论文部分内容阅读
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。
Objective To express pneumococcal surface protein A (PspA) in prokaryotic cells and add it to the culture medium of human monocytes to detect the effect of this protein on the release of inflammatory cytokines and chemokines from human monocytes. Methods The recombinant plasmid pET-32a (+) / PspA was transformed into E. coli BL21 (DE3). The TRx-His-PspA fusion protein was expressed and purified by IPTG induction. The fusion protein TRx-His Part, to obtain PspA protein. Then the PspA protein was added to the culture medium of human monocyte for co-incubation. The difference of the concentration of IL-6, TNF-α, IL-1β, CXCL8, CCL2, CCL3, CCL4 and CCL5 in the supernatant of the culture medium was detected by ELISA. Actinomycin D or cycloheximide was also added to the cell culture medium, and the effect of PspA protein on the synthesis of inflammatory cytokines by mononuclear cells was detected by ELISA. Results The relative molecular mass of TRx-His-PspA fusion protein was about 70 000. After purification, the TRx-His portion of the fusion protein was excised by enterokinase to obtain the PspA protein (relative molecular mass of 50,000). The ELISA assay showed that PspA protein stimulated After monocyte human monocytes, the levels of IL-6, CXCL8, CCL2, CCL4 and CCL5 synthesized and released into the supernatant of the monocytes increased significantly (P <0.05); while the addition of actinomycin D or actinomycetes The ability of PspA protein to stimulate human monocytes to release IL-6, CXCL8, CCL2, CCL4 and CCL5 was significantly attenuated after ketones. CONCLUSION: Recombinant PspA protein can induce human monocytes to repopulate and secrete inflammatory cytokines IL-6 and chemokines CXCL8, CCL2, CCL4 and CCL5, revealing that innate immune effector cells, monocytes and S. pneumoniae The relationship between the factors that will help develop a new strategy to control pneumococcal invasive disease.