摘要
目的:
细菌DNA是革兰阳性细菌和革兰阴性细菌共有的遗传物质,是导致全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)发生的主要致病因子之一,未甲基化的CpG序列即CpG基序(CpG motif)是细菌DNA免疫刺激的最小作用单位。Toll样受体家族的TLR9(Toll-Like Receptor 9)是巨噬细胞识别CpG DNA的模式识别受体,它可通过激活单核/吞噬细胞系统信号转导,活化NF-κB、AP-1,从而大量释放TNF-α、IL-1、IL-6和IL-12等多种前炎症细胞因子,引起急性炎症反应、SIRS甚至休克、死亡。
TLR9是跨膜蛋白质,分为胞外区、跨膜区和胞内区三部分。TLR9胞外区由25个富含亮氨酸的重复序列(leucine-rich repeats,LRRs)组成,与识别CpG DNA有关。其中的LRR2、5、8、11序列后跟随有插入氨基酸序列,可能是与CpG DNA的结合位点或参与结合CpG DNA。
但是,迄今为止,TLR9是如何识别CpG DNA、其识别位点在何处还不清楚,更不清楚TLR9识别CpG DNA的分子机制。因此,明确TLR9与CpG DNA的结合位点对阐明CpG DNA介导炎症的发生机制,并将其作为可能的新的药物靶点进行疾病防治具有重要意义。
本课题深入分析TLR9胞外段与胞内段空间结构和生物学活性;应用细菌生长抑制实验推测LRRs在识别CpG DNA中的作用;合成LRR多肽,观察其与CpG DNA的亲合力以及生物学活性;为明确TLR9与CpG DNA结合位点、阐明TLR9识别机制奠定基础。
方法:
一、TLR9胞外段与CpG DNA识别、结合LRR序列的确定
(一)TLR9胞外段LRR2、5、8、11序列在大肠杆菌中的表达构建含有TLR9胞外段LRR2、5、8、11序列的原核表达pET28系列载体,在大肠杆菌中IPTG诱导表达相应的蛋白。
(二)利用细菌生长变化推测TLR9胞外段与CpG DNA结合位点
含有TLR9胞外段LRR2、5、8、11序列的pET28和pQE30原核表达载体,转化大肠杆菌BL21感受态或M15感受态,1 mM IPTG分别诱导8h,并在第0h、2h、4h、6h、8h分别取菌液测定细菌OD600,绘制生长曲线。
(三)应用生物传感器技术检测LRR多肽与CpG DNA亲和力
人工合成LRR2、5、8、11多肽,将生物素标记的CpG DNA包被于生物素化样品池,利用生物传感器检测各个LRR多肽与CpG DNA的亲和力,结果以结合峰值表示。在此基础上,测量、计算LRR多肽与CpG DNA的解离常数Kd值。
(四)应用细胞因子释放抑制实验观察LRRs在结合CpG DNA中的作用
分离小鼠腹腔巨噬细胞,加入48孔板中,每孔5×106个细胞,培养4h,预先准备各LRR多肽与CpG DNA的混合物,加入培养孔内,继续培养4h后取培养上清,ELISA法检测TNF-α含量。
(五)应用细胞内化实验观察LRRs对CpG DNA在细胞中聚集的影响
分离小鼠腹腔巨噬细胞,加入24孔板中,在细胞培养体系中加入不同的6-FAM标记的CpG DNA与LRR多肽的混合物后,培养1 h,应用流式细胞术观察细胞内荧光强度的变化。
对各多肽进行理论分析,计算多肽的各种理化参数,如分子量、等电点、氨基酸构成、原子组成等。
二、TLR9胞内段抑菌作用的发现和实验研究
(一) TLR9胞内段CT序列在大肠杆菌中的表达
构建含有TLR9跨膜段、胞内段的pQE30-CT载体,转化大肠杆菌M15菌株,SDS-PAGE电泳检测目的蛋白表达,Wester-blot法进一步确定目的蛋白的特异性。
(二) IPTG诱导对pQE30-CT转化细菌的生长的影响
1. 1.0 mM IPTG诱导对pQE30-CT载体转化菌生长的影响扩增pQE30-CT载体转化菌至OD600为0.3左右,1 mM IPTG分别诱导8h,并在第0h、2h、4h、6h、8h分别取菌液测定细菌OD600,绘制生长曲线。
2.不同浓度IPTG诱导对pQE30-CT载体转化菌生长的影响
3. pQE30-CT载体不同表达宿主菌的生长变化
4.不同起始诱导细菌浓度对IPTG诱导后生长的影响
5.含抗生素LB培养液对M15-pQE30-CT菌株诱导后生长的影响
分别使用了含双抗(氨苄青霉素和卡那霉素)LB培养液和不含双抗LB培养液,观察M15-pQE30-CT菌株经1.0 mM IPTG诱导后生长变化。
6. M15-pQE30-CT菌株IPTG诱导24 h生长曲线
7. M15-pQE30-CT诱导后超声上清对大肠杆菌ATCC35218生长的影响
8. M15-pQE30-CT与大肠杆菌ATCC35218菌悬液经IPTG诱导后生长变化
9. M15-pQE30-CT经IPTG诱导后细菌形态变化
(三) IPTG诱导对含有不同区域片段pQE30-CT载体转化菌株生长的影响
将CT段进行分解,共分成TM(跨膜段:810 aa～826 aa)、CD(胞内段: 827 aa～1032 aa)、CT1(CT前1/2段:810 aa～923 aa)、CT2(CT中1/2段:861 aa～972 aa)以及CT3 (CT后1/2段:931 aa～1032 aa),分别构建载体观察转化菌生长曲线。
结果:
一、TLR9胞外段与CpG DNA识别、结合LRR序列的确定
(一)TLR9胞外段LRR2、5、8、11序列在大肠杆菌中的表达
pET28-LRR8转化菌经IPTG诱导后有明显目的蛋白表达,而其它载体如pET28-LRR2、pET28-LRR5以及pET28-LRR11转化菌诱导后未见相应的蛋白条带。
pET28-LRR2、pET28-LRR11转化菌在IPTG诱导6 h后,与未诱导组相比细菌数量显著减少,而转化pET28-LRR8的细菌未见此现象。
(二)利用细菌生长变化推测TLR9胞外段与CpG DNA结合位点
pET28a-LRR5转化菌在IPTG诱导后生长曲线无明显变化,而转化有pET28a-LRR11、pET28a-LRR2、pET28a-LRR8的BL21菌株经IPTG诱导后生长明显受到抑制。特别是pET28a-LRR11、pET28a-LRR2转化菌,IPTG诱导后生长完全受到抑制。pQE30表达载体也出现类似结果。
(三)应用生物传感器技术检测LRR多肽与CpG DNA亲和力
在LRR2、LRR5、LRR8、LRR11这4个多肽中,LRR11与CpG DNA 2006的亲和力最高,LRR8与CpG DNA 2006之间几无亲合力,LRR2和LRR5与CpG DNA 2006的亲合力约为LRR11与CpG DNA 2006亲合力的1/4～1/5,其中,LRR11多肽与CpG DNA的解离常数Kd值为13.2μM。
(四)应用细胞因子释放抑制实验观察LRRs在结合CpG DNA中的作用
4个多肽片段提前与CpG DNA孵育后,LRR8几乎没有抑制CpG DNA刺激小鼠腹腔巨噬细胞释放TNF-α的能力,而LRR11抑制巨噬细胞释放TNF-α的能力最强。
(五)应用细胞内化实验观察LRRs对CpG DNA在细胞中聚集的影响
LRR5、LRR8和LRR11能增加6-FAM CpG DNA在小鼠腹腔巨噬细胞内的荧光强度,各组细胞内平均荧光强度增加分别为27.3%、32.0%和69.2%。LRR11多肽能显著6-FAM CpG DNA在细胞内聚集,且呈明显的量效关系。
对LRR11多肽的分析表明:LRR11多肽不含酸性氨基酸,有4个碱性氨基酸,等电点pI 11.10,增强CpG DNA在细胞内聚集可能与其呈强碱性有关。
二、TLR9胞内段抑菌作用的发现和实验研究
(一) TLR9胞内段CT序列在大肠杆菌中的表达
构建了含有TLR9跨膜段、胞内段的pQE30-CT载体,转化大肠杆菌M15菌株,经Western-blot检测,发现该载体菌经IPTG诱导后能微量表达目的蛋白。另外,还发现重组菌在IPTG诱导4 h后,与未诱导组相比细菌数量显著减少,推测表达的蛋白具有一定的抑菌活性。
(二) IPTG诱导对pQE30-CT转化细菌的生长的影响
1. M15-pQE30-CT带有TLR9的跨膜段和胞内段TIR全序列,经1 mM IPTG诱导后,在各个时间段细菌生长均受到非常显著的抑制。
2. M15-pQE30-CT,经0.1 mM、0.3 mM、1.0 mM、3.0 mM IPTG诱导后,在各个时间段细菌生长均受到非常显著的抑制,不同浓度IPTG组细菌生长曲线几乎完全重合,说明M15-pQE30-CT经IPTG诱导后生长受到抑制与IPTG的非特异作用无关。
3.将pQE30-CT转化DH5α菌株,1 mM IPTG诱导,诱导后pQE30-CT DH5α转化菌在各个时间段均受到明显抑制,但是抑制作用不如M15转化菌株,可能与DH5α并非pQE30系列载体最适表达宿主菌有关。
4.将M15-pQE30-CT菌株扩增至OD值2.0时用1.0 mM IPTG诱导2 h,发现IPTG诱导组细菌OD值明显下降,结果证实起始诱导细菌浓度不影响M15-pQE30-CT经IPTG诱导后生长抑制状态。
5.在含有双抗(氨苄青霉素和卡那霉素)和不含双抗的LB培养液条件下,细菌诱导后生长均受到非常显著抑制,排除抗生素对其的影响。
6.未诱导组细菌在第10 h达到生长平台期,IPTG诱导组细菌在前10 h内处于抑制状态,但从第10 h开始恢复生长,直至第20 h达到平台期。
7. M15-pQE30-CT经IPTG诱导后超声上清不影响大肠杆菌ATCC35218生长,说明:①M15-pQE30-CT细菌诱导后细胞内没有“抑菌蛋白”;或者②M15-pQE30-CT细菌诱导后产生“抑菌蛋白”的量不够多,收集后还不足以抑制ATCC35218生长。
8. M15-pQE30-CT和大肠杆菌ATCC35218不同比例菌悬液经过IPTG诱导后,除了单独M15-pQE30-CT组细菌生长受到抑制外,其余各组细菌生长无显著差异。说明M15-pQE30-CT细菌诱导后不能释放能抑制其它细菌生长的蛋白。
9.未诱导组M15-pQE30-CT细菌革兰染色后,细胞形态清晰、完整,无聚团现象,经1.0 mM IPTG诱导2 h后,镜下细胞数量显著减少,细胞形态不完整、模糊。
(三) IPTG诱导对含有不同区域片段pQE30-CT载体转化菌株生长的影响
共构建了含有TM、CD、CT1、CT2、CT3片段的原核表达载体,观察细菌生长曲线变化,发现转化含有CT1段载体的细菌经IPTG诱导后细菌生长完全受到抑制,转化含有CD段载体的细菌经IPTG诱导后细菌生长部分受到抑制,而IPTG诱导对转化含有TM、CT2、CT3段载体细菌的生长没有影响。
结论:
1. TLR9胞外段LRR2、LRR5、LRR8、LRR11序列中,最有可能是CpG DNA结合位点的是LRR11序列,但不排除LRR2联合参与的可能性。
1)原核表达LRR2、5、8、11蛋白,只有LRR8蛋白顺利表达,其它LRR未能正常表达;
2)合成LRR2、5、8、11多肽,LRR11多肽与CpG DNA亲合力最高;
3) LRR11多肽能抑制CpG DNA刺激小鼠腹腔巨噬细胞释放TNF-α以及增强荧光标记CpG DNA在小鼠腹腔巨噬细胞内聚集。
2.构建含有TLR9胞内段的表达载体,表达的蛋白具有抑菌功能。
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 DNA
In 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.
引文
1. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995,374:546-549.
2. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002,20:709-760.
3. Krieg AM. CpG DNA: trigger of sepsis, mediator of protection, or both? Scand J Infect Dis 2003,35:653-659.
4. Brun-Buisson C. The epidemiology of the systemic inflammatory response. Intensive Care Med 2000,26 Suppl 1:S64-74.
5. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000,408:740-745.
6. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001,98:9237-9242.
7. Ishii KJ, Akira S. Toll-like Receptors and Sepsis. Curr Infect Dis Rep 2004,6:361-366.
8. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004,4:499-511.
9. Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol 2004,5:975-979.
10. Sparwasser T, Miethke T, Lipford G, Borschert K, Hacker H, Heeg K, Wagner H. Bacterial DNA causes septic shock. Nature 1997,386:336-337.
11. Dalpke A, Frank J, Peter M, Heeg K. Activation of toll-like receptor 9 by DNA from different bacterial species. Infect Immun 2006,74:940-946.
12. Bell JK, Mullen GE, Leifer CA, Mazzoni A, Davies DR, Segal DM. Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol 2003,24: 528-533.
13. Barton GM, Kagan JC, Medzhitov R. Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat Immunol 2006,7:49-56.
14. Brinkmann MM, Spooner E, Hoebe K, Beutler B, Ploegh HL, Kim YM. The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLRsignaling. J Cell Biol 2007,177:265-275.
15. Jin MS, Lee JO. Structures of the Toll-like Receptor Family and Its Ligand Complexes. Immunity 2008,29:182-191.
16. Watters TM, Kenny EF, O'Neill L A. Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. Immunol Cell Biol 2007,85:411-419.
17. Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, Wang GG, et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 2006,439:204-207.
18. O'Neill LA, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007,7:353-364.
19. Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL, Tong L. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 2000,408: 111-115.
20. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004,5:190-198.
21. Leifer CA, Kennedy MN, Mazzoni A, Lee C, Kruhlak MJ, Segal DM. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 2004,173: 1179-1183.
22. Latz E, Verma A, Visintin A, Gong M, Sirois CM, Klein DC, et al. Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat Immunol 2007,8: 772-779.
23. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov 2006,5:471-484.
24. Bhattacharjee RN, Akira S. Modifying Toll-like Receptor 9 Signaling for Therapeutic Use. Mini Rev Med Chem 2006,6:287-291.
25. Hoebe K, Jiang Z, Georgel P, Tabeta K, Janssen E, Du X, Beutler B. TLR signaling pathways: opportunities for activation and blockade in pursuit of therapy. Curr Pharm Des 2006,12:4123-4134.
26. Mizel SB, West AP, Hantgan RR. Identification of a sequence in human toll-like receptor 5 required for the binding of Gram-negative flagellin. J Biol Chem 2003,278:23624-23629.
27. Choe J, Kelker MS, Wilson IA. Crystal structure of human toll-like receptor 3 (TLR3) ectodomain. Science 2005,309:581-585.
28. Bell JK, Askins J, Hall PR, Davies DR, Segal DM. The dsRNA binding site of human Toll-like receptor 3. Proc Natl Acad Sci USA 2006,103:8792-8797.
29. Bell JK, Botos I, Hall PR, Askins J, Shiloach J, Segal DM, Davies DR. The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc Natl Acad Sci USA 2005,102:10976-10980.
30. Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, Segal DM, Davies DR. Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 2008,320: 379-381.
31.董燕,丁国富,李斌,和生琦,颜伟,周红,王仙园.甲氧西林耐药金黄色葡萄球菌临床分离株青霉素结合蛋白2a的克隆表达及鉴定.中华烧伤杂志2007,23:100-103.
32.和生琦,丁国富,李斌,李军,罗平,董燕, et al.青霉素结合蛋白2aC-末端在大肠杆菌M15中的表达、纯化及鉴定.创伤外科杂志2007,9:28-31.
33. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 1999,112:531-552.
34. Yibin G, Jiang Z, Hong Z, Gengfa L, Liangxi W, Guo W, Yongling L. A synthesized cationic tetradecapeptide from hornet venom kills bacteria and neutralizes lipopolysaccharide in vivo and in vitro. Biochem Pharmacol 2005,70:209-219.
35. Zhou H, Ding G, Liu W, Wang L, Lu Y, Cao H, Zheng J. Lipopolysaccharide could be internalized into human peripheral blood mononuclear cells and elicit TNF-alpha release, but not via the pathway of toll-like receptor 4 on the cell surface. Cell Mol Immunol 2004,1:373-377.
36.丁国富,王良喜,周红,刘玮.氯喹抑制LPS和CpG DNA诱导人外周血单个核细胞释放促炎细胞因子的研究.山东医药2004,44:9-11.
37.周红,罗平,郑江,鲁永玲.氯喹抑制EC DNA、内毒素协同诱导的IL-6释放.解放军医学杂志2003,28:203-205.
38. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006,124:783-801.
39. Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997,388: 394-397.
40. Uematsu S, Akira S. Toll-Like receptors (TLRs) and their ligands. Handb Exp Pharmacol 2008:1-20.
41. Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, et al. Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 2007, 130:1071-1082.
42. Nyman T, Stenmark P, Flodin S, Johansson I, Hammarstrom M, Nordlund P. The crystal structure of the human toll-like receptor 10 cytoplasmic domain reveals a putative signaling dimer. J Biol Chem 2008,283:11861-11865.
43. Ivison SM, Khan MA, Graham NR, Bernales CQ, Kaleem A, Tirling CO, et al. A phosphorylation site in the Toll-like receptor 5 TIR domain is required for inflammatory signalling in response to flagellin. Biochem Biophys Res Commun 2007, 352:936-941.
44. Andersen-Nissen E, Smith KD, Bonneau R, Strong RK, Aderem A. A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin. J Exp Med 2007,204:393-403.
45. Bella J, Hindle KL, McEwan PA, Lovell SC. The leucine-rich repeat structure. Cell Mol Life Sci 2008,65:2307-2333.
46. Matsushima N, Tachi N, Kuroki Y, Enkhbayar P, Osaki M, Kamiya M, Kretsinger RH. Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases. Cell Mol Life Sci 2005,62:2771-2791.
47. Matsushima N, Tanaka T, Enkhbayar P, Mikami T, Taga M, Yamada K, Kuroki Y. Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics 2007,8:124.
48. Dolan J, Walshe K, Alsbury S, Hokamp K, O'Keeffe S, Okafuji T, et al. The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns. BMC Genomics 2007,8:320.
49. Baroni A, Orlando M, Donnarumma G, Farro P, Iovene MR, Tufano MA, Buommino E. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes inresponse to Malassezia furfur. Arch Dermatol Res 2006,297:280-288.
50. Ishii M, Hashimoto M, Oguma K, Kano R, Moritomo T, Hasegawa A. Molecular cloning and tissue expression of canine Toll-like receptor 2 (TLR2). Vet Immunol Immunopathol 2006,110:87-95.
51. Lee J, Wu CC, Lee KJ, Chuang TH, Katakura K, Liu YT, et al. Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Proc Natl Acad Sci USA 2006,103:1828-1833.
52. Wang Y, Abel K, Lantz K, Krieg AM, McChesney MB, Miller CJ. The Toll-like receptor 7 (TLR7) agonist, imiquimod, and the TLR9 agonist, CpG ODN, induce antiviral cytokines and chemokines but do not prevent vaginal transmission of simian immunodeficiency virus when applied intravaginally to rhesus macaques. J Virol 2005,79:14355-14370.
53. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004,101:5598-5603.
54. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004,303:1526-1529.
55. de Bouteiller O, Merck E, Hasan UA, Hubac S, Benguigui B, Trinchieri G, et al. Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH. J Biol Chem 2005,280: 38133-38145.
56. Sun J, Duffy KE, Ranjith-Kumar CT, Xiong J, Lamb RJ, Santos J, et al. Structural and functional analyses of the human Toll-like receptor 3. J Biol Chem 2006,281:11144- 11151.
57. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001,413:732-738.
58. Sarkar SN, Elco CP, Peters KL, Chattopadhyay S, Sen GC. Two tyrosine residues of toll-like receptor 3 trigger different steps of NF-kappa B activation. J Biol Chem 2006.
59. Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, et al. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 2003,171:3154-3162.
60. Husebye H, Halaas O, Stenmark H, Tunheim G, Sandanger O, Bogen B, et al. Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity. Embo J 2006,25:683-692.
61. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007,13:1050-1059.
62. Lombardo E, Alvarez-Barrientos A, Maroto B, Bosca L, Knaus UG. TLR4-Mediated Survival of Macrophages Is MyD88 Dependent and Requires TNF-{alpha} Autocrine Signalling. J Immunol 2007,178:3731-3739.
63. Horng T, Barton GM, Medzhitov R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol 2001,2:835-841.
64. Kagan JC, Su T, Horng T, Chow A, Akira S, Medzhitov R. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nat Immunol 2008,9: 361-368.
65. Fitzgerald KA, Chen ZJ. Sorting out Toll signals. Cell 2006,125:834-836.
66. Kagan JC, Medzhitov R. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 2006,125:943-955.
67.王良喜,周红.细菌DNA激活免疫细胞的分子机制.中国病理生理杂志2003,19:1129-1132.
68.周红,罗平,潘文东,鲁永玲.大肠杆菌DNA诱导全身性炎症反应综合征的作用机制研究.中国病理生理杂志2004,20:1046-1050 1043.
69.潘文东,周红,郑江,夏培元,秦孝建,鲁永玲,刘晓禄.大肠杆菌DNA对小鼠致死作用的初步研究.第三军医大学学报2001,23:395-397.
70.周红,郑江,鲁永玲,罗平. CpG基元在细菌DNA诱导小白鼠THP-1释放细胞因子中的作用.第三军医大学学报2003,25:750-752.
71. Krieg AM. Immune effects and mechanisms of action of CpG motifs. Vaccine 2000,19:618-622.
72. Krieg AM, Kline JN. Immune effects and therapeutic applications of CpG motifs in bacterial DNA. Immunopharmacology 2000,48:303-305.
73. Krieg AM. CpG motifs: the active ingredient in bacterial extracts? Nat Med 2003,9:831-835.
74. Jurk M, Kritzler A, Debelak H, Vollmer J, Krieg AM, Uhlmann E. Structure-Activity Relationship Studies on the Immune Stimulatory Effects of Base-Modified CpG Toll-Like Receptor 9 Agonists. ChemMedChem 2006,1:1007-1014.
75. Leifer CA, Brooks JC, Hoelzer K, Lopez J, Kennedy MN, Mazzoni A, Segal DM. Cytoplasmic targeting motifs control localization of toll-like receptor 9. J Biol Chem 2006,281:35585-35592.
76. Kim YM, Brinkmann MM, Paquet ME, Ploegh HL. UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 2008,452:234-238.
77. Tabeta K, Hoebe K, Janssen EM, Du X, Georgel P, Crozat K, et al. The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat Immunol 2006,7:156-164.
78. Kashuba VI, Protopopov AI, Kvasha SM, Gizatullin RZ, Wahlestedt C, Kisselev LL, et al. hUNC93B1: a novel human gene representing a new gene family and encoding an unc-93-like protein. Gene 2002,283:209-217.
79. Casrouge A, Zhang SY, Eidenschenk C, Jouanguy E, Puel A, Yang K, et al. Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 2006,314:308-312.
80. Hong Z, Jiang Z, Liangxi W, Guofu D, Ping L, Yongling L, et al. Chloroquine protects mice from challenge with CpG ODN and LPS by decreasing proinflammatory cytokine release. Int Immunopharmacol 2004,4:223-234.
81.丁国富,李斌,吴翀,罗平,李军,周红. CpG ODN核心序列的改变对其生物学活性的影响.中国病理生理杂志2008,24:2025-2028.
82. Heeg K, Dalpke A, Peter M, Zimmermann S. Structural requirements for uptake and recognition of CpG oligonucleotides. Int J Med Microbiol 2008,298:33-38.
83. McCoy SL, Hausman FA, Deffebach ME, Bakke A, Merkens LS, Bennett RM, Hefeneider SH. Quantification of DNA binding to cell-surfaces by flow cytometry. J Immunol Methods 2000,241:141-146.
84. Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA, Barton GM. The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 2008.
85. Lee JH, Voo KS, Skalnik DG. Identification and characterization of the DNA bindingdomain of CpG-binding protein. J Biol Chem 2001,276:44669-44676.
86. Weber AN, Morse MA, Gay NJ. Four N-linked glycosylation sites in human toll-like receptor 2 cooperate to direct efficient biosynthesis and secretion. J Biol Chem 2004, 279:34589-34594.
87.程娟,郑江,周红,蒋栋能,吴翀,陈益国, et al.以CpG ODN为靶点应用生物传感器技术筛选抗炎中药.中国临床药理学与治疗学2005,10:1240-1244.
1. Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997,388: 394-397.
2. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006, 124:783-801.
3. Uematsu S, Akira S. Toll-Like receptors (TLRs) and their ligands. Handb Exp Pharmacol 2008:1-20.
4. Bella J, Hindle KL, McEwan PA, Lovell SC. The leucine-rich repeat structure. Cell Mol Life Sci 2008,65:2307-2333.
5. Bell JK, Mullen GE, Leifer CA, Mazzoni A, Davies DR, Segal DM. Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol 2003,24: 528-533.
6. Barton GM, Kagan JC, Medzhitov R. Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat Immunol 2006,7:49-56.
7. Brinkmann MM, Spooner E, Hoebe K, Beutler B, Ploegh HL, Kim YM. The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. J Cell Biol 2007,177:265-275.
8. Jin MS, Lee JO. Structures of the Toll-like Receptor Family and Its Ligand Complexes.Immunity 2008,29:182-191.
9. Watters TM, Kenny EF, O'Neill L A. Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. Immunol Cell Biol 2007,85:411-419.
10. Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, Wang GG, et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 2006,439:204-207.
11. O'Neill LA, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007,7:353-364.
12. Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL, Tong L. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 2000,408: 111-115.
13. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004,5:190-198.
14. Leifer CA, Kennedy MN, Mazzoni A, Lee C, Kruhlak MJ, Segal DM. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 2004,173: 1179-1183.
15. Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, et al. Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 2007,130:1071-1082.
16. Nyman T, Stenmark P, Flodin S, Johansson I, Hammarstrom M, Nordlund P. The crystal structure of the human toll-like receptor 10 cytoplasmic domain reveals a putative signaling dimer. J Biol Chem 2008,283:11861-11865.
17. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004,4:499-511.
18. Ivison SM, Khan MA, Graham NR, Bernales CQ, Kaleem A, Tirling CO, et al. A phosphorylation site in the Toll-like receptor 5 TIR domain is required for inflammatory signalling in response to flagellin. Biochem Biophys Res Commun 2007,352:936-941.
19. Mizel SB, West AP, Hantgan RR. Identification of a sequence in human toll-like receptor 5 required for the binding of Gram-negative flagellin. J Biol Chem 2003,278: 23624-23629.
20. Andersen-Nissen E, Smith KD, Bonneau R, Strong RK, Aderem A. A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin. J Exp Med 2007,204: 393-403.
21. Baroni A, Orlando M, Donnarumma G, Farro P, Iovene MR, Tufano MA, Buommino E. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur. Arch Dermatol Res 2006,297:280-288.
22. Ishii M, Hashimoto M, Oguma K, Kano R, Moritomo T, Hasegawa A. Molecular cloning and tissue expression of canine Toll-like receptor 2 (TLR2). Vet Immunol Immunopathol 2006,110:87-95.
23. Lee J, Wu CC, Lee KJ, Chuang TH, Katakura K, Liu YT, et al. Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Proc Natl Acad Sci USA 2006,103:1828-1833.
24. Wang Y, Abel K, Lantz K, Krieg AM, McChesney MB, Miller CJ. The Toll-like receptor 7 (TLR7) agonist, imiquimod, and the TLR9 agonist, CpG ODN, induce antiviral cytokines and chemokines but do not prevent vaginal transmission of simian immunodeficiency virus when applied intravaginally to rhesus macaques. J Virol 2005, 79:14355-14370.
25. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004, 101:5598-5603.
26. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004,303:1526-1529.
27. de Bouteiller O, Merck E, Hasan UA, Hubac S, Benguigui B, Trinchieri G, et al. Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH. J Biol Chem 2005,280: 38133-38145.
28. Sun J, Duffy KE, Ranjith-Kumar CT, Xiong J, Lamb RJ, Santos J, et al. Structural and functional analyses of the human Toll-like receptor 3. J Biol Chem 2006,281: 11144-11151.
29. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-strandedRNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001,413:732-738.
30. Sarkar SN, Elco CP, Peters KL, Chattopadhyay S, Sen GC. Two tyrosine residues of toll-like receptor 3 trigger different steps of NF-kappa B activation. J Biol Chem 2006.
31. Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, et al. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 2003,171: 3154-3162.
32. Husebye H, Halaas O, Stenmark H, Tunheim G, Sandanger O, Bogen B, et al. Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity. Embo J 2006,25:683-692.
33. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007,13:1050-1059.
34. Lombardo E, Alvarez-Barrientos A, Maroto B, Bosca L, Knaus UG. TLR4-Mediated Survival of Macrophages Is MyD88 Dependent and Requires TNF-{alpha} Autocrine Signalling. J Immunol 2007,178:3731-3739.
35. Horng T, Barton GM, Medzhitov R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol 2001,2:835-841.
36. Kagan JC, Su T, Horng T, Chow A, Akira S, Medzhitov R. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nat Immunol 2008,9: 361-368.
37. Fitzgerald KA, Chen ZJ. Sorting out Toll signals. Cell 2006,125:834-836.
38. Kagan JC, Medzhitov R. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 2006,125:943-955.
39. Choe J, Kelker MS, Wilson IA. Crystal structure of human toll-like receptor 3 (TLR3) ectodomain. Science 2005,309:581-585.
40. Bell JK, Askins J, Hall PR, Davies DR, Segal DM. The dsRNA binding site of human Toll-like receptor 3. Proc Natl Acad Sci USA 2006,103:8792-8797.
41. Bell JK, Botos I, Hall PR, Askins J, Shiloach J, Segal DM, Davies DR. The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc Natl Acad Sci USA 2005,102:10976-10980.
42. Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, Segal DM, Davies DR. Structuralbasis of toll-like receptor 3 signaling with double-stranded RNA. Science 2008,320: 379-381.
43. Heeg K, Dalpke A, Peter M, Zimmermann S. Structural requirements for uptake and recognition of CpG oligonucleotides. Int J Med Microbiol 2008,298:33-38.
44. Dunzendorfer S, Lee HK, Soldau K, Tobias PS. Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS responses. Faseb J 2004,18:1117-1119.
45. Dunzendorfer S, Lee HK, Soldau K, Tobias PS. TLR4 is the signaling but not the lipopolysaccharide uptake receptor. J Immunol 2004,173:1166-1170.
46. Visintin A, Mazzoni A, Spitzer JH, Wyllie DH, Dower SK, Segal DM. Regulation of Toll-like receptors in human monocytes and dendritic cells. J Immunol 2001,166: 249-255.
47. Lee JH, Voo KS, Skalnik DG. Identification and characterization of the DNA binding domain of CpG-binding protein. J Biol Chem 2001,276:44669-44676.