[Cloning and expression of a biotinylated multiple-epitope HCV fusion antigen gene].
The aim was to develop a single multiple-epitope fusion antigen which incorporates all of the major immunodominant epitopes from the six functional regions of the HCV genome. A nucleic acid sequence consisting of viral core, E1, E2, NS3, NS4, and NS5 regions was constructed and inserted into the Promega Pinpoint Xa-1 T vector for inducing expression. The protein was expressed in JM109 (DE3) as a fusion protein with a 13 kD biotinlated tag to be used for detection and affinity purification. Immunogenicity and biotinylated tag of the fusion protein were detected by Western blot analysis with positive anti-HCV serum and streptavidin alkaline phosphatase. After purified by Promega SoftLink Soft Release Avidin Resin, the protein was pre-coated on microwell and detected with anti-core, anti-NS3, anti-NS4 and anti-NS5 positive sera by EIA, respectively. The results indicated that the recombinant soluble protein was expressed and labelled with biotin successfully, it reacted with anti-HCV positive serum, and exposed all of the major immunogenic epitopes chosen. In conclusion, this recombinant antigen may be used to design an double antigen sandwich anti-HCV immunoassay.
Li
Bao-Chang
BC
Institute of Blood Transfusion, Academy of Military Medical Sciences, Beijing 100850, China.
Sun
Ping
P
Yang
Shu-Hua
SH
Wang
Quan-Li
QL
D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D019921:Biotinylation; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D016174:Hepacivirus / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology
D000956:Antigens, Viral; D018937:Hepatitis C Antibodies; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins