马腺疫链球菌SrtA的表达及其免疫原性评价

Expression and Immunogenicity of the SrtA of Streptococcus equi subsp. equi

旨在表达马腺疫链球菌(Streptococcus equi subsp.equi)的SrtA蛋白并分析其免疫效果。以该菌基因组为模板,用PCR方法扩增SrtA基因,构建重组质粒pET-28a-SrtA,诱导表达SrtA,并以纯化蛋白免疫小鼠,间接ELISA检测小鼠血清中抗体水平效价及抗体亚型。首次免疫后第45天,对免疫小鼠用S.equi攻毒,测定免疫保护力、菌体载量和脾细胞表面CD4^+T和CD8^+T分子的表达,并对脏器进行病理组织学观察。结果表明,成功表达了SrtA蛋白,SDS-PAGE显示该蛋白分子量为27 kDa。初次免疫45 d后,小鼠血清抗体效价达1∶218 700,抗体亚型以IgG1和IgG2b为主;脾淋巴细胞中CD4^+T和CD8^+T细胞表达量升高;攻毒保护率可达75%;SrtA免疫可降低攻毒小鼠肺、脾、肝、肾组织的荷菌量。SrtA免疫可诱导小鼠产生特异性体液和细胞免疫应答。

This study aims to express the SrtA of Streptococcus equi subspecies equi and evaluate its immunogenicity.The genome of the strain was used as a template and the SrtA gene was amplified by PCR and the recombinant plasmid pET-28 a-SrtA was constructed. The SrtA protein was expressed by induction and the purified protein SrtA was used to immunize mice. The serum antibody level,antibody titer and antibody subtype of immunized mice were detected by indirect ELISA. At 45 d post-first immunization, the immunized mice were challenged with S. equi and challenge protection rate,bacteria loading,cellular immune and histopathology observation were investigated. The results indicated that the 27 kDa recombinant protein was successfully expressed and the mice antibody level was significantly higher than that of the control group. After 45 days of initial immunization,the IgG specific antibody titer was 1∶218 700. The antibody subtype of immunized mice mainly was IgG1 and IgG2 b, and mice had 75% survival rate after challenge. SrtA immunization could reduce the amount of bacteria in lung,spleen,liver and kidney of mice and induce specific humoral and cellular immune responses in mice.