肺炎链球菌表面蛋白C对人嗜中性粒细胞释放CXCL8的影响

Effect of pneumococcal surface protein C on release of CXCL8 by human neutrophils

目的原核表达肺炎链球菌表面蛋白C(Pneumococcal surface protein C,PspC),并分析该蛋白对人嗜中性粒细胞释放CXCL8的影响。方法将重组质粒pET-32a(+)/PspC转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达TRx-His-PspC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PspC蛋白,免疫小鼠,获得抗PspC抗体。分离人外周血嗜中性粒细胞,经MTT法检测PspC蛋白对嗜中性粒细胞增殖活力的影响;QRT-PCR检测PspC蛋白及抗PspC抗体对人嗜中性粒细胞释放CXCL8的影响。结果表达的TRx-His-PspC融合蛋白相对分子质量约为80 000,目的蛋白约占菌体总蛋白的21%,可与His Tag McAb特异性结合。获得的PspC蛋白相对分子量约为60 000,免疫小鼠获得了高滴度的抗PspC中和抗体(1∶12 000)。分离的人外周血嗜中性粒细胞纯度>98%,活细胞比例大于96%,经不同浓度的PspC蛋白刺激人嗜中性粒细胞,不会引起宿主细胞的细胞毒作用;不同浓度的PspC蛋白刺激人嗜中性粒细胞8 h后,CXCL8基因mRNA转录水平显著上调(P<0.01),且呈剂量依赖性;CXCL8的释放均呈剂量依赖性增强(P<0.01),且24 h高于12 h;抗PspC抗体可显著抑制PspC蛋白刺激人嗜中性粒细胞释放CXCL8。结论 PspC蛋白可上调人嗜中性粒细胞趋化因子CXCL8的合成和释放,揭示了嗜中性粒细胞和肺炎链球菌致病因素之间的关系,为控制肺炎链球菌侵入性疾病奠定了基础。

Objective To express pneumococcal surface protein C（PspC）in prokaryotic cells and analyze its effect on release of CXCL8 by human neutrophils.Methods Recombinant plasmid pET-32a（＋）/ PspC was transformed to E.coli BL21（DE3）and induced with IPTG.The expressed fusion protein TRx-His-PspC was purified and digested with enterokinase,and the obtained PspC was immunized to mice to prepare the antibody against PspC.Neutrophils were isolated from human peripheral blood,and the effect of PspC on proliferation of neutrophils was determined by MTT method.The effects of PspC and its antibody on release of CXCL8 by human neutrophils were evaluated by quantitative real-time PCR（RT-PCR）.Results The expressed fusion protein TRx-His-PspC,with a relative molecular mass of about 80 000,contained about 21% of total somatic protein and showed specific binding to His Tag McAb.The obtaiend PspC,with a relative molecular mass of about 60 000,induced high neutralizing antibody titers（1 ∶ 12 000） against PspC in mice.The neutrophils isolated from human peripheral blood reached a purity of more than 98%,in which the percentage of live cells was more than 96%.No cytotoxicity was caused in neutrophils after stimulation with PspC at various concentrations.The transcription level of CXCL8 mRNA in neutrophils 8 h after stimulation with PspC at various concentrations increased significantly in a dose-dependent mode（P 〈 0.01）.The release of CXCL8 increased in a dose-dependent mode（P 〈 0.01）,which was higher in neutrophils 24 h than in those 12 h after stimulation.However,the antibody against PspC inhibited the stimulatory effect of PspC on release of CXCL8 by neutrophils.Conclusion PspC up-regulated the synthesis and release of CXCL8 by neutrophils,indicating the relationship between neutrophils and Streptococcus pneumoniae.It laid a foundation of controlling the pneumococcal invasive diseases.