The Molecular Mechanism Of TLR2 Regulating The NF-κB Activity Of IPEC-J2/DC Cells Under Heat Stress

Objective: Heat stress,is one of the major concerns for livestock breeding and production in hot climates;it could lead to immunosuppression and the occurrence of inflammatory bowel disease.Little is known about the pathogenesis of heat stress.Therefore,this study aimed to understand the mechanism of heat shock response mediated by heat stress in pig;based on the fact that toll-like receptors(TLRs)are important pattern recognition receptors.Methods: The TLR2,GM-CSF and IL-4 genes were cloned,and their structure and function were analyzed by bioinformatics method.With the help of p IRES2-EGFP-p GM-CSF and p IRES2-EGFP-p IL-4,eukaryotic expression recombinant plasmid were constructed and transfected into porcine intestinal epithelial cells(IPEC-J2),while,a stable cell line expressing porcine GM-CSF and IL-4 were obtained by screened using G418.The differentiation of porcine bone marrow cells into dendritic cells(DCs)was induced by the culture supernatant of the cell line.The eukaryotic expression vectors pc DNA3.1 / v5-His-p TLR2 and pc DNA3.1/ v5-His-p HSP70 were co-transfected with IPEC-J2 and DC cells.The expression of the gene and NF-κB signal were analyzed by the fluorescence quantitative and luciferase activity assay.Results:Biological information analysis showed that the porcine TLR2 gene encoded 785 amino acids and a molecular weight of 89.6 k Da,and PI was 7.52,which shared high amino acids sequence identity with that of Bos taurus(80.2%),Ovis aries(80.1%),Homo sapiens(77.9%),Canis lupus familiaris(76.8%),and Mus musculus(68.0%),respectively.The porcine TLR2 protein is a trans-membrane protein composed of three parts: extracellular domain,transmembrane region and intracellular domain,with O-glycosylation sites and N-glycosylation sites.The porcine GM-CSF gene encoded 144 amino acids and a molecular weight of 16.3 k Da,and PI was 7.15,which shared high amino acids sequence identity with that of Ovis aries(77.1%),Equus caballus(73.6%),Felis catus(72.2%),Bos taurus(71.5%),Canis lupus familiaris(71.3%)and Homo sapiens(70.8%),respectively.The expression of GM-CSF m RNA in IPEC-J2 cells was significantly higher than that in control cell or empty vector cells at 24 h after transfected(P <0.01),and also showed high green fluorescent expression level.After G418 selection and positive clone expansion for 30 times;the expression of GM-CSF at the gene level was increased by 200 times than that in untransfected cells.The porcine IL-4 gene encoded 133 amino acids and a molecular weight of 15.02 k Da,and PI was 8.64,which shared high amino acids sequence identity with that of bos taurus(80.5%),Ovis aries(76.7%),Equus caballus(72.7%),Felis catus(72.9%),Canis lupus familiaris(72.0%),Homo sapiens(69.2%)and Mus musculus(41.5%),respectively.The expression of IL-4m RNA in IPEC-J2 cells was significantly higher than that in untransfected vector or empty vector cells at 24 h after transfected(P<0.01),and also showed high green fluorescent expression level.After G418 selection and positive clone expansion for 28 passages;the expression of IL-4 at the gene level was increased by 300 times than that in untransfected cells.The porcine bone marrow DC was induced by culture supernatant of GM-CSF and IL-4 stable cell lines.After the eukaryotic expression vector pc DNA3.1/V5-HIS-TLR2 was constructed successfully and transfected into IPEC-J2/DC,the expression of TLR2 and HSP70 m RNA was detected in the cells by q RT-PCR and can activate NF-κB that was detected by luciferase activity.Conclusions:(1)The pig TLR2,GM-CSF and IL-4 genes were successfully cloned.(2)The eukaryotic expression vector p IRES2-EGFP-p GM-CSF and p IRES2-EGFP-p IL-4 were constructed successfully and transfected into IPEC-J2,a stably cell lines that express the porcine GM-CSF and IL-4were obtained,respectively.(3)The porcine bone marrow DC were successfully inducted.(4)The TLR2 gene up-regulates activation of NF-κB signal pathway.