Posttranslational Modification And Biological Activity Of Exotoxin ApxⅣA Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is a severe, contagious pulmonary disease. PCP can lead to mass death of pigs and has brought serious economic loss to the global pig industry. Apx extoxins are the most important pathogenic factors of APP. APP can produce four types of Apx extoxins, including ApxⅠ, ApxⅡ, ApxⅢ and ApxⅣ. The activity of ApxⅠ, ApxⅡ and ApxⅢ depend on the protein that encoded by apxⅠC, apxⅡC and apxⅢC. The activity and activation mechanism of ApxⅣ are lack of study, while the protein encoded by the upstream gene orf1 may activate ApxⅣ. The object of this study is ApxⅣA which is the structure protein of ApxⅣ and the biological activity of ApxⅣA under the condition of posttranslational modification was researched. The following results were obtained successfully.1. Expression and purification of ApxⅣA:The prokaryotic expression recombinant plasmid pET-apxⅣA, pET-orf1-apxⅣA, pET-apxⅠC-apxⅣA and pET-apxⅡC-apxⅣA were constructed by homologous recombination. The recombinant protein ApxⅣA, ORF1-ApxⅣA, ApxIC-ApxⅣA and ApxⅡC-ApxⅣA were purified respectively.2. The hemolytic activity of ApxⅣA:Hemolysis and synergistic hemolysis test were taken in this study. Meanwhile, sheep erythrocytes and porcine erythrocytes were used as contrast. As the result, ApxⅣA, ApxⅠC-ApxⅣA and ApxⅡC-ApxⅣA had no hemolytic activity. ORF1-ApxⅣA showed CAMP effect and weak hemolytic activity, and the hemolytic activity to porcine erythrocytes was a little stronger than that to sheep erythrocytes. Through this study, it revealed that ApxⅣA showed hemolytic activity when co-expressed with ORF1.3. The effect on the vitality of SJPL cells by ApxⅣA:SJPL cells were treated with recombinant protein ApxⅣA, ORF1-ApxⅣA, ApxIC-ApxⅣA and ApxⅡC-ApxⅣA, respectively. As the result, ORF1-ApxⅣA could inhibit the growth of SJPL cells and make SJPL cells shrink and split. Furthermore, ORF1-ApxⅣA was able to reduce the vitality of SJPL cells significantly and this action weakened as the concentration decline of ORF1-ApxⅣA. Through this study, it revealed that ApxⅣA could make effect on the vitality, growth and morphology of SJPL cells when co-expressed with ORF1.4. The effect on the apoptsis of SJPL cells by ApxⅣA:SJPL cells were induced by recombinant protein ORF1-ApxⅣA in different time and concentration, then stained by FITC and PI. The apoptosis of SJPL cells was detected by a flow cytometer. The results showed that ORF1-ApxIVA could not induce apoptosis of SJPL cells.