Immune Efficacy Of The Vaccine Formulation Of Corynebacterium Pseudotuberculosis After Sonication Associated With RPLD And RCP40

In recent years,with the rapid development of the domestic sheep and goat industry,Caseous lymphadenitis(CLA)has been spreading in many domestic breeding areas,and its prevalence continues to rise.The etiologic agent Corynebacterium pseudotuberculosis is a facultative intracellular bacterium,besides,antibiotics cannot penetrate the capsule formed around the CLA’s granulomatous lesions,in this way,the antibiotic treatment of the disease is diffcult.Surgical drainage treatment is easy to contaminate the animal body surface and environment,which is an important risk factor for the spread of the pathogen,so vaccination can be considered the most efective preventive measure.However,there is no commercial vaccine available in China.The aim of this study is to prepare a multi-component vaccine based on Corynebacterium pseudotuberculosis lysate combined with recombinant phospholipase D(rPLD)and recombinant CP40(rCP40),and then evaluate the immunoprotective efficacy of the vaccine in mice and goats,to verify whether it can be used as an effective vaccine strategy to protect goat CLA,which could provide experimental data for the development of CLA vaccine.In this study,pus samples of suspected CLA were collected from a large-scale dairy goat farm in Baoji,Shaanxi Province for isolation and identification of Corynebacterium pseudotuberculosis,and the pathogenicity of the isolated strains was tested in mice.On this basis,pld and cp40 were cloned and the recombinant expression vectors were constructed and identified,and then transferred into Escherichia coli BL21(DE3)for protein expression.After rPLD and rCP40 were successfully expressed,protein purification,renaturation,western blot analysis,protein concentration and concentration determination were performed.Finally,Corynebacterium pseudotuberculosis lysate was prepared and combined with rPLD and rCP40 to prepare vaccines,and the immune effects were evaluated by immunizing mice and goats,respectively.Results were as follows:1.Three strains of Corynebacterium pseudotuberculosis were successfully isolated from 5pus samples by bacterial isolation,gram staining,microscopy,biochemical identification and16 S r RNA identification,which were pathogenic to mice.Intraperitoneal injection of 0.2 m L of 1.5 × 108 cfu/m L bacterial solution caused acute death of mice,and subcutaneous injection of the same amount of bacterial solution caused abscess lesions similar to CLA’s granulomatous lesions at the injection site.2.The recombinant plasmids p ET-32a-pld and p ET-32a-cp40 were successfully constructed and transferred into Escherichia coli BL21(DE3)receptor bacteria.The positive recombinant plasmid expression strains BL21(DE3)/p ET-32a-pld and BL21(DE3)/p ET-32a-cp40 were screened out.The recombinant proteins rPLD and rCP40 were successfully expressed in the form of inclusion bodies after induction.The expression conditions were optimized,and the recombinant proteins were induced and expressed in large quantities under the best expression conditions.Then the purified recombinant proteins with high purity and good reactogenicity were obtained by Ni-Sepharose affinity chromatography and western blot.The purified rPLD and rCP40 proteins were renatured by urea gradient dialysis and concentrated by ultrafiltration centrifugation.The concentrations of rPLD and rCP40 proteins were 1052 μg/m L and 1150 μg/m L,respectively,determined by BCA protein concentration assay.3.When mice were immunized with different components of vaccines,the corresponding antibodies against the vaccine components were produced in the mice,and there was no significant difference in the level of antibodies against the same immunogenic component among the different vaccine groups(P<0.05),Among them,the three-component vaccine of Corynebacterium pseudotuberculosis lysate,rPLD and rCP40 had the highest immune protection rate(77.8%),which was higher than that of the single lysate component vaccine(55.6%)and the two-component vaccine of rPLD+rCP40(64.7%),in addition,the protection rate of rPLD+rCP40 vaccine was also higher than that of single rPLD vaccine(50.0%)and single rCP40 vaccine(52.9%).High levels of antibodies against the three immunogenic components of the vaccine were produced in goats after twice immunization with the three-component vaccine of lysate +rPLD+rCP40.And the results of challenge test showed that the abscess size of goats in the immunized group(1.90±0.36 cm)was significantly smaller than that of goats in the control group(4.26 ± 1.05 cm)(P<0.05),and the goats immunized with the vaccine could limit the transfer and colonization of the pathogen to body surface tissues other than the challenge injection site and produce abscesses,indicating that the vaccine had a good immune protective effect on goats.In conclusion,Corynebacterium pseudotuberculosis strains were successfully isolated and recombinant PLD and recombinant CP40 were obtained through prokaryotic expression.And the prepared Corynebacterium pseudotuberculosis lysate + rPLD + rCP40three-component vaccine immunized mice and goats achieved good immune protection effect,which can be used as an effective vaccine strategy to protect goat CLA and provide reference for the development of goat CLA vaccine.