| NLRs were found recently, they are cytoplasmic pattern recognition receptors(PRRs). NLRs play an important role in defending against pathogen infection by recognising pathogen-associated molecular patterns (PAMPs) and initiating the innate immune response, leading to an adaptive immune response through signal transduction. NODI is a key member of the NLR family, and is involved in the inflammatory response and in apoptosis, and it is also involved in some inflammatory diseases. It has been reported that NLR family includes 23 members identified in humans and more than 34 in mice. However, there is little research report in birds and how many members of NLRs in birds are not well-defined. At present, it is still in the initial research stage about the NODI of poultry. And chicken has become an important model animal, so, in this study, the aim is to cloning and bioinformatic analysis the full-length cDNA sequences of NOD1 gene of chicken, explorate chicken NOD1, RIPK2, NF-κB and MAPK11 mRNA expression in the tissues, discussion regulatory mechanism of NODI to downstream transduction molecules and inflammatory factors through the iE-DAP and NOD1 shRNA vectors treatment the HD11 cells, and investigating the role of NOD1 signaling pathway in the progress of Salmonella infection in vivo and in vitro.1. Cloning and bioinformatic analysis of chicken NOD1 geneIn the current study, the full-length cDNA sequence of NOD1 was cloned using reverse transcription polymerase chain reaction(RT-PCR)and rapid-amplification of cDNA ends(RACE). The full-length of chicken NODI cDNA is 4502bp, containing 124bp of 5’UTR,2856bp open reading frame (ORF) and 1497bp of 3’UTR, followed by 22bp of polyAtail. The complete open reading frame (ORF) of NOD1 contains 2856 bp localized at 125-2980 nucleotide and encodes a 951 amino acid protein. Analysis chicken NOD1 protein using ProtParam show that the protein with a predicted molecular weight of 108676.2 Da and a theoretical isoelectric point (PI) of 6.58, total number of atoms of 15308, formula is C4885H7669N1285O1421S48. The instability index (II) is calculated to be 36.77, the grand average of hydropathicity (GRAVY) is-0.160.There is no signal peptide and no transmembrane helices of chicken NODI protein. The NODI nucleotide sequence is localized at 41824884-41845174 of romosome 2, full length of chicken NODI nucleotide sequence is 20290bp. Structurally, it is comprised of one caspase recruitment domain (CARD) at the N-terminus, seven leucine-rich repeat regions at the C-terminus, and one NACHT domain between the N and C-termini, these is similar with the NOD1 domain of other species, such as, duck, turkey, human, mice and fish. Multiple sequence alignment was done by ClustalW software, and phylogenetic tree was constructed using MEGA software model based on Poisson Correction Phylogenetic analyses. The result showed that chicken NOD1 (NOD1) clusters with duck and turkey, the relationship with mammalian animal near with the fish. There are a high homology among the chicken, mammalian and fish NOD1.2. Development of real-time PCR for detection of the chicken NOD1 signaling pathway molecules mRNA expressionIn the research, we used SYBR Green I dye method to detect chicken NOD-like receptor NODI and its signal transduction molecule RIPK2,TRAF6,CARD9,NF-κB (p65), MAPK11 and inflammatory cytokines IL-1, IL-8 mRNA expression.The results showed that the real-time fluorescence quantitative PCR melting curve of nine genes were all a single peak, solution temperature were 81.5～89.0℃, amplification efficiency were 96.3%～102.3%, the correlation coefficient were 0.966～0.996, and the slope were -3.269 to -3.413; coefficient of variation of CT value were 1.85%～3.84%. The established real-time quantitative PCR methods were highly specific and high repeatability. The results indicate that the methods could be used for quantification of NOD1, RIPK2, TRAF6, CARD9, NF-κB (p65), MAPK11, IL-1β and IL-8, this provide a technical support for the following experimental study.3. Expression profile analysis of NOD1, RIPK2, NF-κB and MAPK11 in different tissues of chickenNODI is an important member of the NLR receptor. RIPK2 is its adaptor molecule, activation RIPK2 can lead to inducing of downstream NF-κB and MAPK signal transduction pathway. Therefore, detection expression of NOD1, RIPK2, NF-κB, MAPK11 of chicken in different tissues or organs benefit to understanding of the innate immune response and immune process during the body against pathogens. For determine the expression of NOD1, RIPK2, NF-κB and MAPK 11 mRNA in different tissues, total of 13 kinds of tissue, including heart, liver, spleen, lung, kidney, thymus, bursa of fabricius, small intestine, brain, testis, ovary, rectum and blood were collection from one-day old chickens. The results showed that NOD1, RIPK2, NF-κB and MAPK11 mRNA transcription were detected in 13 different tissues. However, the expression of the same gene was different in different tissues. The highest expression of NOD1 and NF-kB genes were detected in blood. The highest expression of RIPK2 and MAPK11 genes were detected in brain. These results showed that NOD1, RIPK2, NF-κB and MAPK11 were widely expressed in chicken tissues.4. The regulatory effect of chicken NOD1 gene on downstream signaling moleculesIn the study, three PLL3.7-NODl-shRNA vectors were used to inhibit the expression of NOD1, and iE-DAP was used to stimulus the expression of NOD1 in HD11 cells, the aim was to investigate the role of NOD1 in regulating the expression of downstream signaling pathway genes. Real-time PCR was used to detect the expression of RIPK2, NF-κB, TRAF6, CARD9, MAPK11, IL-1β and IL-8. Firstly, PLL3.7-NOD1-shRNA1, PLL3.7-NOD1-shRNA2 and PLL3.7-NOD1-shRNA3 expression vectors and one PLL3.7-NOD1-control-shRNA vector were constructed. Secondly, the inhibition efficiency of three expression vectors in HD11 cells was detected. The results showed that the inhibition efficiencies of PLL3.7-NOD1-shRNA1, PLL3.7-NOD1-shRNA2 and PLL3.7-NOD1-shRNA3 were 34.1%,19.3% and 81.4% respectively at 48h, and were 37.9%,58.1% and 69.1% respectively at 72h compared with control. Finally, the expression level of RIPK2, NF-κB (p65), TRAF6, CARD9, MAPK11, IL-1β and IL-8 were detected after HD11 cells transfected with PLL3.7-NOD1-shRNA3 or treated with iE-DAP. The results showed that except IL-1β, the expression of these signal transduction molecules and inflammatory factors were up regulated when HD11 cells were treated by iE-DAP, and down regulated when HD11 cells were interfered by NOD1-shRNA3 vectors. These results suggest that NOD1 can positive regulate the expression of the downstream transduction molecules RIPK2, NF-κB (p65), TRAF6, CARD9, MAPK11 and IL-8, can’t regulate the expression of IL-1β in chicken.5. Recognition effect of chicken NOD1 signaling pathway on SalmonellaStudies have shown that Salmonella can activate the NOD1 signaling pathway. NOD1 can interact with receptor protein 2 (RIPK2) molecular, leading to the activation of NF-κB and MAPK signaling pathway. However, it is not clear whether the NODI signaling pathway is activated by the infection of Salmonella in poultry. In order to study the role of the NODI signaling pathway genes in Salmonella infection, ten-day-old chickens subcutaneous injection of 1×10 CFU/ml 0.2ml Salmonella enteritidis and Salmonella pullorum respectively in vivo, at the same time, control group was subcutaneous injection of 0.2ml saline. NODI, RIPK2, NF-κB, MAPK11, IL-1β and IL-8 mRNA expression in spleen and blood were detected at Id,3d,5d, and 7d during Salmonella infection. The results showed that NOD1, RIPK2, NF-κB, MAPK11, IL-1β and IL-8 mRNA expression were fluctuates from 1day to 7day infected with Salmonella pullorum and Salmonella enteritis bacteria, and significantly increased at 3d,5d or 7d. Salmonella pollorum were used to infect HD11 cells and the expression of NOD1 signalling pathway genes were detected. The results showed that the expression of NOD1 was significantly reduced at 3.5h and 6h, while, significantly increased at 1h,24h during Salmonella pullorum infection; that of RIPK2 and NF-κB were significantly increased during Salmonella pullorum infection except of RIPK2 was decreased significantly at 6 hours, NF-κB was decreased significantly at 1 hour; the expression of MAPK11 was decreased slightly at 1h,6h and increased significantly at 24h; the expression of IL-1β and IL-8 were significantly increased at 1h,3.5h,6h and 24h during Salmonella pullorum infection. iE-DAP was used to evaluate the difference of invasion and proliferation of Salmonella pullorum in HD11 cells. The result showed that there are no significantly different of invasion rate between iE-DAP treatment group and no iE-DAP treatment group. The numbers of bacterial of iE-DAP treatment group significantly lower to no iE-DAP treatment group at 4h and 6h in HD11cells.The results suggest that NOD1 signaling pathway plays an important role in the process of removing Salmonella from the body. |
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