Cloning, Expression Of Porcine Toll-like Receptor 7 Gene And Prediction Of Protein Structure And Function

In 1997 Toll-like receptors (TLRs) were identified as a pathogen-associated molecular pattern receptor, mediated mainly innate and acquired immunity and recognized pathogen-associated molecular pattern (PAMP). Among the TLRs family, TLR7 was discovered at 2000, which played a central role in the recognization of ssRNA and in triggerring anti-viral signalling pathways. At present, few works have been done on roles of human and mouse TLR-7s in induction of innate and acquired immunity, and there were no relevant reports about the structure and function on porcine, cattle and poultry TLR-7s in China. Researches on the porcine TLR7 (p TLR7) gene have important scientific significance as they show not only similarity and divergence between species.In this study, porcine Toll-like receptor-7 gene (pTLR-7) was cloned and identified by RT-PCR from porcine spleen lympholeukocyte stimulated by lipopolysaccharide (LPS) and phytohemagglutinin (PHA). The sequence analysis showed that the total pTLR-7 gene was 3153 bp in length, encoding 1050 amino acids, which contains a signal peptide composing 26 amino acids and 16.2% of leucines. The similarity of porcine TLR-7 to the published (DQ₃33222) was up to 98.8%. The structural prediction demonstrated that the pTLR7 was a typical typeâ… transmembrane protein containing extracellular, transmembrane and intracellular regions. The homology of nucleotide sequence of pTLR7 with those of cattle and sheep is much higher than as of equine, cat, human and mice. The pTLR7 gene and the pcDNA3.1/CT-GFP-TOPO vector were ligated together and transformed into E.coli cells,after identified by sequencing, the recombinant plasmid was extracted and transfected into CHO-K1 cells by Lipofectin.The result oberserved under fluorescent microscope showed that pTLR7-GFP fusion protein was expressed.The extracellular region of the pTLR-7 is a critical region containing LRR motifs (XLXXLXLXX), which recognizes PAMPs and triggeres signalling pathways.After double digestion with BamHâ… and Hindâ…¢, the extracellular region of the pTLR-7 and the pMAL-c2x vector were ligated together and transformed into E.coli cells. Expression of the recombinant E.coli was induced using IPTG, resulting in a high level of protein expression. The expressed product was analyzed by SDS-PAGE, showing that the 136 kDa pTLR7-MBP fusion protein was expressed successfully.After double digestion with BamHâ… and Hindâ…¢, the extracellular region of the pTLR-7 and the pcDNA3.1 vector were ligated together and transformed into E.coli cells, this construct was also sequenced to ensure fidelity.The recombinant plasmid was extracted and pTLR7-MBP fusion protein was renaturated, then used them to immunize BAL B/c mice. Antibodies were subsequently detected by indirect ELISA, showing that the antibody titeration was obviously higher than the control group. This indicated that the fusion protein induces a high level of humoral response in mice, and we have obtained mouse-anti-porcine polyclonal antibody successfully, providing the test material to do researches on the structures and the functions of the TLR7 protein.