J Korean Soc Virol.  1998 Sep;28(3):215-224.

Cloning, Sequencing and Expression in Escherichia coli of Herpes simple virus Type-1 Thymidine Kinase Gene

• Lee HB
• Kim JW
• Kang H
• Cha SC

Abstract

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.

Keyword

Herpes simplex virus type 1; Thymidine kinase gene

MeSH Terms

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*
DNA
Isopropyl Thiogalactoside
Thymidine
Thymidine Kinase