Molecular Cloning Of TLR4and Its Functional Analyse In Duck

Toll-like receptors (TLRs) are one kind of pattern recognition receptors (PRRs) that mediate innate and acquired immunity for the host, It can identify pathogen-associated molecular patterns (PAMPs) that express on the pathogenic microbes and is highly conservative, and the signal is conducted to the downstream, then body’s immune response is started and regulated. TLR4is the most popular member of the TLRs family studied. The alternative splicing of TLR4gene has been found in pigs, sheep, mice and other species. It has been not reported whether there are alternative splicing in duck and what is its function in immune response.This experiment was conducted to clone and identify the spliced variants of the TLR4gene in duck, and analyze the regulation of their expression. The temporal and spatial expression pattern was detected by qRT-PCR to analysis the levels of transcript variants in different tissues by constructing the LPS infection duck model. pcDNA3.1+-TLR4overexpression vector is built and transiently transfected into duck embryo fibroblasts. Viral and bacterial infections model is created by using polyI:C and LPS analog viruses and bacteria to investigate the changes in gene expression of TLR4signal pathway, for example IL-1β, IL6and MHC II. The study was designed to find duTLR4gene splice forms, research its structure, and expression characteristics. Then preliminary expound the functions of TLR4gene in resistance to bacterial infections and viral infections, and provides duck genetic resources of natural disease-resistant varieties.A. In this study, the CDS and DNA sequence of duTLR4gene is amplificated by using full-length RT-PCR and LD-PCR techniques, and found that length of duTLR4gene is2532bp (named TLR4a), containing3exons and2introns, One new transcript variants (named TLR4b) of duTLR4gene was found, and sequences analysis showns that it is an alternative exon between the first and the second exon, and the length is3367bp.B. By constructing LPS infected cell model, the expression of TLR4a and TLR4b mRNA is detected in the control group and LPS infection treatment group at different time points, the results showed that the expression of TLR4gene is mainly the wild-type (TLR4a), and after LPS stimulation, CEF cells of TLR4a mRNA expression level of overall first increased, then decreased, expression of TLR4b changed a little, it indicates that TLR4’s main function depends on the TLR4a.C. To further validate the function of TLR4a, duck pcDNA3.1+-TLR4eukaryotic expression vector is built and transiently transfected into duck fibroblasts. After LPS and polyI:C stimulation, qRT-PCR results showed that IL-1beta, IL6and MHCII expression levels increased, and with response-efficacy relationship. By constructing polyI:C infected ducklings vivo models, the expression of TLR4a mRNA was detected in thymus, liver, spleen, lung and kidney of the blank group and polyI:C infection treated group, the results show that TLR4a mRNA expression level of overall first increased, and then decreased in different organizations after polyI:C stimulating. The experimental results further confirmed that duTLR4played an important role not only in the body resistance to bacterial infections but also in the process of anti-viral infection.