The Method Was Established By Screening Of Toll-like Receptors To Target Molecules Of Anti-infective Medicine Ingredients

TLRs were firstly discovered in Drosophila , these receptors were named for their extracellular domain homologous with the Toll protein in Drosophila . TLRs are an important class of pattern recognition receptors (PRRs), which is widely distri- buted in immune cells such as dendritic cells and macrophages. Natural immune response which is the first line of defense against pathogenic microorganisms is started after the identification of pathogenic microorganisms specific pathogen associated molecular patterns (Pathogen-associated molecular patterns, PAMPs ) by TLRs. So far, 11 species were found in human TLRs and 13 mouse TLRs. They can selective recognize different PAMPs, such as TLR1, 2,4,5 and 6 identify bacterial PAMPs in LPS, peptidoglycan, lipoprotein and flagellin protein; and TLR3, 7,8 and 9 identify the nucleotide derivatives, they can also active nuclear factor kappa B (nuclear factor-kappa B, NF-κB) and other signal pathways after recognizing these PAMPs.After specific recognition of their ligands, TLRs can active a series of downstream protein through the dependent or independent pathway of MyD88 (myeloid differentiation factor). Following induce the synthesis and release of corresponding interleukin (IL), tumor necrosis factor (TNF-α) and interferon (IFN), which attend the body immune response against infection.Some research indicated that Chinese herb can increase immunity against infection through many different pathways, also we all know that TLRs play important roles in body immune against infection, so there must be some natural connection between them.Our research mainly focus on the function of anti-infect Chinese herb to TLRs and their downstream signal facters. To evaluate whether TLRs can be used as target facers to screen anti-infect Chinese herb, furthermore we want to set a pattern above. Now describe my research details: First part: Clone and express TLR3 genes of mouse.Primers were designed by the primer 5 software according to the sequence published in Genebank. Using the total RNA of Ana-1 an sample, obtain the whole ORF of gene TLR3 by RT-PCR. p3XFLAG-CMV-7.1-TLR3 vector were built and transinfected into 293T, finally using western blot to check the protein. Results suggested we obtained the target protein successfully. This was basic to Part two: clone, express and Identify of its activity.Total RNA from macrophages of mice were extracted and amplified by RT-PCR, eukaryotic expression plasmid p3XFLAG-CMV-7.1-mTLR9 were built and transfected into 293T cells, finally using western blot to check the protein. By two-luciferase reporter gene system, the impact of mTLR9c to its downstream transcription factor NF-κB were dected. The results show that we successfully obtain the target protein, and the TLR9 gene products really can activate the transcription of downstream transcription factor NF-κB.Part three: The mRNA level impact of Glycyrrhetinic Acid and Icraiin to TLRs and its downstream factors myd88, trif and IFN-β.Primers of TLR2,TLR3,TLR4,TLR7,TLR9 genes of mice for real-time PCR were designed by primer 5 according to the sequence published in genebank. Total RNA were extrated from Ana-1 which were stimulated by Glycyrrhetinic Acid and Icraiin ,using Real-time PCR to detect the impact of to TLRs. Results show that Glycyrrhetinic Acid and Icraiin can significantly increase the express of TLR2,TLR3,TLR4,TLR7和TLR9 in different period. After stimulating for 12h and 24h the express level of MYD88和TRIF were significantly improved, while the express level of IFN-βwere highly increased after 24h.