[Expression of the P1B, P2A, P3AB and P3D protein of hepatitis A virus in prokaryotic cell and antigenicity analysis].
OBJECTIVE
To 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.


METHODS
By 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.


RESULTS
Four 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.


CONCLUSIONS
The 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.
Ning
Shang-yong
SY
Laboratory for Hepatitis, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Yi
Yao
Y
Chen
Si-yong
SY
Bi
Sheng-li
SL
D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D030041:Hepatitis A virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D011994:Recombinant Proteins; D014764:Viral Proteins