| Toll-like receptors are important transmenbrane receptor proteins. It contains extracellular domain, transmembrane domain and cytoplasmic region. TLRs are expressed mainly in monocytes/macrophages, dendritic cells and epithelial cells. It can recognize foreign antigens and activate innate immune responses through MyD88-dependent and MyD88-independent signaling pathway. In addition, it could send signals to T lymphocytes and B lymphocytes to induce the acquired immune responses. Studies showed that mutations or deletions of the TLRs could decrease the resistance of host to bacterial infection and make more sensitive to pathogens. TLR5could recognize flagellin of bacteria such as Salmonella. Campylohacter jejuni zoonotic pathogens. In order to study the pathogenesis and preventive measures of these bacteria to livestock and human, and to provide biological materials for the preparation of TLR5monoclonal antibody, the stable cell line expressing porcine TLR5were established in this study.1. Cloning and expression of porcine TLR5geneGenome was extracted from the whole blood of Jiangquhai porcine, and then its TLR5gene was amplified by PCR, and cloned into pCR2.1vector to construct recombinant plasmid pCR2.1-TLR5. Recombinant plasmid pCR2.1-TLR5was digested by EcoR V and Sal I, and then inserted into pET vertor to construct recombinant plasmid pET-TLR5. The recombinant plasmid pET-TLR5was transformed into Escherichia coli BL21(DE3), and the protein of TLR5was induced to express. Western blotting showed that the expressed TLR5protein could specifically bind with rabbit anti-mouse TLR5monoclonal antibody at the size of95.2KD. The title of polyclonal antiserum from mice immunized with TLR5protein was detemiined up to1:25600. These results suggested that the expressed TLR5protein had good immunogenicity. TLR5gene without stop codon was further amplified by PCR with a specific pair of primers, and then inserted it into pCR2.1vector to construct recombinant plasmid pCR2.1-TLR5. The recombinant plasmid pCR2.1-TLR5was digested by Nhe I and Xba I, and inserted into pcDNA3.1-FLAG vector to construct recombinant plasmid pcDNA3.1-TLR5-FLAG. The fragment of TLR5-FLAG was digested by Nhe I and Apa I from the recombinant plasmid pcDNA3.1-TLR5-FLAG and then inserted into pcDNA5/FRT vector to construct recombinant plasmid pcDNA5/FRT-TLR5-FLAG.COS-1cells were transfected with recombinant plasmid pcDNA3.1-TLR5-FLAG and pcDNA3.1-FLAG, respectively. Expressed TLR5protein was detected by indirect immunofluorescent assay at48h after transfection. The result displayed that fluorescence could only been found in the cells transfected with plasmid pcDNA3.1-TLR5-FLAG and the TLR5protein was located in the cell membrane, which revealed that TLR5protein could be expressed in COS-1cells.2. Establishment and preliminary application of Flp-in-293cell line stably expressing porcine TLR5The recombinant plasmid pcDNA5/FRT-TLR5-FLAG and recombinase expression plasmid pOG44were cotransfected into Flp-in-293cells at a mass ratio of1:9using lipofectamine. Cells were expanded by fedding complete medium at a ratio of1:3at48h after transfection until cell confluence reached70%-80%, and then were fed with selective meidium containing120μg/ml Hygromycin B for resistance screening. Monoclears were digested by trypsin and expanded in cell culture plate. Recombinant cells were identified by FACS. The result showed that TLR5protein was successfully expressed in the recombinant cells. The cells were subcloned to obtain pure cell line, which was named as pTLR5-Flp-In-293(3-3).It was showed that β-galactosidase activity and Zeocin resistance were lost in pTLR5-Flp-In-293(3-3) cell line but Hygromycin B resistance was obtained. RT-PCR was used to detect the transcription of TLR5gene in cell line and the result showed that TLR5fragment was amplified from the recombinant cell line, indicating that TLR5gene had been transcribed. The results of indirect immunofluorescent assay displayed that TLR5protein was successfully expressed in pTLR5-Flp-In-293(3-3) cell line and located in the cell membrane. Western blotting analysis showed that there was target protein band at the size of95.2KD from the cell line indicating TLR5and FLAG tag were fusionally expressed, while negative in negative control. Stability analysis by FACS showed that compared to Flp-In-293cells as negative control, the first,10th and20th generations of the recombinant cell line could express the same level of TLR5protein.Recombinant pTLR5-Flp-In-293(3-3) cells were stimulated by flagellin at different concentrations, and then the secretion of IL-8was quantitatively detected using ELISA assay. The result displayed that the recombinant cell line could expressed high leverl of IL-8, which indicated that TLR5protein expressed in pTLR5-Flp-In-293(3-3) cell line could recognize and interact with flagellin. |
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