J Korean Soc Virol.  1997 Dec;27(2):115-128.

Cloning and Expression of NS5 Region of Korean Type Hepatitis C Virus

• Han DP
• Chang MY
• Yang JM
• Lee TY
• Kim WB
• Kim BM

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.

MeSH Terms

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
DNA, Complementary
RNA, Viral