Cloning and Expression of Hemolytic-toxin from Actinobacillus pleuropneu-moniae and the Immunoprotection in Mice

Abstract:

The ApxⅡA、ApxⅢA、ApxⅣA genes from Actinobacillus pleuropneumoniae serotype 3 and the ApxⅠA gene from Actinobacillus pleuropneumoniae serotype 5 were respectively cloned into the prokaryotic expression vector pGEX-5X-3.Then the recombinant expression plasmids were respectively transformed into E.coli BL 21 and fusion pro-tein expression were induced by IPTG. The expression products were purified by precipitation with ammonium sulfate and chromatography on Sephadex G-200. SDS-PAGE indicated that the productsexpressed at a high level when the recombi-nant E.coli BL21 was induced 2h, joining IPTG to final concentration 1mmol/L. Western blot analysis showed that the expression products had immunogenicity and specificity.Subunit vaccines were made by different purified expression products and Freund's adjuvant. Mice were immunized at 30 days and 45 days with the subunit vaccines. Then the mice were challenged with the APP of serotype 1, 3, 5,7or 10 at 60 days. The result of animal immunoprotection test showed that subunit vaccines (ApxⅠA +ApxⅣA, ApxⅠA + ApxⅢA +ApxⅣA ,ApxⅠA + ApxⅡA + ApxⅢA + ApxⅣA) could offer 58.4%、66.6%、91.7% protection in mice against the challenge of serotype 1, 5 and 7 APP, respectively. These results suggested that the recombinant proteins had good immunogenicity and the subunit vaccine containing four kind of re-combinant proteins could induce better immunoprotection.