【摘要】 目的探讨原核表达肺炎链球菌表面蛋白A(PspA)对人类嗜中性粒细胞释放CXCL8的影响。方法将重组质粒pET-32a(+)/PspA转化到大肠埃希菌BL21(DE3)中,经异丙基-β-D-硫代吡喃半乳糖苷诱导重组菌表达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。用PspA蛋白免疫小鼠,获得抗PspA抗体。再将PspA蛋白加入到人类嗜中性粒细胞的培养基中共孵育,检测CXCL8释放水平的差异;最后,用抗PspA抗体检测其对PspA蛋白促人类嗜中性粒细胞释放CXCL8的影响。结果中性粒细胞细胞内合成和释放到培养基上清液的CXCL8显著增加(P<0.05);而加入了抗PspA抗体后,PspA蛋白刺激人中性粒细胞释放CXCL8的能力明显减弱。结论 PspA可以上调人类嗜中性粒细胞趋化因子CXCL8的合成和释放,揭示了中性粒细胞和肺炎链球菌致病因素之间的关系。更多还原

【Abstract】 Objective To investigate the influence of prokaryotic express pneumococcal surface protein A(PspA) on CXCL8 releasing from human neutrophils.Methods The recombinant plasmid pET-32a(+)/PspA was transformed into E.coli BL21(DE3).The fusion protein TRx-His-PspA was expressed and digested by enterokinase after purification,in order to get protein PspA.Then mice were immunized with protein PspA to get the anti-PspA antibodies.Protein PspA single or mixed with anti-PspA antibodies was added in the medium of human neutrophils for coincubation.Later the CXCL8 producing and releasing from neutrophils were detected.Results The fusion protein TRx-His-PspA was expressed and purified successfully.The protein PspA was obtained after digested by enterokinase.The anti-PspA antibodies with high titer were gotten from the immunized mice.After the protein PspA adding in the medium of human neutrophils,the neutrophils were detected to produce and release more CXCL8 by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay(P<0.05).On the other side,the anti-PspA antibodies could reduce the ability of protein PspA to stimulate the CXCL8 producing and releasing.Conclusion The protein PspA could induce human neutrophil synthesizing and releasing chemokine CXCL8,which reveals the relationship between neutrophils and Streptococcus pneumoniae.更多还原