Cloning, sequencing and expressing in E. coli of the thymidine kinase (TK) gene of Herpes simplex virus type-1 (HSV-1) strain F was investigated. The TK gene, located in the BamHI 3.74 kb DNA flagment of the plasmid PHLA-12, was amplified by polymerase chain reaction (PCR). The 1,131 kb PCR product was cloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The TK gene was subcloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The nucleotide sequence of the 1,131 kb TK gene was determined, and the GC content was 65.13%. There were deduced 367 amino acid residues with a total molecular weight of 43 kDa. The weight was confirmed by the protein produced by E. coli M15/pQE-TK on the SDS-PAGE and Western blot. The production of the TK protein in the IPTG induced cells was measured over 4 h. At the end of 1, 2 and 3 h the level increased by 146,204 and 242%, respectively. The amount of the protein at the highest fraction Purified with Ni-NTA resin chromatography was 0.68 ug Per ml. The soluble state TK protein was present in the cytoplasm. In these results the F strain was different in base sequence and amino acid sequence from that of the CL101 strain, which caused difference in their strains.
Amino Acid Sequence ; Base Composition ; Base Sequence ; Blotting, Western ; Chromatography ; Clone Cells* ; Cloning, Organism* ; Cytoplasm ; DNA ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli* ; Escherichia* ; Herpesvirus 1, Human ; Isopropyl Thiogalactoside ; Molecular Weight ; Plasmids ; Polymerase Chain Reaction ; Simplexvirus ; Thymidine Kinase* ; Thymidine*