Cloning And Establising Of A Cell Line Stably Expressing Porcine Toll-like Receptor 2

In 1997 Toll-like receptors were identified as a pathogen-associated molecular patterns receptor, mediated mainly innate and acquired immunity and recognize microbes-associated molecular patterns. Among the TLRs family, TLR-2 is considered as a central pattern recognization receptor in innate immunity. There have been a lot of researches on the importance of human and mouse of TLR-2 and their ligands in inducing immune related events. TLR-2 can recognize a wide variety of pathogen-associated molecular patterns (PAMPs). At present, few works have been done on roles of pig, cattle and sheep TLR-2s in induction of innate and acquired immunity, there were no relevant reports on porcine TLR-2 in China.In this study, porcine Toll-like receptor-2 gene (pTLR-2) was cloned and identified by RT-PCR from porcine mesenteric lymph nodes (MLNs) stimulated by lipopolysaccharide (LPS) and phytohemagglutinin (PHA). The sequence analysis showed that the total pTLR-2 gene was 2358 bp in length, encoding 785 amino acids, which contained a signal peptide composed by 23 amino acids and 15.01% of leucines. The similarity of porcine TLR-2 to the published (NM₂13761) was up to 99.2%. The structural prediction demonstrated that the pTLR2 was a typical typeâ… transmembrane protein containing extracellular, transmembrane and intracellular region. The similarity of pTLR-2 to cattle, horses, sheep and human was much higher than that of mice, up to 80%, 60-70% respectively; the lowest was that of chicken and fish, which were 51.2% and 33.8%.After identified by sequencing, the recombinant plasmid was extracted and transfected into CHO-K1 cells by Lipofectin.The positive cell clones were selected using G418. A single clone highly expressing pTLR2-GFP fusion protein was obtained by seeding the cells into 96-well plates with one cell per well. Finally, a cell line expressing the pTLR2-GFP fusion protein stably was established. There was no significant difference between the transfected and untranfected CHO-K1 cells on cell growth rate, indicating that expression of pTLR2-GFP fusion protein was not toxic for CHO-K1 cells. Immunohistochemistry analysis showed that pTLR2-GFP fusion protein was localized mainly in the cell membrane. The production of pTLR2-GFP fusion protein could react with human TLR-2 polyclonal antibodies in western blot analysis, demonstrating that pTLR2-GFP fusion protein has an immunological activity structure.