Construction of recombinant mycobacteriophage TM4-RpfE / 上海交通大学学报（医学版）

ABSTRACT

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 mycobaeteriophage 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.