Expression of the P1B, P2A, P3AB and P3D protein of hepatitis A virus in prokaryotic cell and antigenicity analysis.
- Author:
Shang-yong NING
1
;
Yao YI
;
Si-yong CHEN
;
Sheng-li BI
Author Information
- Publication Type:Journal Article
- MeSH: Blotting, Western; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Escherichia coli; genetics; Gene Expression; Hepatitis A virus; genetics; immunology; metabolism; Humans; Polymerase Chain Reaction; Recombinant Proteins; immunology; metabolism; Viral Proteins; genetics; immunology; metabolism
- From: Chinese Journal of Experimental and Clinical Virology 2007;21(1):50-52
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo express P1B, P2A, P3AB and P3D cDNA gene fragments in prokaryotic system using thioredoxin fusion expression system; to investigate the antigenicity and application of recombinant protein.
METHODSBy using PCR technique, P1B, P2A, P3AB and P3D gene fragments were cloned. Choosing M47 as the expressive vector, the recombinant plasmid P1B, P2A, P3AB and P3D was constructed and expressed in Escherichia coli after inducing by IPTG. By anion exchange and affinity chromatography, purified recombinant protein was obtained. By using Western Blot analysis and indirect ELISA to detect its antigenic activity.
RESULTSFour recombinant plasmids was proved to be constructed successfully by sequencing and the correct molecular weight of their expression products. Recombinant proteins were obtained in BL21 (DE3) and purified after Ni2+ affinity chromatography. Western Blot analysis and indirect ELISA showed that P2a had specific antigenicity.
CONCLUSIONThe P2a protein expressed in prokaryotic system was proved to have specific antigenicity. The indirect ELISA distinguished 24 positive sera from 24 negative sera. It is very likely that P2a can be an antigen to diagnose acute patients of hepatitis A and differentiate inactivated vaccine-induced immunity from an infection.
