| ObjectiveSwine erysipelas(SE)caused by Erysipelothrix rhusiopathiae(ER)is an acute septicaemic infectious disease and a zoonotic disease,manifesting adult swine septicaemia,characteristic skin diseases and chronic polyarthritis,bringing huge economic losses to the pig industry.The incidence has been on the rise in recent years.In order to establish specific detection and efficient prevention and control techniques,the main protective antigen of Erysipelothrix rhusiopathiae,Spa A-N,was selected and purified by prokaryotic recombinant expression to prepare monoclonal antibodies.On this basis,an animal model of swine erysipelas mucosal infection and immunohistochemistry methods of Erysipelothrix rhusiopathiae were established,and the dynamic distribution of Erysipelothrix rhusiopathiae in different organs and tissues was studied by histopathological staining(HE),immunohistochemical staining(IHC)and realtime real-time PCR,which provided a basis for further exploring the infection mechanism and pathogenic mechanism of Erysipelothrix rhusiopathiae,and provided a basis for the establishment of specific detection methods for erysipelas infection,safe and efficient vaccine development and evaluation of immune effects.MethodsIn this study,the genomic DNA of Spa A of Erysipelothrix rhusiopathiae was used as a template,and the 1053 bp gene was obtained after sequence optimization after biosequencing,and p UC/Spa A-N was cloned into the expression vector p GEX-4T-1 after digestion by restriction enzymes Bam H I and Xho I,and p GEX-4T-1/Spa A-N recombinant plasmids were constructed.After the recombinant plasmid was transformed into E.coli BL21(DE3),the recombinant bacteria induced expression by adding IPTG with a final concentration of 0.5 mmol/L,and the protein Spa A-N finally obtained soluble expression and obtained higher purity Spa A-N protein by affinity chromatography purification.The wall protein of Erysipelothrix rhusiopathiae was extracted,and the wall protein was used as an immunogen to immunize BALB/C female mice,using cell fusion,cloning and screening.Then,the specificity tests were performed with Erysipelothrix rhusiopathiae wall protein,p GEX-4T-1 empty carrier protein and whole bacteria protein of Pasteurella bovaceae.The pigs were infected by oral infection of Erysipelothrix rhusiopathiae,and the pigs were dissected 1 d,3 d,5 d,and 7 d after the challenge,and the heart,liver,spleen,lung,kidney,duodenum,tonsils,mesenteric lymph nodes,inguinal lymph nodes,submandibular lymph nodes and other tissues were taken to establish immunohistochemical methods,and RT-PCR,IHC and other methods were used to study the temporal and spatial dynamic distribution of Erysipelothrix rhusiopathiae.Results1.The recombinant strain E.coli BL21(p GEX-4T-1/Spa A-N)was successfully constructed,and the purified Spa A-N protein was obtained after induction expression of column purification.Verified by SDS-PAGE electrophoresis analysis and Western-Blot analysis,successful expression and purification of GST-tagged recombinant proteins were demonstrated.2.A monoclonal antibody against Spa A recombinant protein Spa A-21 D was successfully obtained.The monoclonal antibody can specifically recognize the Spa A-N protein in the body wall protein of Erysipelothrix rhusiopathiae,and does not have a non-specific reaction with Pasteurella bobis whole bacteria protein and GST tags,and the Spa A-21 D heavy chain has been identified as IgG2 b.3.Successfully established an animal model of Erysipelothrix rhusiopathiae mucosa infection in pigs.Experimental pigs aged 3~4 months were orally perfused with 100 ml of 3.1×109 CFU/m L of fresh bacteria solution of Erysipelothrix rhusiopathiae(preservation number CVCC43008),and the infection rate was 100%.4.The IHC detection method of Erysipelothrix rhusiopathiae was successfully established.The conditions were that the monoclonal antibody was diluted 100-fold as a primary antibody and incubated overnight at 4 °C;HRP-labeled goat’s anti-murine Ig G secondary antibody dilution factor was1:400;The closing time is 30 min;The color rendering time is 5 min.5.The distribution characteristics of Erysipelothrix rhusiopathiae in various tissues and organs in pigs were observed 1~7 days after infection.The target organs of the pathogen are duodenum and spleen,mainly localized in the lymph nodes and trabeculae of the spleen,duodenal loose connective tissue and other areas,typical clinical symptoms appear from the 3rd day of challenge,the pathogen load is the highest on the 5th day,and then gradually returns to normal level.Conclusions1.A monoclonal antibody against Spa A recombinant protein Spa A-21 D was successfully obtained,which can be used for the detection of swine erysipelas infection,vaccine development and evaluation of immune effect,and the function of Spa A protein2.Successfully established an animal model of swine erysipelas mucosal infection,which provided a basis for the pathogenesis study of porcine erysipelas and the development of prevention and control drugs.3.The IHC detection method of Erysipelothrix rhusiopathiae was successfully established.This method can be applied to clinical diagnosis or laboratory animal negative pig screening,and serves the research work of epidemic disease diagnosis and further pathogenesis.4.The spatiotemporal dynamic distribution law and tissue tropic characteristics of Erysipelothrix rhusiopathiae in pigs were explored,and in the course of infection,they were mainly localized in the lymph nodes and trabeculae of the spleen,and the duodenal loose connective tissue. |
