Toll-like receptor 3 chicken cloning and preliminary study involved IBDV infection
|School||Guangxi University for Nationalities|
|Course||Biochemistry and Molecular Biology|
|Keywords||Off the chicken Toll-like receptor 3 Quantitative PCR Infectious bursal disease virus Chicken peripheral blood lymphocytes Cytokines|
Toll-like receptors (Toll-like receptor, TLRs) are important in innate immunity receptors and signal transduction transmembrane receptors, but also identify the pathogen-associated molecular patterns (PAMP) is one of the important receptors, it is not only involved in the natural immunity, and is connected to innate and acquired immunity important bridge. Toll-like receptor 3 (TLR3) primarily identified double-stranded RNA. Infectious bursal disease virus (Infectious bursal disease virus, IBDV) genome double-stranded RNA, and IBDV caused by infectious bursal disease (IBD) is currently the largest poultry industry hazards immunosuppressive diseases, current understanding of the pathogenesis of IBDV is not comprehensive, especially in the disease process in the host role of information. This study aimed to investigate the Guangxi Sanhuang TLR3 structural characteristics, a preliminary study of peripheral blood lymphocytes, IBDV infection with chicken between TLR3 signaling pathway for the pathogenesis of IBDV deeply revealing, prevention and treatment of IBD further explore ways to provide new ideas. Research by first landing PCR from peripheral blood lymphocytes Guangxi Sanhuang TLR3 gene fragment was amplified, and its cloning, functional areas prokaryotic expression and polyclonal antibody preparation. The results obtained in Guangxi Sanhuang TLR3 (GX-sh-chTLR3) gene length is 3036bp, with the reported sequence homology with chicken TLR3 96.5 to 99.7%, the difference mainly in the extracellular ago 173 amino acids; and cattle, rats, sheep, pigs, horses, rabbits, chimpanzees, people, gorillas amino acid homology was lower, at 68 ~ 74.8%; in phylogenetic analysis, birds and other animals and human TLR3 TLR3 belong to two branches , at different evolutionary position. Structure prediction showed that, GX-sh-chTLR3 obvious transmembrane protein structure, extracellular proteins from the outer multiple α helices and β-strands of multiple recessed inside a solenoid-shaped saddle structure. Construction of critical areas of antigen recognition chicken TLR3 prokaryotic expression vector, the expression product of the induction in mice immunized mouse prepared polyclonal anti-chicken TLR3 has good specificity, and with the chicken fibroblast proteins with specificity in TLR3 reaction. The results for the in-depth study of the structure and function of TLR3 chicken and chicken TLR3 role of disease in poultry provide important material foundation. The study also by fluorescence quantitative PCR (real time RT-PCR) technology, the chicken peripheral blood lymphocytes in IBDV infection and the relationship between the expression of TLR3 were studied: We first established based IBDV-VP2 gene of IBDV quantitative detection standard curve experiments confirmed by the sensitivity and reproducibility of the standard curve specificity, high sensitivity, and has a good stability, can be used for quantitative detection of IBDV. Secondly, the IBDV infected chicken peripheral blood lymphocytes in vitro after viral replication, TLR3 mRNA expression and its downstream effector cytokine expression were detected. The results show that the IBDV infection 6h harvested cells, the highest number of copies of the viral genome, TLR3 expression is the highest. The TLR3 downstream effector of IL-8 and IFNβ change is different, the IBDV infected cells were harvested after 3h IFNβ expression was significantly increased at this time, IL-8 is slightly raised, and 6h, the expression of IL-8 reached highest. IBDV infection, TLR3 downstream effectors IFNβ and IL-8 expression pattern inconsistent with that, IBDV infection, TLR3 through different paths may start a different cellular responses, and this reaction results will directly affect the virus in the cell on destiny. Finally, we use different virulence IBDV infected cells, and select the number of viral copies reached the peak, cells were harvested virulence of different IBDV replication on cells quantitative detection of differences and detecting TLR3 expression of its downstream effectors difference . Result, very virulent strain NN0704 infection caused by TLR3 signaling pathway strongest, significantly higher than middle-virulent strains NN040124 and medium virulence vaccine strain B87 (in) infection. The virulence of different strains of the level of replication in cells also appeared in the same rule. Showed that IBDV infected lymphocytes, TLR3 pathway strength and virulence was positively correlated with virulence stronger, causing more intense cellular responses. In summary, the peripheral blood lymphocytes, IBDV TLR3 pathway triggered by a corresponding cell immune response, and signal strength and induce cell responses and the strength of virus virulence. These findings will be further studied TLR3 IBDV pathogenic molecular mechanisms involved in the foundation, but also for exploring new ways to prevent IBDV provide new ideas.