分枝杆菌噬菌体重组体TM4-RpfE的构建

Construction of recombinant mycobacteriophage TM4-RpfE

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。

Objective · To construct recombinant mycobacteriophage TM4-RpfE to lay a foundation for experimental research about how to eradicate Mycobacterium tuberculosis in combination with anti-tuberculosis drugs, and how to shorten treatment for tuberculosis ultimately. Methods · Electrotransformation was used to introduce pJV53 plasmid into Mycobacterium smegmatis to prepare recombinant engineering bacteria. After amplification of hsp60-RpfE fusion gene by overlap PCR, a long gene fragment （homologous ＋hsp60-RpfE＋homologous, HHRH） was amplified by multi-step overlap PCR. The DNA of mycobacteriophage TM4 and HHRH fragment were cotransfected into the recombinant engineering bacteria by electrotransformation, then the recombinant phage from the single primary plaques were confirmed by PCR and sequencing. SDS-PAGE was used to analyze the protein expression in recombinant phage. Results · The hsp60-RpfE fusion gene at the length of 901 bp and HHRH fragment at the length of 1 873 bp were identified by overlap PCR. The PCR product produced 955 bp and 301 bp DNA bands in the first generation plaques colony. SDS-PAGE analysis showed a specific protein band at 21 000 in the recombinant phages. Conclusion · The recombinant mycobacterium phage TM4-RpfE was successfully constructed and the expression of target gene RpfE was initially verified.