摘要
目的制备高纯度的猪链球菌烯醇化酶(SsEno)重组蛋白并通过抗体封闭实验评价SsEno对猪链球菌抗吞噬能力的影响,鉴定其与人纤维蛋白原(hFg)结合的活性。方法构建hisSsEno重组表达质粒,将其转入大肠杆菌BL21(DE3)菌株,IPTG诱导表达后,通过SDS-PAGE分析蛋白表达情况,镍柱亲和纯化目的蛋白并用Western blot法进行验证。用hisSsEno免疫兔制备其高滴度的抗血清并通过人全血杀伤模型评价Eno对猪链球菌抗吞噬能力的影响。采用ELISA和Far-Western blot法鉴定hisSsEno与hFg结合活性。结果成功构建了His标签蛋白原核表达质粒并获得了高纯度的hisSsEno重组表达蛋白。SsEno免疫的抗血清能显著降低强致病的猪链球菌05ZYH33在人全血中的存活能力。hisSsEno能特异地与hFg结合。结论获得了hisSsEno重组表达蛋白,通过抗体阻封和全血杀伤模型发现SsEno是猪链球菌潜在的抗吞噬因子,且hisSsEno能特异性结合hFg。
Objective To prepare the recombinant enolase of Streptococcus suis（ SsEno）,analyze the effect of SsEno on the antiphagocytosis by antibody-blocking assay in the human blood bactericidal model,and identify the human fibrinogen（ hFg）-binding activity of SsEno protein. Methods SsEno gene was amplified using the primers designed according to05ZYH33 genome sequences and cloned into the expression vector pET28 a to construct recombinant plasmids. The plasmids were transformed into E. coli BL21（ DE3） and induced to express by IPTG. The expression level was analyzed by SDS-PAGE. The recombinant proteins were purified by nickel affinity chromatography and identified by Western blotting.High-titer specific antiserum against SsEno was prepared by immunizing rabbits with the purified recombinant proteins. The impact of SsEno on antiphagocytosis was analyzed by antibody-blocking assay in the human blood bactericidal model. In addition,the specific binding activity with hFg was identified by Far-Western blotting and ELISA. Results The prokaryotic expression vector of hisSsEno（ SsEno with His tag） was constructed and high-purity recombinant expressed protein was purified. In specific antibody blocking assay,antiserum against the SsEno significantly decreased the percent of survival bacteria as observed in high virulent strain 05ZYH33. Additionally,hisSsEno was proved to have the specific binding activity with hFg. Conclusion SsEno was found to be a potential antiphagocytic factor of S. suis with a specific binding to hFg,suggesting that SsEno play an important role in antiphagocytosis of S. suis.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第11期1146-1149,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30870091)
