Molecular Cloning And Function Studies On Duck TLR3 Gene

【Objective】Innate immunity is the first line of host sensing and resisting invasion of pathogen. Toll-like receptors are important part of pattern recognition receptors, which sense pathogen associated molecular pattern first and induce innate immune response. Recently, the knowledge of duck TLR3 about its strucrure and function has remained to be understood. To research the structure and function of TLR3 in Peking duck, Peking duck TLR3(named duTLR3) gene were cloned and predicted structure and function by utilizing Bioinformatics Technology. First the real-time PCR assay for detecting duTLR3 was established and duTLR3 polyclonal antibodies were prepared, then antiviral function of endogenous TLR3 was confirmed by using above two methods.【Method】(1) Peking duck TLR3(named duTLR3) gene were cloned from Peripheral blood mononuclear cell of duck by using RT-PCR and were predicted structure and function by utilizing Bioinformatics Technology.(2)The extracellular domain of duck TLR3 gene was amplified by PCR and cloned into expression vector pET-32 a,and the recombinant plasmid pET-32a/TLR3 was transformed into E.coli BL21(DE3) cells before being induced by IPTG. Duck TLR3 protein was purified from Inclusion Body. PCR amplified 972 bp of the extracellular domain of TLR3 gene, the recombinant protein vaccinated into rabbit to produce polyclonal antibodies. The anti-serum was collected and detected by western blot and ELISA for specificity and titer.(3)According to the gene conserved sequences of duck TLR3 available in GeneBank, the specific primers were designed, and β-actin mRNA was as internal control, aiming to develop a real-time fluorescent quantitative PCR(qRT-PCR) assay for detection of duck toll-like receptor 3 mRNA.(4) The antiviral function was verified in cell and tissue level using the above established method.【Result】(1) DuTLR3 gene were cloned successfully and were predicted with characteristic of typical structure of TLR family.(2) TLR3 extracellular region was successfully expressed and the specific polyclonal antibodies against the recombinant protein were also prepared.(3) The standard curves showed this method had good liner relationships(r2>0.99) and was efficiency about 0.9 in the range of 1×102 to 1×108 copies/μL. The melting curve analysis showed that the qRT-PCR assay was specific for detecting TLR3 mRNA. In addition, the reproducibility showed that the minimum detectable concentration of positive plasmids of TLR3 and β-actin were 100 copies/μL. Both the intra-assay and inter-assay coefficient of variation values were less than 3 %. The tissue distribution of TLR3 was detected by this qRT-PCR assay, and the results showed TLR3 is widely expressed in various tissues of duck, with the highest expression level in trachea and the lowest expression level in spleen.(4) The relationship was positively correlated between TLR3 expression level and DRV titer, as well as downstream cytokines.【Conclusion】The study preliminary confirms that duck TLR3 has a direct interaction with DRV and plays its antiviral function by the downstream signaling molecules and antiviral protein.