OBJECTIVETo fusionaly express the HAV 3a (located in 1403-1456 aa) and 3d located in 1719-1764 aa cDNA gene fragments in prokaryotic system; to investigate the antigenicity and application of recombinant protein.

METHODSBy using PCR technique, 3a and 3d gene fragments were cloned. Choosing pET-30a as the expressive vector, the recombinant plasmid Pet-3ad was constructed and pET-3ad was expressed in Escherichia coli after inducing by IPTG. By affinity chromatography, purified recombinant protein was obtained. By using Western blot analysis and indirect ELISA to detect its antigenic activity.

RESULTSRecombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion and sequence measurement. Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography. Western blot analysis and indirect ELISA showed that P3ad had specific antigenicity.

CONCLUSIONSRecombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion (Nco I/Hind III) and sequence measurement. Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography. Specific immunoblotting appeared at 18,000 by western blot analysis, which showed the recombinant protein P3ad had specific antigenicity, indirect ELISA further proved its antigenicity.

Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Hepatitis A virus ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology