Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system.
Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase.
Esumi
M
M
First Department of Pathology, Nihon University School of Medicine, Tokyo, Japan. mesumi@med.nihon-u.ac.jp
Hayashi
N
N
Takahashi
H
H
Shikata
T
T
Moriyama
M
M
Arakawa
Y
Y
Eto
T
T
Nishihara
T
T
Nozaki
C
C
Mizuno
K
K
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D004279:DNA, Viral; D004926:Escherichia coli / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006526:Hepatitis C / Q000097:blood / Q000276:immunology; D018936:Hepatitis C Antigens; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000956:Antigens, Viral; D004279:DNA, Viral; D018936:Hepatitis C Antigens; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C499352:core protein p22, Hepatitis C virus; C069130:nucleocapsid protein, Hepatitis C virus