Toxicity And Immunogenicity Of Recombinant ApxⅠ, ApxⅡ, ApxⅢ Toxin Of Actinobacillus Pleuropneumoniae In Mice

Porcine contagious pleuropneumonia (PCP) is an infectious porcine respiratory tract disease causing severe economic losses worldwide in the swine industry, caused by Actinobacillus pleuropneumoniae (APP).Apx I Apx II ApxIII, which can be expressed in vitro and in vivo, are necessary for Actinobacillus pleuropneumoniae to bring clinical symptom and typical lung pathological changes by studing on it's virulence factors. They are main virulent factors and virulent to alveolus macrophages, polymorphonuclear leukocytes, alveolus epithelia and endothelial cells. The study to the virulence factors of the APP showed that the exotoxins (Apx toxins) secreted by APP played an important role in the pathogenesis of porcine pleuropneumonia and were important protective antigens equally.Apx is encoded by the apx operon, which consists of the activator gene apxC, the structural gene apxA and the secretion-apparatus-encoding genes apxB and apxD. Without the apxC, the proteins of the apxA have no hemolytic and cytotoxic, so we clone the gene apxA only and made it express in BL21, then study of toxicity and immunogenicity of recombinant ApxI, ApxII, ApxIII Toxin of APP was performed, Which can provides theoretic evidence for the development App vaccination.The experimentations were done based on the study of our laboratory:1. Cloning and expression the structural gene encoding ApxI, ApxII and ApxIII of APPProkaryotic expression plasmid pET-apxIA was constructed in our laboratory by others, and we got protein in the form of inclusion body after induced by IPTG .Cloning the structural gene of ApxII by PCR according to the sequence of apxIICA (AY232288)of App-7, prokaryotic expression plasmid pET28aIIA was constructed. The N terminal and the C terminal of the structural gene of apxIIICABD were cloned by PCR according to the sequence (L12145) of serotype 2, and we obtained the plasmid pET-28aIIIN-C. After induced by IPTG a high expression of fusion protein was obtained in the form of inclusion body.2. Toxicity of Actinobacillus pleuropneumoniae ApxI, ApxII and ApxIII toxins recombinant proteins to mouseBecause of the virulence of the natural Apx, toxicity of Actinobacillus pleuropneumoniae (APP) ApxI, ApxII, ApxIII toxin recombinant proteins (in tow forms: crude inclusion bodies and purified renatured recombinant protein) were evaluated in mice, and were compared with the toxicity of natural toxin respectively. All kinds of...