论文部分内容阅读
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 high purity SsEno recombinant protein and evaluate the effect of SsEno on the anti-phagocytosis of Streptococcus suis by antibody blocking assay to identify its binding activity to human fibrinogen (hFg). Methods The recombinant plasmid HisSsEno was constructed and transformed into E. coli BL21 (DE3). The expression of protein was analyzed by SDS-PAGE after IPTG induction. The target protein was affinity purified by nickel column and verified by Western blot. The rabbit was immunized with hisSsEno to prepare a high titer antisera and the effect of Eno on the anti-phagocytosis of Streptococcus suis was evaluated by the human whole blood cytotoxicity model. HisSsEno and hFg binding activities were identified by ELISA and Far-Western blot. Results The His tag protein prokaryotic expression plasmid was successfully constructed and the highly purified hisSsEno recombinant protein was obtained. SsEno-immunized antisera significantly reduced the viability of the strongly pathogenic Streptococcus suis 05ZYH33 in human whole blood. hisSsEno specifically binds to hFg. Conclusion HisSsEno recombinant protein was obtained. SsEno was found to be a potential anti-phagocytosis factor of Streptococcus suis by antibody blocking and whole-blood-killing model, and hisSsEno could specifically bind to hFg.