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目的通过原核表达分枝杆菌噬菌体TM4 motif3蛋白和构建结核分枝杆菌标准株H37Rv休眠菌模型,检测motif3蛋白对结核休眠菌的复苏作用。方法利用聚合酶链式反应(PCR)技术扩增噬菌体TM4 motif3基序,将PCR产物连接至表达载体p ET-28a(+),重组质粒测序正确后,在大肠杆菌BL21(DE3)中诱导表达,采用非变性方法对表达蛋白进行纯化,并将不同浓度的motif3蛋白加入结核休眠菌模型中,观察其对结核休眠菌的复苏效果及异烟肼对复苏后结核菌的杀灭作用。结果得到融合组氨酸残基的motif3重组蛋白,在12.65 ku处出现目的条带,浓度为1.1×104pmol·L~(-1)的motif3蛋白对结核休眠菌具有明显的复苏作用且复苏后结核菌能被异烟肼杀灭。结论噬菌体TM4motif3蛋白对结核休眠菌具有复苏作用。
Objective To detect the effect of motif3 protein on the recovery of Mycobacterium tuberculosis by expressing TM4 motif3 protein of mycobacterium phage and constructing H37Rv dormant bacterium model of Mycobacterium tuberculosis. Methods The motif TM4 motif3 was amplified by polymerase chain reaction (PCR). The PCR product was ligated into the expression vector p ET-28a (+). After sequencing, the recombinant plasmid was induced to express in E. coli BL21 (DE3) , The expressed protein was purified by non-denaturation method, and different concentrations of motif3 protein were added into the model of tuberculosis dormant bacteria to observe its resuscitation effect on the tuberculosis dormant bacteria and the killing effect of isoniazid on the tuberculosis bacilli after the recovery. Results The motif3 fusion protein with histidine residue was obtained, and the target band was found at 12.65 ku. The motif3 protein with a concentration of 1.1 × 104 pmol·L -1 had a significant recovery effect on N. tuberculosis and the tuberculosis Bacteria can be killed by isoniazid. Conclusion The phage TM4motif3 protein has a resuscitation effect on the tuberculosis dormant bacteria.