pComb3XTT vector

Clinical and experimental findings indicate that endotoxin/Toll-like receptor 4 (TLR4)-mediated activation of pro-inflammatory and pro-fibrotic signaling is critical in the development of liver fibrosis. Here, researchers 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. The prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was thus constructed and transformed into Escherichia coli (E.coli) BL21. The characteristics of hTLR4-Fab01 were detected by SDS-PAGE, Western blotting, ELISA, affinity and kinetic analysis. Data indicate that hTLR4-Fab01 can specifically bind TLR4, and its treatment significantly attenuates the pro-inflammatory effects, characterized by reduced LPS-induced production of TNF-α, IL-1, IL-6, and IL-8 in human macrophages. Therefore, this suggests that hTLR4-Fab01 may be a potential candidate for the treatment of liver fibrosis.

In this study, total RNA was extracted from positive clones and cDNA was synthesized. The variable regions of the heavy chain (V_H) and light chain (V_L) were amplified by PCR using degenerate primers. Conserved regions of heavy chain domain 1 (C_H1) and light chain (C_L) were amplified from pcomb3XTT. The PCR products of V_H and V_L were purified and then cloned into pETDuet-1 at Nde I/Xho I and Nco I/Hind respectively. Heavy chain Fd and light chain L were amplified from V_H combined with C_H1 and V_L combined with C_L using forward primer L1 or F1 in combination with reverse primer L4 or F4, respectively. The PCR products of Fd and L were cloned into pETDuet-1 at Nde I/Xho I and Nco I/Hind III, respectively. The recombinant vector pETDuet-1/hTLR4-Fab01 was sequenced and further analyzed using the VBASE2 database.

Figure 1. Screening of strains positive for hTLR4-Fab01. (A) PCR products of positive phages. (B) The heavy chain Fd and light chain L were spliced. (Wang, Maorong, et al., 2016)