网络关键节点。Western blot验证结果显示免疫蛋白印记与定量比较蛋白组学实验结果有较好的可重复性。结论 LVA-SDS对结核分枝杆菌确实存在很好的杀灭作用；差异蛋白的发现有助于了解LVA-SDS对结核分枝杆菌的杀菌机制，为新型抗结核药物和消毒剂的研发提供新的分子标识。

The evaluation of disinfection efficacy of levulinic acid plus sodium dodecyl sulfate for Mycobacterium tuberculosis and its proteomics study of bactericidal mechanism

Objective To investigate the germicidal efficacy of levulinic acid plus sodium dodecyl sulfate (LVA-SDS) for H37Rv and to study its bactericidal mechanism at the protein level. Methods According to the 2002 edition of the Technical Specification for Disinfection, suspension quantitative germicidal test was used to evaluate the germicidal efficacy of LVA-SDS for H37Rv. Then Label-free quantitative proteomics technology was used to screen differential expression proteins from 3 independently replicated experimental groups (LVA-SDS and Mycobacterium tuberculosis H37Rv contacting 30 min) and control groups. Bioinformatics analysis was performed to analyze the interactions between the differential proteins. Differential expression proteins were further verified by Western blot. Results The results showed that 20% LVA+2% SDS had a good killing efficacy on Mycobacterium tuberculosis H37Rv; 414 differential expression proteins were screened (P<0.05), of which 44 proteins were up-regulated and 370 were down-regulated. Protein interactions revealed that 14 proteins such as ICL were at key nodes in the interaction network. The results of Western blot validation showed that the results of immune imprinting and quantitative proteomics were reproducible. Conclusion LVA-SDS has good efficacy for killing H37Rv. The discovery of differential expression proteins can help us understand the bactericidal mechanism of LVA-SDS against Mycobacterium tuberculosis and provide reference for the development of new anti-tuberculosis drugs and disinfectants.