Cloning and expression of a biotinylated multiple-epitope HCV fusion antigen gene.
- Author:
Bao-Chang LI
1
;
Ping SUN
;
Shu-Hua YANG
;
Quan-Li WANG
Author Information
1. Institute of Blood Transfusion, Academy of Military Medical Sciences, Beijing 100850, China.
- Publication Type:Journal Article
- MeSH:
Antigens, Viral;
genetics;
immunology;
Biotinylation;
Cloning, Molecular;
Enzyme-Linked Immunosorbent Assay;
Hepacivirus;
immunology;
Hepatitis C Antibodies;
blood;
Humans;
Immunodominant Epitopes;
Recombinant Fusion Proteins;
genetics;
immunology
- From:
Journal of Experimental Hematology
2004;12(3):359-362
- CountryChina
- Language:Chinese
-
Abstract:
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.
