Chicken Toll-like Receptor4Involvement In APS Regulation Of Chicken Intestinal Immune Function

This test regulate intestinal immune function chickens start from Astragalus polysaccharide, Study on The role of chTLR4its signal transduction pathway in this process.First, using Trizol extraction of total chickens thymus RNA, reverse transcribed into cDNA, According to the GenBank sequences chTLR4been published, containing more antigenic sites in the area of design specific primers, According to the GenBank sequences chTLR4been published, containing the antigenic sites in the area of design more specific primers, cloned chTLR4including intracellular, extracellular, transmembrane part of fragment size is1332bp, and construct prokaryotic plasmid pET30a-chTLR4, After induction, the expression of a size of about58.5KD recombinant proteins, The use of purified recombinant proteins successfully prepared a rabbit anti chTLR4polyclonal antibody, After indirect ELISA, Western-blot and indirect immunofluorescence method for identifying rabbit anti-chTLR4polyclonal antibody, and found that it has good reactivity and specificity.Then, The7days chickens were randomly divided into four groups, that is APS high dose group (A), APS middle dose group (group B), APS low-dose group (group C) and control group (group D). A, B, C group of chickens are fed a high, medium and low-dose APS at7days,1times/d, continuous5d, After the first dose1,3,5,7d, take chickens duodenum, jejunum, ileum and Fabricius, using real-time quantitative PCR, immune enzyme-histochemistry method, indirect ELISA technique to detect changes of chTLR4of chickens intestinal and its signal transduction pathways associated signaling molecules (chMyD88, chTRIF, NF-κB, chIRF3) activation of cytokines (chIFN-β, TNF-a), the dynamic of slgA. The results showed that after7days chicks fed for serving APS five days,8days old, APS high-dose group (A) duodenum chTLR4mRNA expression levels were significantly higher in the (group D)(P<0.01), then decreased, and significantly lower than the control group (P<0.01); Middle dose group APS (group B) chTLR4mRNA expression in the duodenum were significantly lower than the control group (P<0.01) In10to14days; low-dose group (group C) chicks the expression was significantly higher amount (P <0.01).at14days old; A group of Fabricius chTLR4mRNA expression was significantly higher than that in group D (P<0.01) at12days old; Group B of chTLR4mRNA expression in the Fabricius continued to rise, reaching a peak was significantly higher than that in group D (P<0.01) when14days old;10-14days old, C Group of chTLR4mRNA expression levels in the Fabricius consistent with the trend in group B, and14days old was significantly higher than that in group D (P<0.01).; and14days old was significantly higher than that in group D(P<0.01); In chicks duodenum, jejunum, ileum, The trends chTLR4protein of expression is consistent with the duodenum of chTLR4mRNA. In the duodenum,Fabricius of Chicken The adapter protein molecules chTRIF, downstream transcription factor (NF-κB, chIRF3) and inducing product (chIFN-β, TNF-α) mRNA the expression of chTLR4signal transduction pathway compared to chTLR4is basically the same; Another adapter protein molecule mRNA expression chMyD88of chTLR4only in the8days old (B, Group of C),10days old (A Group of) was significantly higher than that in group D (P<0.01or P<0.05), then decreased, When the14days old, the difference with the D Group of was not significant. This suggests that After the chicks fed APS, chTLR4signal transduction pathway activation pathway of intestinal is mainly chTRIF way. In addition, Continuously fed to the chicks after five days APS, Group of A, B chicks, the sIgA levels in the duodenum after the first rise showed a downward trend since8days old to14days old, A group of chicks the sIgA levels peaked in group B chicks sIgA levels peaked at10days old; C group the trends of sIgA levels in the intestinal decreased after an initial increase, followed by14days old and then rises to a peak and sIgA were higher than A, B, D three groups. showed that APS can improve the content of sIgA in the chick duodenum.The study from the gene and protein expression levels confirmed chTLR4its signal transduction pathways involved in the APS regulation the process of chicken intestinal immune, Further in vivo chicks pattern recognition and signal transduction perspective illustrates the APS regulate immune function of the molecular mechanisms, Clinically, For more on the science and clinical application of APS and increase resistance to disease and provide scientific basis for chicken.