分枝杆菌噬菌体重组体TM4-RpfE的构建

发布时间：2018-06-13 10:52

本文选题：分枝杆菌噬菌体 + 噬菌体重组　；参考：《上海交通大学学报(医学版)》2017年07期

【摘要】：目的·构建分枝杆菌噬菌体(TM4)与复苏促进因子(Rpf)的重组体(TM4-RpfE),为联合抗结核药物实现杀灭结核休眠菌、缩短结核化疗疗程奠定实验基础。方法·电转化法将pJV53质粒转入耻垢分枝杆菌,制备重组工程菌;提取TM4基因组DNA;重叠PCR扩增目的融合基因hsp60-RpfE,多步重叠PCR扩增插入长片段HHRH(homologous+hsp60-RpfE+homologous);电转化法将噬菌体DNA与插入长片段HHRH共同转入重组工程菌,挑取单噬菌斑进行PCR和测序验证;SDS-PAGE分析重组噬菌体的蛋白表达。结果·重叠PCR扩增出全长901 bp大小的目的融合基因hsp60-RpfE和1 873 bp大小的插入长片段HHRH;PCR验证重组后的噬菌斑,分别在955 bp和301 bp处出现特异性条带;SDS-PAGE分析显示重组型噬菌体在21 000处出现特异性蛋白条带。结论·分枝杆菌噬菌体重组体TM4-RpfE构建成功,初步验证目的基因RpfE得到表达。
[Abstract]:Objective to construct the recombinant of Mycobacterium bacteriophage TM4) and the resuscitation promoter RpfE (TM4-RpfEN), so as to establish the experimental foundation for the combination of anti-tuberculosis drugs to kill dormancy bacteria of tuberculosis and shorten the course of tuberculosis chemotherapy. Methods the plasmid pJV53 was transferred into Mycobacterium smegmatis by electrical transformation to prepare recombinant engineering bacteria. The genomic DNA of TM4 was extracted, the fusion gene hsp60-RpfE was amplified by overlapping PCR, and the long fragment HHRHH homologous HSP60-RpfE homologousus was amplified by multi-step overlapping PCR. The protein expression of recombinant phage was analyzed by SDS-PAGE. Results the target fusion gene hsp60-RpfE with length of 901bp was amplified by overlapping PCR, and the inserted long fragment HHRHU of 1873 BP was amplified by PCR to verify the plaque of the recombinant phagocytic plaque. SDS-PAGE analysis of specific bands at 955 BP and 301 BP showed that the recombinant phage displayed specific protein bands at 21 000 sites. Conclusion Mycobacterium bacteriophage recombinant TM4-RpfE was successfully constructed and the expression of the target gene RpfE was preliminarily verified.
【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;北京市结核病胸部肿瘤研究所;
【基金】：国家自然科学基金(81570010)~~
【分类号】：R378.911

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