The Study On Chicken Toll-Like Receptors

Toll-like receptors(Toll-like receptors,LTRs), a new class of immune receptors that discovered recentlly can identify the invasion of pathogenic microganisms specially, activate the innate immune cells through coupling the singaling pathways, and lead to a series of inflammatory reactions. TLRs can not only activate the innate immune system,but also provide a costimulatory signal for the acquired immune system. At present,13kinds of TLRs have been identified and it’s expression profiles have been widely found in the body and the surface of about20species cells. Now9kinds of TLRs have been detected in various tissues, immune cells and the cultured cells which separated from the chicken. This study aimed to detect the main chTLRs that imedated the singaling pathways in the process of IBDV invading, and explore the mechanism of immune response, which the chTLRs had took part in during the process of virus invading, through the IBDV infection model. On this basis, the chTLRs were regulated by the foreign substances, which was applied to the IBDV vaccine as the adjuvants and hoped to enhance the immunogenicity of IBDV vaccine.1The study of types of Toll like receptors on the Hy-Line Brown hens11pairs of specific primers were desigened for9kinds of Toll-like receptors(TLRs) genes in chicken according to the recently published sequences.11fragments of chicken TLRs genes were amplified in this research and compared with the sequences that reported in the Genbank, and the matching rate is100%. At the same time,3pairs of specific primers were designed for Toll-like receptor9(TLR9) according to the gene sequences of human TLR9, murine TLR9, swine TLR9, then the chTLR9was tried to amplified from the total cDNA, but the chicken TLR9was not found in the total cDNA in the end.2The estblishment of SYB Green I real-time fluorescent quantitativity assay for chTLR3, chTLR7, chTLR21mRNAIn this chapter, the SYBGreen I real time fluorescent quantitativity assays were established for chTLR3mRNA,chTLR7mRNA,chTLR21mRNA. Based on the recombinant plasmids pMD19-T-pchTLR3, pMD19-T-pchTLR7, pMD19-T-pchTLR21which recombinanted in the last chapter and used as stand templates,3pairs of specific primers were designed for them and the conditions of reactivity were optimized. The SYBGreen Ⅰ real time fluorescent quantitativity stander curves were established at the optimized condition and the methodology was evaluated.The stand curves that estblished all met the requirements of the parameters. The sensitivity of the assay of chTLR3and chTLR7were100copies/ul, while that of β-actin and chTLR21were10copies/ul, the specificity and repetitiveness of the stander curves were all very good.3The dynamic changes of expression of chTLR3, chTLR7, chTLR21mRNA in bursal after vaccination by virulent IBDVTo investigate the change of chTLR3、chTLR7、chTLR21gene expression in Bursa of Fabricius and spleen and its potential role in earlier period of immune response against infectious Bursa disease virus(IBDV) was condected in vivo experiments in chicken.Sixty14-day-old HY-Line Brown chickens were randomly divided into two groups:control group which had10chickens, while the other30chickens were in experiment group. Experimental group was vaccinated by the virulent IBDV (CEVAC(?)IBD L, W2512), at dose of2birds of each, the control group was injected with0.9%NaCl, and reared isolately. Five chickens were sacrificed at the designed time points, then total mRNA were extracted from lymphocytes, which separated from Bursa of Fabricius and spleen. The expression of chTLR3, chTLR7, chTLR21mRNA in Bursa of Fabricius and spleen lymphocytes was mesured by SYBGreen Ⅰ real time fluorescent quantitativity PCR that had estblished at diferent time points. The results showed that the expression of chTLR3mRNA in Bursa of Fabricius and spleen had the most significant changes at36hours after vaccination, after48hours, the expression of chTLR21mRNA also changed significantly in the Bursa of Fabricius and spleen, but the magnitude of changes less than the chTLR3, the expression of chTLR7mRNA in the two tissues had no significant change during the whole process.4The effection of polyl:C and CpG to the TLR3mRNAexpression of PBMCS in vitro testThe objective of this study in the fourth chapter was to value the effection of polyI:C and CpG to the TLR3mRNAexpression of PBMCS in vitro test. The PBMCS were separated, diluted into106/ml and spreaded into each hole in16ml/hole. Then polyI:C and CpG were added to the cell culture medium, their final concentration were100ug/ml and50ug/ml respectivly. Two repreat holes were set by each concentration, and the control group was setup. The cell plate was put into the cell culter incubator for24h. Then the total RNA was extracted from the lymphocytes and coloned by RT. The expression of chTLR3 mRNA was tested with real-time PCR established in chapter2. The result showed that the expression of chTLR3mRNA reached the peak after3hours cultured by polyI:C, however the expression of chTLR3mRNA was inhibited by CpG. The result showed that polyI:C was the specific adjuvant to chTLR3and could stimulate the lymphocytes to produce chTLR3mRNA.5The impact research of polyI:C to the immune efficiency of IBDV live vaccine14-day-old HY-Line Brown chickens were vaccined by polyI:C and IBDV live vaccine in this research. The antibody level of IBDV were detected to confirm wether polyI:C had enhancement to the immune efficiency of IBDV live vaccine.Thirty14-day-old HY-Line Brown chickens were divided into3groups of10each: the control group; IBDV live vaccine group; polyI:C and IBDV live vaccine group. Each group was raised isolately. From the date of vaccination, the chicken were blooded5times totally and the serum were pooled from the blood. The level of antibody was assessed by IDEXX ELISA test kit.Five weeks after vaccination, the chicken were attacked by IBDV BC6/85at the dose of100BID50/each, observed daily. In the fourth day, all the chicken were put to death. Observing the anatomical lesions. The results showed that the level of antibody of the adjuvant group is equivalent to that of vaccine group,and the protection rate of the adjuvant group had10%higher than the vaccine, but the difference was not significant(p<0.05).