Molecular cloning and expression of hepatitis C virus core protein and production of monoclonal antibodies to the recombinant protein.
The gene encoding nucleocapsid (core) protein of hepatitis C virus (HCV) was isolated from a Thai blood donor infected with HCV genotype 1b. The nucleotide sequence of this clone showed a high degree of homology to that of 4 HCV strains isolated from other Thai blood donors as well as that of the HCV prototypes of genotypes 1a, 1b, 2a and 3a. The entire region of the core gene was cloned into an expression plasmid pGEX-3X to be expressed as a fusion protein with glutathione-S-transferase (GST). E. coli transformants containing this plasmid did not express the fusion protein. However, GST-HCV core fusion protein could be produced when the core gene was truncated at the 3' end resulting in a gene encoding only the first 123 amino acid residues of the core protein. This fusion protein was insoluble in standard buffers, but could be solubilized by sarkosyl and thus subsequently purified using glutathione-Sepharose 4B. The purified fusion protein was immunogenic and could react with antibodies from blood donors infected with all genotypes of HCV found in Thailand. In addition, two murine hybridoma clones secreting monoclonal antibodies specific to the recombinant HCV core protein were produced. The purified HCV core protein and the monoclonal antibodies to the recombinant protein will be useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively.
Songsivilai
S
S
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Dharakul
T
T
Kunkitti
R
R
Thepthai
C
C
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D005838:Genotype; D016174:Hepacivirus; D006526:Hepatitis C / Q000453:epidemiology; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013785:Thailand / Q000453:epidemiology; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014758:Viral Core Proteins