Herpes simplex virus type 1 thymidine kinase gene expression in Escherichia coli

Abstract

The HSV-1 thymidine kinase (TK) gene of Herpes simplex virus was inserted into plasmids pBR322, pMOB45 and pHAlO. The recombinant hybrid plasmids were used to transfect a tdk − mutant of Escherichia coli (Ky895) and the synthesis of the viral TK in the bacterial host was studied. Recombinant plasmids containing the entire BamHI- BamHI DNA fragment carrying the viral TK gene and the upstream sequences containing its promoter were able to produce the thymidine kinase, but removal of the BamHI- BglII DNA fragment containing the viral promoter of the TK gene resulted in enhanced expression of the viral gene, originating from the pBR322 tet r gene promoter. When the BglII- BamHI DNA fragment containing the viral TK gene was cloned in a plasmid with temperature-dependent copy number control (pMOB45), expression of the TK gene was enhanced fourfold by the temperature shift. Cloning of the HSV-1 TK gene in plasmid pHAlO showed that the λ p L promoter allowed transcription of the viral gene but not the synthesis of active TK. The BamHI- BglII DNA fragment containing the viral upstream sequences with the promoter of the TK gene was found to contain transcription-termination sequences that prevented expression of the λ kil gene of pHAlO in the presence of λ N function.