重组表达萤火虫荧光素酶结核分枝杆菌的构建及其用于最低抑菌浓度测定的研究  

The research of constructing the recombinant Mycobacterium tuberculosis strain expressing FFLuc and applying it to determine minimal inhibitory concentration

作　　者：李媛媛 陈曦[1] 王彬[1] 付雷[1] 朱慧[1] 陆宇[1] 

机构地区：[1]首都医科大学附属北京胸科医院,耐药结核病研究北京市重点实验室,北京市结核病胸部肿瘤研究所药物研究室,101149

出　　处：《中国防痨杂志》2017年第11期1193-1198,共6页Chinese Journal of Antituberculosis

基　　金：“十二五”国家科技重大专项（2015ZX09102007-015）;北京市医院管理局“登峰”计划（DFL20151501）

摘　　要：目的构建萤火虫荧光素酶（FFLuc）重组表达的结核分枝杆菌菌株，利用其建立抗结核药物最低抑菌浓度（MIC）检测方法，并应用该方法测定目前主要的抗结核药物的MIC。方法（1）以质粒pMV306hsp＋FFLuc为模板，PCR扩增FFLuc基因。构建重组表达质粒pMV361＋FFLuc，电转化至结核分枝杆菌标准株H37Rv中，应用小动物活体成像系统鉴定FFI。tlC表达。比较结核分枝杆菌与重组结核分枝杆菌生长状态并绘制生长曲线。（2）检测不同浓度的异烟肼（INH）、利福平（RFP）、乙胺丁醇（EMB）、莫西沙星（Mfx）、链霉素（Sin）、左氧氟沙星（Lfx）、利奈唑胺（Lzd）作用不同时间的表达荧光量变化，考察在不同药物作用下FFLuc表达的稳定性，建立以FFLuc标记结核分枝杆菌为基础的测定抗结核药物MIC的方法并测定INH、RFP、EMB、Mfx、Sm、Lfx、Lzd等7种药物的MIC。结果（1）pMV361＋FFLuc重组表达载体构建的稳定表达FFLuc的结核分枝杆菌菌株的菌量与荧光表达量（相对荧光强度）呈线性关系[r^2=0．994，lg（y）=0．905×lgCr）＋0．832]。（2）应用新方法检测的7种药物的MIc与微孔培养显色（MABA）法MIC测定结果基本相同。第4天时荧光检测INH、RFP、Mfx、EMB、Sm、Lfx和Lzd的MIC值分别是0．05、0．05、0．09375、4、0．8、0．15、0．15μg／ml；与MABA法检测结果相比，结果在相差1-2倍MIC值的可接受范围内。结论建立了FFLuc重组表达的结核分枝杆菌菌株检测抗结核药物MIC的方法与MABA法相比，所需时间更短，第4天即可检测结果。Objective To construct the recombinant Mycobacterium tuberculosis （MTB） strain that expresses stably high levels of firefly luciferase, and establish the method of detecting minimum inhibitory concentration （MIC） by using the recombinant MTB strain. Methods （1） FFLuc gene （the firefly luciferase encoding gene） was cloned by PCR with the plasmid pMV306hsp＋FFLuc as a template. Then, the recombinant expression vector plasmid pMV361＋ FFLue was constructed and transformed into H37Rv （a MTB strain） competent cells by the elec- tro transformation method. The expression of FFLue was tested by the in vivo imaging system （IVIS） spectrum. Finally, the growth of MTB and recombinant MTB were compared and the growth curves were drawn. （2） The fluo- rescence quantity under different concentrations of isonicotinyl hydrazide （INH）, rifampin （RFP）, ethambutol （EMB）, moxifloxacin （Mfx）, streptomycin （Sin）, levofloxacin （Lfx）, and Levofloxacin （Lzd） in different time was detected, in order to analyze the effect of different drugs on stabilization of FFLuc expression and establish the method of detecting MIC on the basic of the recombinant MTB stain that expresses FFLuc. In addition, the MICs of the seven drugs were tested. Results （1） The density of the recombinant MTB strain was linearly related to the fluorescent expression of FFLuc in the recombinant expression vector pMV361 ＋ FFLuc （r^2 = 0. 994, lg （y）= 0. 905 × lg（x）＋0. 832）. （2） The detected MICs were similar to those obtained by conventional Micro-plate Alamar Blue Assay （MABA） method. The MICs of INH, RFP, Mfx, EMB, Sin, Lfx, and Lzd detected on the fourth day by the new method were 0.05, 0.05, 0. 09375, 4, 0.8, 0.15, and 0.15 μg/ml, respectively. Compared with the results obtained by the MABA method, changes in MICs were in the range of -（1-2） times of MIC, which were acceptable. Conclusion We established a MIC detection method by using recombinant MTB stain harboring FFLuc. Compa

关 键 词：分枝杆菌 结核 微生物敏感性试验 DNA 重组 荧光素酶类 萤火虫 

分 类 号：R392-33[医药卫生—免疫学]