噬菌体TM4 motif3蛋白制备及其复苏结核休眠菌的作用

发布时间：2018-03-05 16:23

本文选题：噬菌体TM　切入点：基因表达　出处：《中国临床药理学杂志》2016年03期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to construct a model of Mycobacterium tuberculosis standard strain H37Rv by prokaryotic expression of TM4 motif3 protein of Mycobacterium tuberculosis phage. Methods the TM4 motif3 motif of bacteriophage was amplified by polymerase chain reaction (PCR), and the PCR product was ligated to the expression vector pET-28a (pET-28a). After the recombinant plasmid was sequenced correctly, the recombinant plasmid was induced to express in E. coli BL21 (DE3). The expressed protein was purified by non-denaturing method, and different concentrations of motif3 protein were added to the dormancy bacteria model. The resuscitation effect of isoniazid on the resuscitation of Mycobacterium tuberculosis and the killing effect of isoniazid on the resuscitation were observed. Results the recombinant protein of motif3 fusion histidine residue was obtained and the target band appeared at 12.65 ku. The motif3 protein at the concentration of 1.1 脳 10 4 pmol 路L ~ (-1) can resuscitate the Mycobacterium tuberculosis dormancy and after resuscitation, it can be killed by isoniazid. Conclusion Phage TM4motif3 protein has a resuscitation effect on the dormancy mycobacterium tuberculosis.
【作者单位】：重庆医科大学附属第一医院呼吸与危重症医学科;
【基金】：国家科技部“十二五”重大专项基金资助项目(2012ZX10003-009) 国家临床重点专科专项经费基金资助项目[卫办医政函2012(949号)]
【分类号】：R378

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