Cloning and Expression of NS5 Region of Korean Type Hepatitis C Virus.
- Author:
Dong Pyou HAN
;
Mi Yoon CHANG
;
Jai Myung YANG
;
Taek Youl LEE
;
Won Bae KIM
;
Byong Moon KIM
- Publication Type:Original Article
- MeSH:
Amino Acid Sequence;
Base Sequence;
Blotting, Western;
Clone Cells*;
Cloning, Organism*;
DNA, Complementary;
Electrophoresis;
Hepacivirus*;
Hepatitis C*;
Hepatitis*;
Humans;
Plasmids;
Polymerase Chain Reaction;
RNA, Viral
- From:Journal of the Korean Society of Virology
1997;27(2):115-128
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative a analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and PRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-P-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETA5-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.
