Detection And Evaluation Of The Transcription Levels Of Relative Molecules Associated With Toll-Like Receptors And NOD-Like Receptors Signal Pathway In The Theileria Annulata-Infected Cells

Innate immunity,including immune cells and molecules of innate immunity,is an important immune defense mechanism of the resistance of host to pathogen invasion.Molecules of innate immunity in signaling pathways play an important role in immune defense.Innate immune signaling pathways include Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,Retinoic acid-inducible gene I-like receptor signaling pathway and C-type lectin receptor signaling pathway.The Toll-like receptor signaling pathway is mainly recognition protozoon,viruses and bacteria,and plays an important role in resistance to pathogen invasion.The NOD-like receptor signaling pathway mainly identifies intracellular antigens and plays an important role in immune defense by regulating the release of cytokines.Theileria annulata is a kind of protozoan which parasitized in lymphocytes and red blood cells of cattle.It main invading immune cells such as dendritic cells,B lymphocytes and macrophages,and can make them immortalized and change the original characteristics of them.So,the research of the transcription levels of the molecular in Toll-like receptor and NOD-like receptor signaling pathway in cells infected with Theileria annulata not only has an important significance in disease prevention and control,but has important biological significance.To establish Real-time quantitative PCR assay for detection of bovines 48 molecules in bovine Toll-like receptor(TLR)signal pathway and NOD-like receptor(NLR)signal pathway including TLR1-10,NOD1,NOD2,IL6 and so on,we design the primers of this gene according the sequence which reported on Gene Bank,and detection and evaluation of the transcription levels of molecules associated with TLR-like receptors signal pathway and NOD-like receptors signal pathway in the Theileria annulata-infected cells using 48 Real-time quantitative PCR assay.The main results of our research are as follows:1.Establish of Real-time quantitative PCR assay: The establish of Real-time quantitative PCR assay for48 molecules in bovines,we design the primers of this gene according the sequence which reported on Gene Bankabout TLR1-10,NOD1,NOD2,IL6 and so on.The result showed that the Real-time PCR methods of this 48 genes have signal peak in melting curve,which means these methods are good in specific.The plasmid concentration which can be test in 48 methods is less than 100 copies/?L,which means the methods have good sensitivity.The coefficient of variation was not more than 3.52%,and the coefficient of variation was not more than 3.92%.All the evaluating indicator of the detection methods in this study are good,which are suitable for the transcription levels detection of the molecular.2.Detection and evaluation of the transcription levels of relative molecules associated with Toll-like receptors and NOD-like receptors signal pathway in the Theileria annulata-infected cells:The detection and evaluation of the transcription levels of 48 genes associated with Toll-like receptors signal pathway and NOD-like receptors signal pathway in the Theileria annulata-infected cells.This study test the F10 generation of 3 Theileria annulata-infected cells include Ta XJ-1?Ta NM and Ta XJ-2 cell lines,using the 48 Real-time quantitative PCR assay and using peripheral blood mononuclear cell as control group.The result showed that TLR1-TLR10 were significant down-regulated(P<0.01),and TLR1?TLR5?TLR7and TLR10 were more significant then other Toll-like receptors.NOD1?NOD2?NAIP?NLRP1 and NLRC4 were significant down-regulated(P<0.01),NLRP3 washard test intheexperiment samples.IL6 and IL1? transcription levels were up-regulated(P<0.01)in the molecules of associated with Toll-like receptors signal pathway and NOD-like receptors signal pathway in the Theileria annulata-infected cells,and the fold change of IL6 is more big then IL1?,the other 30 molecules were significant down-regulated(P<0.01),ASC?IFN??NF-?B1 and FOSwere more significant then other molecules.Our experiment provide date support for the futher research of immune action of the infect of Theileria annulatain bovine cells.