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。
Objective To investigate the activity of motif3 of mycobac- teriophage TM4 in resuscitating dormant mycobacterium tuberculosis on the basis of constructing bacteriophage TM4 motif3 recombinant protein and the dormant model of mycobacterium tuberculosis respectively. Methods The objective motif3 fragment from bacteriophage TM4 was amplified by polymerase chain reaction, and then cloned into the pET- 28a（ ＋ ） vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21 （DE3） , then puri- fied and identified by Western blot. Different concentrations of motif3 were added into dormant cells of mycobacterium tuberculosis respective- ly, then its resuscitation activity and the sterilization effect of isoniazid on resuscitated mycobacterium tuberculosis cells were detected. Results The recombinant motif3 which fused histidine residue was obtained. The objective protein was located at 12.65 ku. Additionally, the resuscitation activity was remarkable when the concentration of motif3 was 1.1 ×104pmol· L-1 . The resuscitated mycobacterium tuberculosis cells were killed by isoniazid. Conclusion The recombinant protein motif3 had the resuscitation activity on the dormant mycobacterium tuberculosis.
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2016年第3期241-244,共4页
The Chinese Journal of Clinical Pharmacology
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)]
关键词
噬菌体TM4
基因表达
结核休眠菌
复苏
bacteriophage TM4
gene expression
dormantmycobacterium tuberculosis
resuscitation
