Prokaryotic Expression Of Actinobacillus Pleuropneumoniae Exotoxin Apx? And Its Mediated Cytotoxicity Study

Actinobacillus pleuropneumoniae(APP)is a pathogen causing infectious pleuropneumonia in pigs.The aim of this study was to obtain a biologically active recombinant ApxI protein using E.coli prokaryotic expression system and to conduct a preliminary study on its mediated host cell toxicity.First,bioinformatics analysis was pe rformed on the APP ApxIA gene.ApxIA protein contains 1023 amino acids,and it is a stable hydrophilic protein without transmembrane helix and signal peptide;predicted its secondary and tertiary structure,rich in alpha helix(469,45.85%),irregular curl(284,27.76%),there are also extended chains(193,18.87%)and ?-turns(77,7.53%);it was predicted that the amino acids at position 631-841 are B-cell epitope dense regions.Secondly,APP ApxI polyclonal rat serum antibody was prepared by immunization.The ApxIA' protein was recombinantly expressed,and the immunized mice were subcutaneously immunized three times to obtain immune serum.The serum titer of the serum was 1:102400 by indirect ELISA,and the ApxIA' recombinant protein was specifically identified by western blotting.Finally,expression of recombinant ApxI protein was obtained with he molytic activity and me diated cytotoxicity.The recombinant protein ApxI was obtained by expressing E.coli BL21(DE3)pLysS as a expressing strain and expressing pET32a-ApxICA recombinant plasmid.90?g/ml this protein can completely dissolve 1% sheep red blood cells by hemolysis test,which proves that it has good hemolytic activity;A cell model of cytotoxicity mediated by recombinant ApxI protein using PAM as a research object was established by comprehensive cell morphological changes and CCK-8 assay results;After 50 ?g/ml of ApxI recombinant protein was infected with PAM for 24 h,apoptosis was observed by AO-EB staining.At the same time,the protease activity of Caspase-3 and the transcription level of mRNA were significantly increased.It was confirmed that a low concentration of this protein can mediate cytotoxicity by means of apoptosis.