Prokaryotic expression of SclE protein from Streptococcus equi ssp. zooepidemicus and evaluation on its immune efficacy

【Objective】To investigate the immune efficacy of the surface protein SclE of Streptococcus equi ssp.zooepidemicus(SEZ), to provide theoretical basis for prevention of streptococcal disease caused by SEZ.【Method】The SclE gene of SEZ was amplified by PCR and cloned into prokaryotic expression vector pCold I. The vector was transformed into the competent cells of Escherichia coli BL21(DE3), and finally induced by isopropyl-beta-D-thiogalactopyranoside(IPTG), after SDS-PAGE analysis, BALB/c mice were immunized with recombinant protein SclE(r SclE) (100μg/mL, 0.5 mL) by intraperitoneal injection(n=10, the cycle of first and second immunization was 14 d). Serum was collected 14 d after the first and second immunization, and antibody levels and specificity were detected by ELISA and Western blotting. After secondary immunization, the mice were tested for immune protection, and the bacterial load of liver, spleen, lung and kidney after challenge was detected and histopathology was observed.【Result】SDS-PAGE identification showed that rSclE was successfully expressed, and the size of rSclE was about 93 kD, which was consistent with the theoretical value. Western blotting showed that the recombinant protein could bind to SEZ positive control group mice serum specifically(intraperitoneal injection of 0.5 m L of SEZ inactivated vaccine), indicating that rSclE had good immunogenicity. ELISA showed that the specific antibody levels in the serum increased significantly after the secondary immunization(P<0.05, the same below), and the mice could produce high IgG titers, the antibody level of IgG1(0.436±0.031) was significantly higher than that of IgG2a(0.161 ± 0.009). These results indicated that IgG1 was the main subtype of rSclE induced antibody. rSclE immunized mice could significantly inhibit the colonization ability of SEZ in each organ, liver and spleen hemorrhage and inflammatory cell infiltration were alleviated, lung and kidney bleeding and other pathological injuries were alleviated;in the mouse immune challenge protection experiment, 70% mice were protected from lethal dose of SEZ strain.【Conclusion】The successfully expressed rSclE can induce mice to produce SCLEspecific antibodies, and has a good protective effect, and can be used as a potential vaccine candidate antigen for SEZ.