KMID : 0357419980280030215
|
|
Korean journal of Virology 1998 Volume.28 No. 3 p.215 ~ p.224
|
|
Cloning, Sequencing and Expression in Escherichia coli of Herpes simple virus Type-1 Thymidine Kinase Gene
|
|
Hyung Boan Lee
Jung Woo Kim/Hyun Kang/Sung Chul Cha
|
|
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 0.68 p,g 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,
|
|
FullTexts / Linksout information
|
|
|
|
Listed journal information
|
|
|
|