Cloning Of Human TLR9 Gene And Construction Of Eukaryotic Expression Vector

TLR9 belongs to Toll like receptors which are a family of pattern recognition receptors. Among the TLR family, TLR9 recognizes bacterial unmethylated cytosine-guanine dinucleotide (CpG) motifs. After binding with the ligand, TLR9 signal pathway leads to subsequent downstream activation of the NFκB and MAPK signaling pathways, which may be responsible for the proinflammatory. TLR9 provides a crucial link between the recognition of pathogens by the innate immune system and subsequent activation of adaptive immunity. TLR9 plays a media role,a variety of immune cells can be activated by CpG, such as B cells, DC cells, macrophages.It increased the expression of costimulatory molecules of cell surface such as CD40,CD80,CD86 and MHCII molecules. The activated TLR9 gene leads dendritic cells (DCS) to maturation and promote DCs release of pro-inflammatory TH1 cytokines such as IL-6, IL-12, TNF, IFN-y and IFN-a. It is also counter-regulatory to the induction of T regulatory cells.CpG mediated by TLR9 activation could alter the presentation capacity of DCs.The process by which exogenous antigen are presented to the CD8+T cells is known as cross-presentation. Polymorphisms in TLR9 genes may influence protein expression and functional activity of the TLR9 receptor, inhibite the body's natural immune function. TLR9 gene polymorphisms are related with body's susceptibility to infectious diseases.In this study, we extracted total RNA from human peripheral blood cells and obtained TLR9 gene using reverse transcriptase-polymerase chain reaction (RT-PCR) method. PCR product was inserted into PMD18T vector by TA clone. We got the positive clone using blue-white screening. After plasmid extraction screening and identification of restriction enzyme, the TLR9 gene was subcloned to pcDNA3.0 to construct recombinant eukaryotic expression vector TLR9-pcDNA3.0. The recombinant plasmid was transformed into copetent cells E. coli JM109,we obtained the engineering bacteria containing recombina-nt plasmid. The sequencing analysis showed that Genbank provided the same sequence,determine the success clone of TLR9 gene. There are four bases in the replacement. The first one at 1635bp,the second at 3036bp, both of them are degenerate bases without amino acid chang.The third is "t" at 209bp replaced with "c", encoding tryptophan into a serine.The fourth is "c"at 3057bp replaced with"g",encoding histidine into glutamic. Results of this study contri-bute to constitute cell lines containing pcDNA3.0-TLR9 and further study of the expression and biological activity of TLR9.It could provide a new idea associated with immune and inflammatory about disease research and treatment.