Immunoprotective Efficacy Of Escherichia Coli-derived Outer Membrane Vesicles Displaying GalT Protein Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP)is the causative agent of porcine contagious pleuropneumonia(PCP),a severe respiratory disease which has caused serious economic losses in the swine industry worldwide.This study aims at using Escherichia coli outer membrane vesicles(OMVs)to deliver APP GalT protein antigen and assessing the potential of recombinant OMVs as novel vaccine candidate by evaluating their immunoprotective efficacy.Firstly,we constructed the recombinant E.coli strain expressing ClyA-GalT fusion protein.ClyA(909bp)and galT(1050bp)gene fragments were inserted into pBAD18-Cm and subsequently transformed into Escherichia coli strain JC8031 as the host bacteria.After the 5h induction of 0.2% L-arab,an overexpression band of ~75KD could be detected in SDS-PAGE and Western-blotting assays.Mass spectrometry identified the band as the fusion protein of ClyA and GalT.Secondly,we established the protocol of isolating E.coli OMVs delivering APP GalT protein.The suspension of recombinant OMVs could be harvested from the supernant of recombinant E.coli culture induced about 20 h,then after low-speed centrifugation,100 KD ultra-filtration and 120,000×g ultracentrifugation of 3h processes.The photograph of TEM showed that the diameter of OMVs are between 20 nm and 150 nm,which is consistent with previous reports.Western-blotting showed that GalT protein was successfully delivered to the OMVs.Finally,we evaluated the immunoprotective efficacy of recombinant OMVs delivering GalT protein.We assessed the humoral immune,cellular immune,protective rate and pathological changes in mouse model.Results showed that immunized mice could secret high titer specific IgG antibody(1:204800),lymphocyte proliferation and cytokine secretion level increased significantly.APP challenge assay showed that GalT-OMVs could confer 87.5% and 50% protective rate in active immune and passive immune assays,respectively.Results of pathological and IHC analysis showed that GalT-OMVs could reduce the pathological changes and neutrophil infiltration in mice lungs.