Cloning, Sequencing and Expression in Escherichia coli of Herpes simple virus Type-1 Thymidine Kinase Gene.
- Author:
Hyung Boan LEE
;
Jung Woo KIM
;
Hyun KANG
;
Sung Chul CHA
- Publication Type:Original Article
- Keywords:
Herpes simplex virus type 1;
Thymidine kinase gene
- MeSH:
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*
- From:Journal of the Korean Society of Virology
1998;28(3):215-224
- CountryRepublic of Korea
- Language:English
-
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.
