Expression And Antigenicity Analysis Of Aer Toxin Of Aeromonas Hydrophila

Aeromonas hydrophlia is a bacterial pathogen which is infective to a wide range of hosts including mollusks, freshwater fishs, amphibians, reptiles, birds and mammals and causes death of animal. Aeromonas hydrophlia also cause human infection, such as acute gastroenteritis, septicaemia, wound infection, otitis media and peritonitis, etc. Following the development, Aeromonas hydrophlia has become a serious problem of fishery farming cultivation, which cause the infection of soft-shelled, carp, eel, river crab, catfish, bullfrog , etc. The pathogenicity of Aeromonas hydrophlia is related to co-virulence factors, such as exogenous toxin, proteinase, fimbria, outer membrane protein, etc, but many reports suggested that exogenous toxin of Aeromonas hydrophlia is the most important virulence factor. At present, it has been confirmed as aerolysin, hemolysin, hemolytic toxin, cytolytic enterotoxin. The names of related exogenous toxins are different, but the structures of the genes are in high homology. Now it is generally recognized as aerolysin. In this study, the Aer toxin gene was cloned and expressed in prokaryotic system and the antigenicity of its recombinant protein was analyzed.Cloning of Aer toxin gene and DNA sequence analysis Sequence encoding the matureprotein (443 aa) of Aer toxin gene of Aeromonas hydrophlia strain AHCS02. which was isolated in Sijiazi reservoir, JI Lin, China, was amplified by PCR, the primers were designed from the aer gene sequence in Genbank (accession No. Ml6495). After being cloned into pMD18T-vector, the amiplified aer gene was sequenced and analyzed.Construction of prokaryotic expression vector and expression in E.coli BL21 (DE3) The amplified Aer toxin gene was linked into prokaryotic expression vector pET28b. Then the recombinant pET28b-aer was transformed into host strain E.coli BL21 (DE3) induced by IPTG . The expressed protein of 53.9KDa was identified by SDS-PAGE. The amount of the expressed protein increased with time extending and the fusion protein was expressed at high level, amount to48.57% of the total protein of E.coli BL21 (DE3) after induced by IPTG(lmmol/L) for 4 hours.Purification of recombinant Aer toxin and antigenicity analysis of recombinant and natural Aer toxins The inclusion body of fusion protein was purified by SDS-PAGE and electrophoresis elution. The natural Aer toxin of Aeromonas hydrophlia strain AHCS02 was purified by ammonium sulfate pricipatition. Both recombinant and natural Aer toxins were used to immunize rabbits to prepare anti-serum. Western blotting showed that the rabbit antibodies raised against the recombinant fusion protein could recognize the natural Aer toxin and the antibodies raised against natural Aer toxin could recognize the recombinant Aer toxin. The results indicated that natural and recombinant Aer toxins had similar antigenicity.