Prokaryotic Expression Of Duck TLR5 And Preparation Of Its Monoclonal Antibodies

Toll-like receptor (TLR) is condisered as a class of transmembrane protein family recognizing pathogen associated molecular pattern (PAMP) and initiate immune response. TLR is one of the best-characterized innate receptors which play a crucial role in protection of the host from pathogen infection. Various TLRs recognize its characterized their ligands and initiate the innate immune responses. For example, the TLR2-TLR3 complexes or TLR2-TLR6 complexes can recognize lipopeptide or lipoprotein, virus dsRNA for TLR3, lipopolysaccharide (LPS) for TLR4, flagellin for TLR5, single RNA for TLR7 and TLR8, unmethylated DNAs for TLR9.TLR5 is expressed on the surface of epithelial cells, monocytes/macrophages and dendritic cells (DCs). Flagellin is the unique ligand for TLR5. Flagellin can activiate the TLR5-mediated pathway, and then initiate the MyD88-dependent pathway activating transcription factors (NF-κB), and finally induce the production of proinflammatory cytokines. At present, the research on the innate immune response mainly focused on mice and humans, but for poultry research is relatively rare. The shortage of related biological materials and reagents heavily restricted the research on poultry. Therefore, we constructed and expressed the extracellular domain (ECD) of duck TLR5 protein and prepared the monoclonal antibodies against it. These antibodies could be useful for the ananlysis of innate immune on poultry.In this study, we designed the primers for amplification of TLR5 extracellular domain (ECD) from the duck cDNA of TLR5 stored in our laboratory. The high fidelity DNA polymerase was used to clone the fragment of 1608 bp, which was then ligated to the cloning vector pMD20-T. After identification by sequencing, the target gene TLR5(ECD) were cloned into the prokaryotic expression vector pCold-1 and pGEX-6p-l to give rise to pCold-TLR5 and pGEX-6p-1-TLR5, respectively. The two recombinant prokaryotic plasmids pCold-TLR5 and pGEX-6p-1-TLR5 were transformed into the host cell E.coli BL21 to develop the recombinant bacteria pCold-TLR5/BL21 and pGEX-6p-1-TLR5/BL21, respectively.The recombinant bacteria pCold-TLR5/BL21 and pGEX-6p-l-TLR5/BL21 were inoculated into liquid LB medium with Amp, respectively. After induction by IPTG for 4-6 hours, the two bacteria pellets were collected separately and lysed by ultrasonic cracking. The supernatant and precipitation were separated by centrifugation and analyzed by SDS-PAGE. According to the electrophoresis results, both of the recombinant fusion protein rHis-TLR5 and rGST-TLR5 were expressed as inclusion body in precipitation.A set of 6-8 weeks old BABL/c mice was immunized with the recombinant fusion protein rHis-TLR5. Mouse B lymphocyte hybridoma technique was used to make monoclonal antibody. Using the indirect ELIS A and subclone screening method, we obtained six hybridoma cell lines secreting TLR5 monoclonal antibodies stably, and named 1C10,4B5,5B9,5B11,5F9 and 6D7, respectively.The antibody titers of these 6 mAbs were detemined through indirect ELISA. The results showed that the titers of four mAbs were about 104 and the others were below 104 in their culture supernatants. Five mAbs was about 107and one mAbs was less than 106in ascitic fuluids. Antibody subtype identification showed that 5F9 mAbs was IgGl, the others were IgM.The collected ascites were treated by dialysis and prepared using the rProtein A Sepharose 4B affinity chromatography technique. The results of SDS-PAGE analysis of all the purified antibodies showed that all the mAbs were purified.Western blotting analysis showed that all of the monoclonal antibodies showed that all of six mAbs could react specifically with the protein TLR5(ECD) expressed by recombinant bacteria pCold-TLR5/BL21 and pGEX-6p-l-TLR5/BL21 induced by IPTG. In addition, the eukaryotic recombinant plasmid pcDNA3.1-TLR5 was transfected into HEK293T cells. Indirect immunofluorescence assay was then used to identify the reactivity between mAbs and the expressed TLR5. The results showed that all of them could react with the expressed TLR5 in the HEK293T cells harboring pcDNA3.1-TLR5 plasmid.Prepared the duck spleen lymphocytes and labeled the monoclonal antibody 5F9 as the first antibody, FITC labeled Goat anti mouse IgG as the second antibody, then for flow cytometry analysis. The results show that the monoclonal antibody 5F9 can specially recognized duck spleen lymphocyte.In this study, TLR5(ECD)from duck was successfully constructed and expressed in the prokaryotic expression system. All of obtained six hybridoma cell lines can stably secrete monoclonal antibodies against TLR5 (ECD). Western blotting and indirect immunofluorescence assay showed that all the mAbs can sepecifically bind to TLR5 expressed in the prokaryotic expression system and eukaryotic expression system. The monoclonal antibodies 5F9 can be used for flow cytometry analysis.