A Human Anti-Toll Like Receptor 4 Fab Fragment Inhibits Lipopolysaccharide-Induced Pro-Inflammatory Cytokines Production in Macrophages

PloS One
Maorong WangJin Zhu

Abstract

The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (VH) chain and light chain (VL), then coupled with highly conserved regions of the heavy chain domain 1 (CH1) and the light chain (CL), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and I...Continue Reading

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Methods Mentioned

BETA
ELISA
PCR
Assay
Protein Assay
Surface
chip
electrophoresis
Flow cytometry
phage display

Software Mentioned

Biacore X100 Plus Package evaluation
Excel

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