肺炎链球菌蛋白PspA诱导人类单核细胞生产炎症介质和趋化因子的研究

发布时间：2018-08-24 17:37

【摘要】：目的原核表达肺炎链球菌表面的毒性蛋白(pneumococcal surface protein A,PspA)并加入到人类单核细胞的培养基中,检测该蛋白对人类单核细胞释放炎性细胞因子和趋化因子的影响。方法将重组质粒pET-32a(+)/PspA转化到大肠杆菌BL21(DE3)中,经IPTG诱导重组菌表达TRx-His-PspA融合蛋白并纯化;通过肠激酶切掉融合蛋白的TRx-His部分,获得PspA蛋白。再将PspA蛋白加入到人类单核细胞的培养基中共孵育,ELISA检测培养基上清中IL-6、TNF-α、IL-1β、CXCL8、CCL2、CCL3、CCL4、CCL5浓度的差异;最后,将放线菌素D或者放线菌酮也加入到细胞培养基中,ELISA方法检测其对PspA蛋白促单核细胞合成炎性细胞因子的干预作用。结果重组菌表达TRx-His-PspA融合蛋白相对分子质量约为70 000,纯化后用肠激酶切除融合蛋白的TRx-His部分,获得PspA蛋白(相对分子量为50 000);ELISA试验表明PspA蛋白刺激人类单核细胞后,单核细胞系合成和释放到培养基上清的IL-6、CXCL8、CCL2、CCL4和CCL5显著增加(P0.05);而加入了放线菌素D或者放线菌酮后,PspA蛋白刺激人类单核细胞释放IL-6、CXCL8、CCL2、CCL4和CCL5的能力明显减弱。结论重组的PspA蛋白可以诱导人类单核细胞重新合成和分泌炎性细胞因子IL-6和趋化因子CXCL8、CCL2、CCL4、CCL5,揭示了天然免疫的效应细胞—单核细胞和肺炎链球菌致病因素之间的关系,将有助于制定新的战略来控制肺炎链球菌侵入性疾病。
[Abstract]:Objective to investigate the effect of prokaryotic expression of (pneumococcal surface protein A PspA on the release of inflammatory cytokines and chemokines from human monocytes. Methods the recombinant plasmid pET-32a () / PspA was transformed into Escherichia coli BL21 (DE3). The recombinant strain was induced by IPTG to express and purify TRx-His-PspA fusion protein, and the PspA protein was obtained by removing the TRx-His part of the fusion protein by enterokinase. Then the PspA protein was added to the culture medium of human monocytes to detect the difference of the concentration of IL-6,TNF- 伪 -nil IL-1 尾 in the supernatant of the medium by co-incubating with CCL2CCL3, CCL4 and CCL5 in the supernatant of the medium. Actinomycin D or actinomycin was added to the cell culture medium to detect its effect on the synthesis of inflammatory cytokines induced by PspA protein. Results the relative molecular weight of the TRx-His-PspA fusion protein expressed by the recombinant strain was about 70,000. After purification, the TRx-His part of the fusion protein was removed by enterokinase, and the PspA protein (relative molecular weight was 50 000) was obtained by Elisa. The results showed that the PspA protein stimulated human monocytes. The synthesis and release of IL-6,CXCL8,CCL2,CCL4 and CCL5 into the supernatant of monocytes increased significantly (P0.05), but the ability of PspA protein to stimulate the release of IL-6,CXCL8,CCL2,CCL4 and CCL5 by human monocytes was significantly decreased after the addition of actinomycin D or actinomycin. Conclusion Recombinant PspA protein can induce the resynthesis and secretion of inflammatory cytokine IL-6 and chemokine CXCL8,CCL2,CCL4,CCL5, in human monocytes, which reveals the relationship between the effector cells-monocytes and Streptococcus pneumoniae. It will help to develop new strategies to control Streptococcus pneumoniae invasiveness.
【作者单位】：重庆医科大学附属第一医院检验科;重庆医科大学基础医学院病原生物学教研室;
【分类号】：R378.14

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