The Study On Biological Characteristics And Immunogenicity Of SpaA Protein Of Erysipelothrix Rhusiopathiae Isolated From Anhui

Swine erysipelas is a zoonotic disease caused by Erysipelothrix rhusiopathiae,has more than 20 years rarely seen,but in recent years,whether domestic and foreign are swine erysipelas occurred has significantly increased.SpaA protein is the main immune protective antigen for the Erysipelothrix rhusiopathiae and the antigen exists almost in all strong toxicity of Erysipelothrix rhusiopathiae.In this study,the biological characteristics of Erysipelothrix rhusiopathiae isolates from 8 different areas in Anhui were oberseved,the immunogenicity of SpaA protein and its protective immunity in mice was determined,and provide reference for the selection of good subunit vaccine candidate units,in order to provide a theoretical basis for the swine erysipelas prevention and swine erysipelas vaccine research.Both strains were identified by its morphological and culture characteristics,biochemical test,PCR and animal regression test,the drug susceptibility of all the strains were tested,the immune protection test of commercial attenuated vaccine of the G4T10 strain of Erysipelothrix rhusiopathiae was performed.The SpaA gene of Erysipelothrix rhusiopathiae was amplified by PCR method and then cloned into prokaryotic expression vector pGEX-6P-1,expressed by induction of IPTG.The products were analyzed by SDS-PAGE and Western-blotting;The mice were injected with recombinant protein,then the IgG and Th1/Th2 cytokines levels were detected through ELISA after the immunization.The immunogenicity of the proteins was analyzed by attacking the clinical isolated from cases of septicaemia and rash block type of swine erysipelas,then the pathological tissue sections were made to observe the change of pathological tissue.In the present study,twenty nine Erysipelothrix rhusiopathiae were isolated,the strains from 8 different areas showed the similar characteristic in morphology and biochemistry.The strains were highly sensitive to Ampicillin and Cefatriaxone(the ratio were 100%),as following,were Penicillin(93%),Erythomycin(89.7%)and Cefotaxime(75.9%),but less sensitive to thirteen other kinds of antibiotics.The eight strains from different areas were highly pathogenic to mice,the LD50 were between 14.3~2.36*102 cfu/mL.The mice were immuned with commercial attenuated vaccine of the G4T10 strain of Erysipelothrix rhusiopathiae through neck hypodermic injection,then be challenged with 100 LD50 of eight strains from different areas by lumbar injection,the commercial attenuated vaccine can protect mice from infection,the protective ratio was 100%.The results showed that the SpaA gene were expressed in E.coil Rosetta and expressed proteins with the size of 75 kD which could react with Swine erysipelas positive serum;High levels of specific antibodies and high levels of Th1/Th2 cytokines of mice were induced by recombinant protein,the recombinant protein protect from the clinical isolated from cases of septicaemia and rash block type of swine erysipelas attained 100%,compared with the control group,the pathological changes of the protein group were significantly different.The results indicate that the swine erysipelas was increasing in Anhui these years,the strains from different areas showed the similar biological characterization and were sensitive to beta lactam antibiotics,the commercial attenuated vaccine of G4T10 strain of Erysipelothrix rhusiopathiae can certain inducing immune ability of protective reaction.The recombinant proteins possess excellent immunogenicity and showed good protection against infection with the clinical isolated from cases of septicaemia and rash block type of swine erysipelas,can be used as a good candidate unit for a subunit vaccine,to offer theoretical basis for the study on new vaccine of swine erysipelas and prevention and control of swine erysipelas.