Identification Of The Fragment On SpaA Responsible For Immunological Protection From Erysipeolothrix Rhusiopathiae And Epitope Chimeric Plasmid Construction

Erysipelothrix spp., a gram-positive bacterium ditributed worldwidely which is commonly associated with erysipelas disease in swine and some other animals. It contains at least the following two species: Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum. Researches suggested that E.rhusiopathiae is the only causative agent of erysipelas, and it also been called E.rhusiopathiae of swine. Because it was an infectious agent of swine bacteremia can lead to highly lethiferous ratio among pigs, and great ecnomic losses at last. Although attenuated vaccines or bacterin have long been used to control this kind of dieases, itself may partly be involved in the occurrence of the chronic form of erysipelas. And the efficiency of existed vaccines was limited by different serotypes of Erysipelothrix rhusiopathiae. So there is a necessity to develop a more efficient and economical vaccine to control erysipelas disease.To build up a speedy and reliable method for swine erysipelas detecting, this research investigated a novel loop-mediated isothermal nucleic acid amplification (LAMP) method targets to the Spa gene which is solely in the genome of E.rhusiopathiae. The specificity of was LAMP was carried out with 15 Erysipelothrix spp., 5 non- Erysipelothrix spp. and bloods of mice infected with E.rhusiopathiae. Results showed that the target gene was amplified specifically with loop-primer of LAMP under 61°C isothermal condition. And when compared to conventional PCR, the sensitivity of LAMP is 10 times than that of conventional PCR. Therefore, LAMP is applicable for E.rhusiopathiae detection.Recent researches have revealed that the surface protective antigen Spa plays an important part in protection of animals infected by E.rhusiopathiae. According to different amino acid sequences and molecular weights, Spa can be classified into three sorts, SpaA, SpaB and SpaC. Since the region of Spa for immunological protection in different E.rhusiopathiae serotypes remains to be elucidated, in this study, we obtained and purified the N-terminal domain or C-terminal domain of SpaA from C43150 (SpaA-N & SpaA-C) and discussed their protective function respectively. The protection experiment showed that it was the SpaA-N rather than SpaA-C bears the effective protection against infection of E. rhusiopathiae C43150 (p<0.01). The results indicated that SpaA-N should be the major protective domain of SpaA from E. rhusiopathiae C43150.In order to develop a more economical and safer DNA vaccine and discuss its immunological activity, a chimeric DNA plasmid pcDNA3-HPV-16 L1-ΔspaA was constructed based on the predicted B cell epitope of SpaA-N. Then the chimeric plasmid was transfected into 293T cells and the successful expression was identified by RT-PCR and Western blot. ELISA analysis showed that the chimeric plasmid could stimulate antibodies specific to SpaA-N in mice. In addition, bactericidal test in vitro revealed the antiserum from immunized mice could mediate efficient killing of E. rhusiopathiae by addition of complements. This results indicated that the DNA vaccine with chimeric human papillomavirus L1 expressing an epitope of SpaA could induce efficient immune response to E.rhusiopathiae infection in mouse.This study built a speedy LAMP method target to Spa for E.rhusiopathiae detection and provided a selective strategy for developing a more efficient and economical DNA vaccine to control E. rhusiopathiae infection.