The Journal of Korean Society of Virology (대한바이러스학회지)
- Volume 28 Issue 3
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- Pages.215-224
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- 1998
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- 1225-2344(pISSN)
Cloning, Sequencing and Expression in Escherichia coli of Herpes simplex virus Type-1 Thymidine Kinase Gene
- Lee, Hyung-Hoan (Department of Biology & Institute for Genetic Engineering, Konkuk University) ;
- Kim, Jung-Woo (Department of Biology & Institute for Genetic Engineering, Konkuk University) ;
- Kang, Hyun (Department of Biology & Institute for Genetic Engineering, Konkuk University) ;
- Cha, Sung-Chul (Department of Biology & Institute for Genetic Engineering, Konkuk University)
- Published : 1998.09.30
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 fragment of the plasmid pHLA-12, was amplified by polymerase chain reaction (PCR). The 1,131 kb PCR product was cloned into the BamHI and EcoRI sites of pBacPAK9 plasmid and then named pBac-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