Identification of B cell epitopes of hepatitis C virus RNA dependent RNA polymerase.
The aim of this study was to identify the B cell epitopes of hepatitis C virus (HCV) NS5B RNA dependent RNA polymerase (RdRp). The truncated HCV NS5B protein NS5B-dc21 was expressed in Escherichia coli and its antigenicity was confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) using 130 HCV-positive human sera and 15 negative sera. Antibodies specific to NS5B-dc21 protein were purified by affinity chromatography using sepharose-4B coupled with the recombinant protein. A 12-mer phage displayed random peptide library was screened four rounds with the purified antibodies. Three epitopes were identified from the phage library, which correspond to amino acids 2444-2452, 2521-2528, and 2915-2925 of HCV RdRp. These epitopes were then expressed in E. coli as fusion proteins with phage M13 pIII protein. ELISA demonstrated that two of these epitopes (P4 and P34, corresponding to amino acids 2443-2452 and amino acids 2512-2528, respectively) have good reactivity and sensitivity. Mutagenesis study of P4 peptide showed that this epitope, which is derived from a phage displayed library, exhibited higher affinity with HCV serum than the corresponding original HCV sequences.
Hou
Lihua
L
Department of Applied Molecular Biology, Institute of Microbiology and Epidemiology, No. 20 Dongdajie Street, Fengtai District, 100071, Beijing, PR China. houlihua@hotmail.com
Du
Guixin
G
Tong
Yigang
Y
Wang
Haitao
H
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology*; D016174:Hepacivirus / Q000201:enzymology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000276:immunology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D019151:Peptide Library; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase