Study On Surface Protective Antigen A Of Erysipelothrix Rhusiopathiae As Subunit Vaccine And DNA Vaccine

Background and Objective: Erysipelothrix rhusiopathiae, the cause of swine erysipelas and human erysipeloid, also can cause a variety of diseases in other animals, which continues to be a major cause of economic loss in wide world pig production and threat to human health. Swine erysipelas can occur as an acute septicemia or chronic disease with development of arthritic lesions and endocarditics. Presently, live-organism vaccine, bacterin, or antiserum has been widely used for prophylaxis and treatment of swine erysipelas. They can greatly avoid the spread of swine erysipelas, but their effectiveness depends on the susceptibility of the animal to the vaccine. The maternal antibody leads the piglet unsusceptible to live-organism vaccine. Recent research has indicated that the current vaccine was not effective on preventing the chronic swine erysipelas and to some extend increased the incidence of chronic swine arthritis. In addition, these vaccines themselves are not very stable .Thereby, it is considered to be necessary to develop more effective, cheap and safe vaccine. This research was to develop a new generation of subunit vaccine and DNA vaccine which can be implicated in Erysipelothrix rhusiopathiae control.Methods: In this study, we successfully cloned N-terminal region of spaA gene from the strain XJ-1249 which was isolated in Xinjiang province. Then, the gene was subcloned into the expression vector pGEX-4T-1, forming the recombinant plasmid pGEX-spaA-N. The recombinant protein was expressed in E.coli BL21 and purified as a GST-tagged protein.Kunming White mice were inocubated subcutaneous at multiple sites with the purified protein at 0, 14 and 28 days. At the same time, the level of antibody was detected by ELISA. Two weeks after the last inoculation, all mice were challenged with the lethal strain 140 in about 1.5×10~5 Erysipelothrix rhusiopathiae, the clinical symptoms and the survival of mice were observed in the following days .For construction of spa DNA vaccine, the spaA gene was cloned and was subcloned into the eukaryotic expression vector pcDNA3.1. Then, the recombinant plasmid of pcDNA3-spaA was transfected into Hela cells Hela in vitro with lipofectin. After 48 hours, the expression of spaA gene on mRNA level was detected by RT-PCR. The Kunming White mice were immunized with the plasmids pcDNA3-spaA intramuscularly, and the immunized effect was detected by ELISA.Result :(1) The investigation of subunit vaccine. The mice immunized with recombinant protein r-SpaA-N were protected from the infection of Erysipelothrix rhusiopathiae, but mice in control groups all died after mice challenge. We can detect the antibody to SpaA-N in r-SpaA-N-immunized group by Western Blot and ELISA. The titer of antibody is about 1:150000. (2) The investigation of DNA vaccine. In this study, we have successfully constructed the DNA vaccine of swine erysipelas and detected the expression of spaA gene on mRNA level .However, after inoculation of the DNA vaccine, the antibody to protein SpaA couldn't be detected in vivo by ELISA.Conclusion: The recombinant protein SpaA-N can effectively protect mice from the infection of Erysipelothrix rhusiopathiae, which shows it has a potential function on the development of vaccine against swine erysipelas. The DNA vaccine just expressed on mRNA level in vitro, it was not show humoral reaction in vivo, which need to be improved in furture study.