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  

Pneumococcal surface protein A induces monocytes to produce inflammatory mediators and chemokines

作　　者：邓济甦[1] 曹炬[1] 徐蕾[2] 

机构地区：[1]重庆医科大学附属第一医院检验科,400016 [2]重庆医科大学基础医学院病原生物学教研室,400016

出　　处：《免疫学杂志》2013年第4期296-300,共5页Immunological Journal

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(P<0.05);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。Pneumococcal surface protein A （PspA） plays a key role in the pathogenesis of invasive pneumococcal infection. In this study, we aimed to investigate the effects of PspA on cytokine and chemokine secretion from human peripheral blood monocytes and the underlying intracellular signaling mechanisms. PspA obtained by prokaryotic expression in E. coli was added alone or mixture with Actinomyein D/cycloheximide in the medium of human monocytes. Then we detected the IL-6, TNF-a, IL-1β, CXCL8, CCL2, CCL3, CCL4, and CCL5 producing and releasing from monocytes. Enzyme-linked immuuosorbent assay showed that protein PspA could promote human monocytes to release more IL-6, CXCL8, CCL2, CCL4, and CCL5 （P〈0.05）, but the Actinomycin D or cycloheximide could antagonize the efficacy of protein PspA. These results indicated that PspA could induce human monocytes synthesizing and releasing inflammatory cytokine IL-6 and chemokines including CXCL8, CCL2, CCL4 and CCL5. which reveals the relationshin hetween neutrophils and Streptococcus pneumoniae.

关 键 词：肺炎链球菌 单核细胞 IL-6 CCL2 CCL4 

分 类 号：R378.14[医药卫生—病原生物学]