Study On The Prepration And Characteirzation Of A Human Anti-toll Like Receptor4Antibody Fab Fragment

TLR4was one of TLRs，which played the critical role in the treatment of anti-infection,because it mediated the signal transduction of LPS by dependment or indepentment of MyD88. Itwas the rate limiting step of the whole process of inflammatory reaction caused by LPS, and wasbottleneck of inflammation. Blocking the signal conduction of TLR4on endotoxin is the mosteffective means used to control biological effect of endotoxin (9,10,11). In our previous studieson murine anti TLR4antibody, a murine anti-human TLR4monoclonal antibody was obtainedwhich showed significant inhibition effect on a-TNF produced by LPS stimulation in vitro.However, human could produce human anti-mouse antibody (HAMA) against murine antibody,resulting in strong immunological reaction, which limited its clinical application (12,13,14).With the development of phage-display library technique, human antibody can be obtainsuccessfully. Accordingly, this study was designed to select a human anti-TLR4Fab fragmentgene by the anti-hTLR4phagemid from a human phage-display library, to construct aprokaryotic expression vector to express and purify the Fab, and to assess its biologicalefficiency.Methods：1. In the research, the anti-hTLR4phagemid from a human phage-display antibody librarywas screened using hTLR by solid-pahse screening method through5rounds and verified byELISA. The positive clones were sequenced.2. Builded recombinant of positive colon about pETDuet-1and expressed by E.col.Origami(DE3). The Fab fragment was expressed induced with IPTG.3. The purified anti-hTLR4Fab was characterized by SDS–PAGE, Western-blotting andELISA assays.4. To evaluate the effects of anti-hTLR4Fab blocking the LPS-induced TNF-α expression in human PBMC cells.Results:1. Through5rounds of affinity panning,11positive phage clones were obtained, amplified,and sequenced. The result was that all positive phage clones exhibited same sequence of FV. Theclone producing anti-hTLR4Fab was named as No.1clone.2. Two fragments L and Fd were obtained and cloned into pETDuet-1. The Fab fragmentwas expressed in form of inclusion bodies induced with IPTG. After denaturing and refolding,the Fab antibody was purified by protein L.3. The Fab antibody exhibited specific reactivity with hTLR4by ELISA and Western-blot.4. The Fab could significantly block the LPS-induced TNF-α expression in human PBMCcells, with the inhibition ratio of76.25%.Conclusions:1. Human anti-hTLR4antibody hTLR4Fab1was successfully selected from human phagedisplay antibody library.2. hTLR4Fab gene can be expressed in Origami2（DE3）. The Fab fragment washigh-efficiently expressed in form of inclusion bodies induced with IPTG. After denaturing andrefolding. The Fab antibody was purified by protein L.3. The Fab antibody exhibited specific reactivity with hTLR4by ELISA and Western-blot.It could also significantly block the LPS-induced TNF-α expression in human PBMC cells, withthe inhibition ratio of76.25%.In conclusion, this study successfully developed an hily efficient expression system of ananti-hTLR4Fab fragment with promising biological activity which may be therapeutic benefitfor TLR4-mediated inflammatory diseases.