| Objective:Systemic inflammatory response syndrome (SIRS) can be caused by bacterial DNA, which is a potent stimulus to immune cells. The immune-stimulatory activity of DNA is assigned to the sequence motifs containing unmethylated CpG dideoxynucleotides. This feature provides a major distinction between bacterial and mammalian host DNA.The Toll-like receptor (TLR) family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition. Mammalian TLRs comprise a large family with extracellular leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin (IL)-1R (TIR) homology domain.TLR9 plays an essential role in initiating the innate immune response against CpG DNA. TLR9 recognizes CpG DNA and activates the signaling cascade leading to production of TNF-α,IL-1,IL-6 and IL-12 via an adaptor protein MyD88.But to date, it is unclear which LRR is the binding site(s) to CpG DNA of TLR9 or involving in the binding site(s). Therefore, making sure the binding site(s) of TLR9 recognizing CpG DNA is useful to understand the TLR9 signal mechanism and to develop new drugs.Methods:1. Study on the binding site(s) to CpG DNA of LRRs in the ectodomain of TLR9(1) To express the LRR2, 5, 8, and 11 of TLR9 in E.coli. The plasmids containing LRR2, 5, 8, or 11 gene sequences of TLR9 were constructed and transformed to E.coli. Then, the recombinant bacteria were induced by IPTG to express the relative proteins.(2) To postulate CpG DNA binding sites of LRRs of TLR9 using bacterial growth curve. The plasmids, pET28a and pQE30, containing LRR2, 5, 8, or 11 gene sequences of TLR9 were constructed and transformed to E.coli competence BL21 or M15. The combinations were induced by 1.0 mM IPTG and the bacterial OD600 were measured at 0 h, 2 h, 4 h, 6 h and 8 h after induction and then the bacterial growth curves were drawn.(3) To detect the direct binding ability of LRR- peptides to CpG DNA using affinity biosensor technology.The biosensor technology was applied to detect the binding of synthesized LRR2, 5, 8 and 11- peptides to CpG DNA immobilized on the biotin cuvette of biosensor.(4) To observe the effect of LRR-peptides on the releasing of TNF-αof mouse macrophages stimulated with CpG DNAIn vitro, mouse macrophages cultured in dishes were stimulated with the mixture of LRR- peptides and CpG DNA pre-incubated for 30 min, and after 4 hours the level of TNF-αin supernatants were tested by ELISA.(5) To observe the influence of LRR peptides on accumulation of CpG DNA in mouse macrophages. LRR- peptides were incubated with 6-FAM labeled CpG DNA for 30 min in vitro.Mouse macrophages cultured in dishes were treatment with the mixture of LRR- peptides and 6-FAM CpG DNA for 1 hour, and the fluorescence intensity and the label rate of cells were measured by flow cytometry.(6) To calculate the molecular weight, theoretical pI, amino acid composition and number of negatively or positively charged residues using ProtParam software on line.2. Study on the new function of the intracellular domain of TLR9(1) To express the transmembrane and intracellular domain (CT) of TLR9 in E.coli.The transmembrane and intracellular domain (CT) of TLR9 was constructed in the plasmid pQE30, subsequently, the plasmid was transformed to E.coli competence M15. Then, the constructed bacteria were induced by IPTG to express the target protein which was detected by SDS-PAGE and Western-blot.(2) To evaluate the influence of IPTG on the growth of the constructed bacteria transformed with pQE30-CT.The bacteria transformed with pQE30-CT were amplified with LB to OD600 at 0.3, and then different concentration of IPTG was added and incubated for 8 h. The bacterial growth curves were drawn based on the measurement of OD600 at 0 h, 2 h, 4 h, 6 h and 8 h after induction.(3) To detect the influence of IPTG on the growth of the bacteria transformed with pQE30 recombinants cloned with different CT sequences.CT sequence was disintegrated into 5 segments: TM (transmembrane segment: from 810 aa to 826 aa), CD (intracellular segment: from 827 aa to 1032 aa), CT1 (the ahead semi-part of CT: from 810 aa to 923 aa), CT2 (the middle semi-part of CT: from 861 aa to 972 aa), and CT3 (the tail semi-part of CT: from 931 aa to 1032 aa). All sequences were cloned into pQE30 and the construct were transformed to competence M15. The recombinant bacteria were induced by IPTG and the growth curve were drawn after the measurement of OD600 at 0 h, 2 h, 4 h, 6 h and 8 h after induction.Results:1. LRR11 is the binding site to CpG DNA.(1) Expression of the LRR2, 5, 8, and 11 of TLR9 in E.coli.LRR8 was cloned into pET28 and the recombinant was transformed into BL21 competence. There are interested protein on PAGE gel. But the BL21 transformed with recombinants, pET28-LRR2, 5 and 11, didn't express the interesting protein after induced with IPTG.Surprisingly, the OD600 of bacteria transformed with pET28-LRR2 or pET28-LRR11 were significantly declined after induced with IPTG..(2) Postulating CpG DNA binding sites of LRRs of TLR9 by using bacterial growth curve.The growth curves of the bacteria transformed with pET28a-LRR5 didn't change after induced by IPTG, but the growth of bacteria transformed with pET28a-LRR2, pET28a-LRR8, or pET28a-LRR11 were obviously inhibited after induced by IPTG. Especially, the bacteria transformed with pET28a-LRR2, or pET28a-LRR11 was almost completely inhibited after induced with IPTG..With cloning the LRR2, 5, 8 and 11 into pQE30 and transforming the recombinants into M15 competence, the similar results were obtained.(3) The direct binding ability of LRR- peptides to CpG DNA using affinity biosensor technology. LRR11- peptide had a highest binding affinity with CpG DNA 2006. In contrast, LRR8 peptide has no very low binding affinity with CpG DNA. The LRR2 and LRR5 had a modest affinity, which was about one forth of LRR11's, with CpG DNA.(4) The effect of LRR peptides pre-incubated with CpG DNA on the releasing of TNF-αof mouse macrophages stimulated with CpG DNA.In vitro, LRR- peptides were pre-incubated with CpG DNA for 30 min and the mixtures were added into cells. The levels of TNF-αin supernatant were tested by ELISA method. LRR8- peptide had no inhibition on TNF-αreleasing from mouse macrophages stimulated with CpG DNA. But, LRR11- peptide had most strong inhibition on TNF-αrelease.(5) The influence of LRR- peptides on accumulation of CpG DNA within mouse macrophages.LRR5-, 8- and 11- peptides could significantly increase the fluorescence intensity of 6-FAM- CpG DNA within mouse macrophages. LRR11- peptide could increase the accumulation of CpG DNA in a dose-dependent manner.(6) The LRR11- peptide didn't possess negative charged residues, and contain 4 positive charged residues. The strong base characteristic of LRR11- peptide possibly contributed to CpG DNA's accumulation within macrophages.2. Expression of the intracellular domain of TLR9 in E.coli could inhibit bacterial growth.(1)Expression the transmembrane and intracellular domain (CT) of TLR9 in E.coli.The CT domain with transmembrane and intracellular fraction of TLR9 was cloned into pQE30, and the recombinant was transformed into M15 competence. There was no interested protein on PAGE gel. But there was an obvious brand appeared using western-blot method.Interestingly, the OD600 of bacteria transformed with pQE30-CT significantly declined after the bacteria were induced with IPTG..(2) Influence of IPTG on the growth of the constructed bacteria transformed with pQE30-CT.The bacteria transformed with pQE30-CT were significantly inhibited when induced for 8 h by IPTG ranging from 0.1 mM to 3.0 mM. The different initial bacterial concentration had no influence on the inhibition of bacterial growth.(3) Influence of IPTG on the growth of the bacteria transformed with pQE30 recombinants cloned with different CT sequences.CT sequence was disintegrated into 5 fragments: TM, CD, CT1, CT2, and CT3. All sequences were cloned into pQE30 and the construct were transformed to competence M15. Only the bacteria transformed with pQE30-CT1 were significantly inhibited when induced by IPTG.
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