Evaluation of immunogenicity of Streptococcus equi ssp. zooepidemicus surface protein FIPP
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摘要: 【目的】评价马链球菌兽疫亚种(Streptococcus equi ssp. zooepidemicus,SEZ)表面蛋白FIPP(纤维蛋白原和Ig结合蛋白前体)的免疫效力,为预防SEZ感染提供新的候选疫苗抗原。【方法】根据NCBI数据库公布的FIPP基因序列设计引物进行PCR扩增,将扩增片段克隆到原核表达载体pColdⅠ,并转化至大肠杆菌BL21(DE3)感受态细胞,FIPP蛋白通过IPTG诱导在大肠杆菌中表达及纯化,以SDS-PAGE进行鉴定。纯化后的重组蛋白FIPP(rFIPP)对BALB/c小鼠进行免疫,二免14 d后对小鼠进行免疫保护试验并采集血清;解剖感染SEZ小鼠的肝脏、脾脏和肺脏进行病理组织学观察。以酶联免疫吸附试验(ELISA)方法检测小鼠血清中的特异性抗体IgG水平,以蛋白质印迹(Western blotting)方法检测重组蛋白的反应原性,通过全血杀菌试验检测血清的杀菌活性。【结果】通过SDS-PAGE电泳可观察到约96 k D的蛋白片段,与FIPP蛋白的理论分子量相符。重组蛋白rFIPP免疫小鼠后对SEZ攻击的保护率达70.0%。ELISA方法检测结果表明,重组蛋白rFIPP免疫小鼠后可诱导产生特异性抗体免疫球蛋白G(IgG),且极显著高于阴性对照(P<0.01,下同),通过IgG亚型分析,发现IgG1的抗体水平显著高于IgG2a(P<0.05),说明重组蛋白rFIPP免疫后产生的抗体亚型以IgG1为主。全血杀菌试验结果表明,重组蛋白rFIPP超免血清对SEZ具有极显著杀灭作用。组织病理学观察结果显示,重组蛋白rFIPP免疫后能有效减弱小鼠各类器官的病理损伤。【结论】经表达纯化的重组蛋白rFIPP在小鼠感染SEZ模型中具有良好的免疫保护效力,可用作制备SEZ亚单位疫苗的有效候选抗原。Abstract: 【Objective】This study aimed to investigate the immune efficacy of the surface protein FIPP(fibrinogenand Ig-binding protein precursor) of Streptococcus equi ssp. zooepidemicus(SEZ) and to provide a new candidate vaccine antigen for SEZ. 【Method】According to the FIPP gene sequence published in the NCBI database, the primer was designed for PCR amplification, and the amplified fragment was cloned into the prokaryotic expression vector pColdⅠ, and then transformed into Escherichia coli BL21(DE3) receptor cells. The FIPP protein was expressed and purified in E. coli through IPTG induction. Identification by SDS-PAGE. BALB/c mice were immunized with purified recombinant FIPP(rFIPP). 14 d later, the mice were immunologically protected and serum was collected. The liver, spleen and lungs of the mice infected with SEZ were dissected for histopathological observation. Enzyme-linked immunosorbent assay(ELISA) was used to detect specific antibody IgG levels in serum of mice, the reactivity of recombinant protein was detected by Western blotting analysis, and the bactericidal activity of serum was detected by whole blood bactericidal test. 【Result】The protein fragments of about 96 kD were observed by SDS-PAGE electrophoresis, which was consistent with the theoretical molecular weight of FIPP protein. The protection rate of rFIPP against SEZ attack was 70.0% in mice immunized with RFIPP. ELISA assay results showed that rFIPP immunized mice with high titer specific antibody immunoglobulin G(IgG), which was extremely significantly higher than that of negative control(P<0.01, the same below). By IgG subtype analysis, the antibody level of IgG1 was significantly higher than that of IgG2a(P<0.05). These results indicated that IgG1 was the main antibody subtype produced after rFIPP immunization. The results of whole blood bactericidal test showed that rFIPP superimmune serum had an extremely significant killing effect on SEZ. The histopathological observation showed that rFIPP immunization could effectively reduce the pathological damage of various organs in mice. 【Conclusion】In this study,recombinant protein rFIPP obtained by expression purification has good immunoprotective efficacy in a mouse infecting SEZ model and can be used as a valid candidate antigen for SEZ subunit vaccine.
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Keywords:
- Streptococcus equi ssp. zooepidemicus /
- FIPP protein /
- immune protection /
- evaluation
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