Gene expression of NS5a strong antigenic regions of hepatitis C virus in E.coli and detection of their antigenicity.
OBJECTIVE
To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic region at position 2212-2313 by using recombinant proteins.


METHODS
Thirteen representative sequences from HCV genotypes 1 to 6 were selected to design synthetic genes encoding for this antigenic region. These genes were assembled by PCR from synthetic olionucleotides and expressed in E.coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n=61) obtained from patients infected with HCV genotypes 1 through 6.


RESULTS
A comparison of sequences derived from different HCV genotypes showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4% to 72.5%. All but one protein immunoreacted with 62% to 93% of serum samples. Although a variable degree of genotype specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies.


CONCLUSIONS
Different genotype HCV genes were successfully cloned, expressed and purified. Sequence variability has a profound effect on the antigenic properties of the HCV NS5a immunodominant region. This finding should be considered in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.
Dou
Xiao-Guang
XG
Department of Infectious Diseases, 2nd Affiliated Hospital of China Medical University, Shenyang 110004, China. guang40@163.com
Feng
Guo-He
GH
Shi
Li-Lan
LL
Fields
Howard
H
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000378:metabolism*; D005784:Gene Amplification; D015870:Gene Expression; D005838:Genotype; D005982:Glutathione Transferase / Q000235:genetics; D016174:Hepacivirus / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D016056:Immunodominant Epitopes / Q000235:genetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000276:immunology*; D012324:RNA-Dependent RNA Polymerase
D005982:Glutathione Transferase; D007106:Immune Sera; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase