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

doi:10.11843/j.issn.0366-6964.2020.10.019

Abstract

Abstract: 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 cfp10 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-α in 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.

Key words: recombinant protein CFP10, A549 cells, TLR signaling pathway, inflammatory cytokines

CLC Number:

XU Zhaokun, WANG Jianhong, LI Wu, WANG Yujiong. The effects of the Recombinant Mycobacterium Tuberculosis CFP10 Protein on the TLR Signal-mediated Inflammatory Responses in A549 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2509-2517.

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

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