The Human Anti-Siglec-9 Antibody Fab Fragment Reduced LPS Induced Inflammatory Response In Vitro

Siglec-9,as a member of immunoglobulin superfamily,was classification to CD33 relative Siglec,play an important role in cell proliferation,differentiation,apoptosis and autophagy by recognized sialylated glycans,and regulated the inherent immunity and adaptive immunity.Siglec-9 was predominantly expressed on immune cells,such as neutrophil and monocyte,and a minor population of lymphocyte and dendritic cell.Sepsis is an infection-induced systemic inflammatory response syndrome,bacterial endotoxin stimulated macrophages produce a large number of inflammatory factors,causing the body's anti-inflammatory factor and pro-inflammatory factor imbalance,evensevere septic shock and multiple organ failure.The purpose of this study was constructing and purifies the human anti-Siglec-9 antibody Fab fragment,which was evaluated and explores its foundation on inflammatory aspect by neutralization effects in vitro.Methods: 1.Construction of the Fab fragment of the human anti-Siglec-9 antibody The variable regions of light and heavy chain from the positive clone was amplified by PCR,of which nucleic acid sequence and amino acid sequence was alignment with data base.The VL and VH were attached with CL and CH1 respectively by overlapping extension PCR with primers was designed in accordance with nucleic acid sequence,then L and Fd were obtained.The L and Fd were connected with the prokaryotic expression vector p ETDUET-1 with restriction enzymes and the recombinant prokaryotic expression plasmid p ETDUET-1-Fab was successfully constructed.The p ETDUET-1-Fab was transformed into Escherichia coli BL21 and was induced with IPTG overnight at 37 oC,which was purified by protein system.2.Identification of the Fab fragment of the human anti-Siglec-9 antibody interaction with antigen and antibody affinity analysis SDS-PAGE and Western blot were used for the identification of human anti-Siglec-9 antibody Fab fragment;ELISA,FACS and Biacore X100 were applied for the high affinity and specifically bind of human anti-Siglec-9 antibody Fab fragment.3.To detect of the effect of the Fab fragment of the human anti-Siglec-9 antibody in vitro The expression of inflammatory cytokines was measured by Q-PCR and ELISA when the human anti-Siglec-9 antibody Fab fragment stimulated THP-1 cell derived macrophages and human peripheral blood mononuclear cellsderived macrophages.Results: 1.The variable regions of light and heavy chain were amplified by PCR.It was confirmed by sequencing that no nonfunctional gene or gene mutation occurred.The VL and VH were attached with CL and CH1 respectively by overlapping extension PCR,then L and Fd were obtained.The recombinant prokaryotic expression plasmid p ETDUET-1-Fab was successfully constructed.The human anti-Siglec-9 antibody Fab fragment was obtained after recombinant prokaryotic expression plasmid transformed into Escherichia coli BL21.2.The human anti-Siglec-9 antibody Fab fragment was obvious band at SDS-PAGE about 27 KD.ELISA,FACS indicated that the human anti-Siglec-9 antibody Fab fragment could specifically bind Siglec-9.Biacore X100 showed that the affinity constant of human anti-Siglec-9 antibody Fab fragment was 6.58× 10-7.3.Q-PCR and ELISA suggested that the human anti-Siglec-9 antibody Fab fragment could reduce the expression level of proinflammatory cytokines while the expression level of anti-inflammatory cytokine was enhanced,Western blot show that the human anti-Siglec-9 antibody Fab fragment could suppress the protein phosphorylation level of TLR4 signal path.Conclusion: We confirmed that the human anti-Siglec-9 antibody Fab fragment was developed.The high affinity and specifically bound of human anti-Siglec-9 antibody Fab fragment with Siglec-9 protein was evaluated.The human anti-Siglec-9 antibody Fab fragment could significantly reduce the expression level of proinflammatory cytokines,such as IFN-??TNF-??IL-1??IL-6,while the expression level of anti-inflammatory cytokine IL-10 was obviously enhanced in vitro,and suppress the protein phosphorylation level of TLR4 signal path,thus our results indicated that the human anti-Siglec-9 antibody Fab fragment have anti-inflammatory affection.Finally,we believed that the human anti-Siglec-9 antibody Fab fragment may lay the foundation for prevention and treatment of inflammatory diseases.