重组结核分枝杆菌CFP10蛋白对A549细胞TLR信号途径介导的炎症反应的影响

The effects of the Recombinant Mycobacterium Tuberculosis CFP10 Protein on the TLR Signal-mediated Inflammatory Responses in A549 Cells

本研究旨在检测重组结核分枝杆菌10 ku培养滤液蛋白(culture filtrate protein 10, CFP10)对肺泡Ⅱ型上皮细胞系A549细胞Toll样受体(TLRs)信号途径及其介导的炎症反应的影响。PCR扩增cfp10目的基因片段,构建重组质粒载体pCzn1-CFP10。将重组质粒pCzn1-CFP10转入BL21(DE3)大肠杆菌感受态细胞,IPTG诱导表达重组蛋白CFP10(rCFP10)并鉴定,纯化rCFP10,去除内毒素及脱盐备用。设置对照组,利用MTT检测rCFP10对A549细胞的存活率的影响;通过qRT-PCR、Western blot及ELISA等技术,分别在转录和翻译水平检测rCFP10对A549细胞TLRs信号途径关键分子及其下游炎症因子表达的影响。结果显示,成功构建重组表达载体pCzn1-CFP10并表达纯化出高纯度的rCFP10。MTT结果显示,随着rCFP10浓度增加和处理时间延长,A549细胞存活率显著降低。与空白对照组相比,rCFP10分别从转录和翻译水平显著(P<0.05)或极显著(P<0.01)上调A549细胞中TLRs途径关键分子TLR2、TLR4、MyD88、TRAF6和NF-κB p65及下游炎症因子IL-6、TNF-α的表达水平。rCFP10蛋白可以通过激活A549细胞TLRs受体信号途径促进细胞炎症因子的分泌,这将为进一步加深理解结核病发病机制提供理论依据。

The purpose of this study was to investigate the effects of recombinant 10 kDa culture filtrate protein(CFP10)of Mycobacterium tuberculosis on the inflammatory responses mediated by Toll-like receptor(TLR)signaling in A549 cells.The recombinant plasmid pCzn1-CFP10 was obtained by amplifying the cfp 10 gene fragment using PCR and cloning it into the prokaryotic expression vector pCzn1.The obtained recombinant plasmid pCzn1-CFP10 was transformed into Escherichia coli BL21(DE3)and induced by IPTG to express the recombinant protein CFP10(rCFP10).The purified rCFP10 protein was preserved after endotoxin was removed and desalted before use.The effect of rCFP10 treatment on the survival rate of A549 cells was detected by MTT assay.A549 cells were treated with rCFP10 to detect changes of key molecules of TLR signaling pathway and downstream inflammatory factors in A549 cells by qRT-PCR,Western blot and ELISA.The results showed that the recombinant expression vector pCzn1-CFP10 was successfully constructed and the high-purity rCFP10 protein was expressed and purified in this study.MTT assay showed that rCFP10 could inhibit the survival rate of A549 cells in a time-and concentration-dependent manner.In addition,rCFP10 could significantly(P<0.05)up-regulate the key molecules of TLR pathways TLR2,TLR4,MyD88,TRAF6,NF-κBp65 and downstream inflammatory cytokines IL-6 and TNF-αin A549 cells as compared to the control group.The recombinant Mycobacterium tuberculosis protein CFP10 could promote the secretion of cytokines by activating TLR receptor signaling pathway in A549 cells,which will provide a theoretical basis for further understanding of the pathogenesis of Mycobacterium tuberculosis.