Prokaryotic Expression And Immunoprotection Of AdhE Protein Of Actinobacillus Pleuropneumoniae Serotype 7

Actinobacillus pleuropneumoniae(App)is the causative agent of porcine pleuropneumonia.Pleuropneumonia is a highly contagious severe pig disease that has caused huge economic losses to the breeding industry around the world.Porcine pleuropneumonia is characterized by acute hemorrhage and chronic fibrinous pleuropneumonia,which can be transmitted through contact.AdhE is a bifunctional enzyme with catalytic activity of acetaldehyde dehydrogenase and alcohol dehydrogenase.In recent years,a large number of studies have shown that AdhE also plays an important regulatory role in the process of bacterial infection of the host.This experiment was based on the RNA-seq technique result that the differentially expressed genes obtained by the transcriptomes of App serotype 7 and the infected mice lung tissue.The gene AdhE with the highest differential expression in the up-regulated gene was selected as the research object.The AdhE protein encoded by the gene was cloned and prokaryotically expressed,and the immunogenicity of the recombinant protein and its immunoprotective effect on mice were investigated.1.Prokaryotic expression and immunogenicity analysis of App7 type AdhE proteinBy amplifying the AdhE whole gene of the App7 strain,bioinformatics analysis of the encoded protein revealed that the protein was highly conserved.In order to analyze the immunogenicity of the AdhE protein,the pET-32a(+)-AdhE E.coli expression vector was constructed and expressed in E.coli BL21 to successfully express a protein of approximately 110 kDa.Polyclonal antibodies were prepared by immunizing mice with purified rAdhE protein.The results showed that higher IgG antibody levels could be detected in the serum of mice after booster immunization,and the logarithm of antibody titer could reach lg=4.609.Western blot specific detection results showed that polyclonal antibodies contained high levels of anti-rAdhE-specific antibodies,further demonstrating that AdhE is highly immunogenic.2.Analysis of immunoprotection of App7 AdhE proteinIn this experiment,we evaluate whether the App AdhE protein can provide effective immune protection to App-infected mice by the mouse challenge protection experiment?Pathological analysis of lung tissue and monitoring the expression level of cytokines in mouse before infection and after non-lethal dose of App infection 4,8,12,24,48 h.The lethal dose of App serotype 7 or App serotype 1 strain was intraperitoneally injected into the challenged mice.The mice in the PBS group all died within 36 h,and the rAdhE group were able to obtain 90%(App7)or 70%(App1)protection respectively,which both slightly lower than 100% of the vaccine group.Histopathological observations show that compared with the PBS group,the severe lung lesions were interstitial pneumonia and there was a large amount of inflammatory cell infiltration in the tissues.The lung tissue lesions of the rAdhE group were mild,and only a small amount of inflammatory infiltration by neutrophils was observed in the alveolar wall and around the bronchi.However,the number of inflammatory cells in the lung tissue of the rAdhE group was still slightly higher than vaccine group.Cytokine detection results showed that the rAdhE immunized mice can induce IFN-? production in the body before infection,and activate the host's natural immunity;Within 4h of the initial infection,the cytokines of PBS group and rAdhE group showed significantly high expression compared with before infection.The mice stimulated the body to produce a large number of inflammatory cytokines in response to acute inflammation.With the development of the infection course,it was observed that the expression of IL-6,TNF-? and IFN-? in the rAdhE group was significantly inhibited compared with the PBS group.The mice were allowed to control the inflammatory response to a lower level within 12 h of infection.It indicates that AdhE has a good immunomodulatory effect,and it can effectively control the inflammatory reaction after bacterial infection by regulating the expression level of IL-6,TNF-? and IFN-? cytokines in mice.This study demonstrates that the highly conserved AdhE protein has good immunogenicity and immunomodulatory effects,and also has cross-immunoprotective effects against App1 and App7 infection.