草鱼脂多糖结合蛋白的分离纯化(英文)

脂多糖结合蛋白(lipopolysaccharide binding proteins ,LBP)是机体对革兰氏阴性菌感染产生的可溶性急性期蛋白,结合并提呈LPS给细胞表面的模式识别受体,激发免疫反应。在本试验中,用低浓度嗜水气单胞菌( Aeromonas hydrophila)感染草鱼(Ctenopharyngodon idellus)24 h后,抽取全血,离心,获得血浆。结合LBP活性检测,经硫酸铵沉淀、CMSephadex C-50阳离子交换层析和DEAE Sephadex A-25阴离子交换层析后,分离纯化得到LBP。该蛋白对异硫氰酸荧光素标记的脂多糖(Fluorescein isothiocyanate labeled li-popolysaccharide ,FITC-LPS)有较强的结合能力。在SDS-PAGE电泳后,考马斯亮蓝染色可见3条明显的带,分子量大约为68、53和48 kDa。同时探索一条简便分离纯化脂多糖结合蛋白的方法,为进一步研究LBP的功能奠定了基础。     

脂多糖刺激对断奶仔猪脾脏、胸腺和外周血白细胞PPARγ蛋白表达的影响

 【目的】研究免疫应激对断奶仔猪脾脏、胸腺和外周血白细胞过氧化物酶体增殖物活化受体γ（PPARγ）蛋白表达水平的影响。【方法】选取12头健康断奶仔猪,分成2组,每组6个重复。试验组注射100 μg•kg-1 BW的脂多糖（LPS）建立免疫应激模型,对照组注射等量生理盐水。注射后3 h,采血分离血浆和白细胞待测,然后立即屠宰仔猪,取脾脏和胸腺,通过Western blot 测定脾脏、胸腺和白细胞中PPARγ蛋白表达水平。【结果】LPS刺激仔猪,导致血浆白细胞介素-6﹑肿瘤坏死因子-α、皮质醇和前列腺素E2含量显著升高（P＜0.05）,胰岛素、类胰岛素生长因子-1含量显著降低（P＜0.05）;LPS刺激导致血浆葡萄糖、总蛋白、白蛋白和球蛋白含量显著降低（P＜0.05）;LPS刺激导致脾脏（P＜0.05）、胸腺（P＜0.1）和白细胞（P＜0.05）PPARγ蛋白表达水平升高。【结论】脂多糖剌激可提高仔猪脾脏、胸腺和外周血白细胞PPARγ蛋白的表达,显示PPARγ可能参与仔猪免疫应激的调控,可能成为缓解仔猪免疫应激的一个新靶点。     

家蚕整合素β2的表达、纯化及其免疫功能

【目的】 分析家蚕（Bombyx mori）整合素β2的序列和结构特征及其在家蚕感染病原菌后血细胞中的表达变化,检测其重组蛋白对不同病原相关模式分子识别和病原菌的凝聚作用,为进一步探究家蚕整合素β2的蛋白功能打下基础。【方法】 利用生物信息学对整合素β2的序列和结构特征进行分析,利用实时荧光定量PCR检测家蚕分别注射大肠杆菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus）后整合素β2在血细胞中的表达变化。通过PCR技术扩增获得整合素β2完整的胞外域片段,构建至pET22b原核表达载体后转化至E.coli Rosetta(DE3)表达菌株。经IPTG诱导获得重组蛋白,利用Ni-NTA亲和层析得到纯化的体外重组蛋白,利用SDS-PAGE和Western blot对纯化获得的体外重组蛋白的纯度和质量进行检测。利用ELISA检测重组蛋白与两种不同病原相关分子模式LPS和PGN的结合情况,运用Western blot检测重组蛋白与不同病原微生物的结合情况,通过凝集试验检测重组蛋白对金黄色葡萄球菌的凝集能力,最后在个体水平探索重组蛋白在体内细菌清理中的作用。【结果】 家蚕整合素β2具有典型的β整合素亚基保守结构特征,即由一个较长的胞外域、一个单次跨膜结构域和一个较短的胞内域构成。家蚕整合素β2具有金属离子结合位点MIDAS、EGF结构域、半胱氨酸重复基序和NPxY等整合素典型的结构特征。实时荧光定量PCR检测结果表明家蚕在受到细菌感染后,整合素β2的表达发生显著变化。通过原核表达和蛋白纯化获得纯度较高的重组蛋白,SDS-PAGE和Western blot检测结果表明纯化的重组蛋白纯度较高,可以用于后续试验。ELISA试验表明重组蛋白对LPS和PGN等病原相关分子模式具有较强的结合能力。细菌结合试验结果显示重组蛋白能够结合多种细菌,但与革兰氏阳性菌的结合能力要高于革兰氏阴性菌。细菌凝聚试验结果显示重组蛋白在Ca ²⁺的存在下对金黄色葡萄球菌具有较强的凝聚作用。细菌清除试验证实重组蛋白可以有效促进机体对外源入侵细菌的清理作用。【结果】 整合素β2具有典型的整合素β亚基的结构特征,可能具有识别病原相关分子模式,如LPS和PGN等的能力,通过与细菌的直接结合而实现对入侵病原微生物的凝聚作用,从而增强有机体的免疫能力,推测在家蚕免疫反应中发挥重要作用。     

不同分子量枸杞多糖体外抗氧化活性研究

[目的]研究不同分子量枸杞多糖的体外抗氧化活性。[方法]采用膜分离技术对枸杞多糖(LBP)进行分离,以得到分子量<1万(LBP1)、1万～3万(LBP2)、3万～5万(LBP3)、>5万(LBP4)的多糖,并对LBP及这4种小分子枸杞多糖的体外抗氧化活性进行测定。[结果]不同分子量的小分子多糖抗氧化活性均高于粗多糖,其中LBP2清除超氧阴离子自由基作用、清除羟自由基活性、总抗氧化能力最强,显著高于LBP。[结论]为合理开发与高效利用枸杞多糖提供了新方法。     

iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS

Innate immune responses are vital for pathogen defense but can result in septic shock when excessive. A key mediator of septic shock is tumor necrosis factor-α (TNFα), which is shed from the plasma membrane after cleavage by the TNFα convertase (TACE). We report that the rhomboid family member iRhom2 interacted with TACE and regulated TNFα shedding. iRhom2 was critical for TACE maturation and trafficking to the cell surface in hematopoietic cells. Gene-targeted iRhom2-deficient mice showed reduced serum TNFα in response to lipopolysaccharide (LPS) and could survive a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to control the replication of Listeria monocytogenes. Our study has identified iRhom2 as a regulator of innate immunity that may be an important target for modulating sepsis and pathogen defense.     

虾青素对牛子宫内膜细胞炎症损伤的保护作用

【目的】利用虾青素较强的抗氧化特性,通过探讨黏膜屏障重塑对脂多糖（LPS）诱导的牛子宫内膜细胞炎症的影响,为牛子宫内膜细胞炎的预防和治疗提供理论基础。【方法】培养液中添加1 μg·mL ^-1 LPS,培养子宫内膜细胞6 h,检测培养液中炎症因子TNF-α和IL-6含量,建立细胞的体外炎症模型。在此基础上去掉培养液,重新加入含1×10 ^-6 mol·L ^-1 虾青素的新鲜培养液,继续培养细胞24 h。对照组为不添加LPS或AST,LPS组为仅添加LPS,AST组为添加LPS和AST。采用ELISA法检测培养液中TNF-α和IL-6炎症因子分泌量以及细胞SOD和CAT酶活性,利用流式细胞仪检测细胞内活性氧簇（ROS）水平,利用免疫荧光染色检测细胞紧密连接蛋白Claudin、CDH1、TJP1的分布情况,利用荧光定量RT-PCR法和Western Blot法分别检测Claudin、CDH1、TJP1基因的mRNA表达水平和蛋白表达水平。【结果】利用1 μg·mL ^-1 LPS培养子宫内膜细胞6 h,检测发现培养液中炎症因子IL-6和TNF-α分泌水平显著高于对照组（P小于0.05）,说明LPS诱导子宫内膜细胞产生炎症反应。与对照组相比,细胞内ROS水平显著增加（P小于0.05）,SOD和CAT酶活性都显著降低（P小于0.05）,说明LPS诱导的子宫内膜细胞炎症造成了氧化损伤。加入1×10 ^-6 mol·L ^-1 虾青素培养细胞24 h后,发现培养液中IL-6和TNF-α因子的分泌水平显著低于LPS组（P小于0.05）,同时ROS阳性细胞比率显著下降（P小于0.05）,SOD和CAT酶活性都显著升高（P小于0.05）。LPS组与对照组相比,细胞膜处Claudin、CDH1、TJP1蛋白的荧光信号减弱,这3种紧密连接蛋白的mRNA和蛋白表达水平也都显著降低（P小于0.05）。加入1×10 ^-6 mol·L ^-1虾青素培养24 h后,与LPS组相比,在细胞边缘和细胞核内可以检测到荧光信号很强的Claudin、CDH1、TJP1蛋白,而且这3种紧密连接蛋白的mRNA和蛋白表达水平也都显著升高（P小于0.05）。【结论】虾青素通过降低子宫内膜细胞因炎症反应产生的氧化损伤,减轻炎症导致的子宫内膜细胞紧密连接结构损伤,对子宫内膜的物理免疫屏障起保护作用,为牛子宫内膜炎的预防和治疗提供理论借鉴意义。     

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.     

枸杞多糖的提取分离及其组成研究

对枸杞多糖的提取分离及其组成进行了研究,结果表明:枸杞经氯仿-甲醇脱脂,水提醇沉,乙醇、丙酮和乙醚洗涤干燥,双氧水脱色,savage法除蛋白后可得枸杞多糖;高效液相色谱测定枸杞多糖分子量为:73 950～138 090 Da;枸杞多糖中单糖分别有葡萄糖、阿拉伯糖、鼠李糖、半乳糖、木糖和甘露糖,其中含量较多的是葡萄糖、阿拉伯糖和半乳糖,其他单糖含量较低。     

Lactic Acid Inhibits NF-κB Activation by Lipopolysaccharide in Rat Intestinal Mucosa Microvascular Endothelial Cells

To investigate whether lactic acid could inhibit the LPS-activation of NF-κB p65 in rat intestinal mucosa microvascular endothelial cells (RIMMVECs), RIMMVECs, cultured in vitro, were pretreated with different concentrations of lactic acid and then exposed to lipopolysaccharide (LPS). Cells and cell culture media were then collected at different time intervals. Production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was examined at the protein level by enzyme-linked immunosorbent assay. The influence of lactic acid on the LPS-activation of NF-κB was examined at mRNA and protein levels by real-time quantitative PCR and Western blot analysis, respectively. TNF-α and IL-6 protein levels were significantly decreased after pretreatment with lactic acid compared with cells exposed to LPS only. After pretreatment with 7.5, 5.0, and 2.5 μL mL-1 lactic acid, NF-κB mRNA levels were increased by 1.51-, 2.62- and 3.00-fold, respectively, compared with levels in control cells without LPS treatment. Western blot analysis indicated that the level of NF-κB p65 in the lactic acidpretreated group was significantly lower than that in the group treated with LPS only (positive control) and was unchanged compared with the group without LPS treatment (blank control). These results suggest that lactic acid may inhibit LPSactivation of NF-κB, leading to the down-regulation of TNF-α and IL-6.     

阴外动脉灌注脂多糖对泌乳奶牛动脉血液中脂肪酸组成、摄取及血液脂质指标的影响

研究阴外动脉灌注脂多糖(LPS)对泌乳奶牛动脉血液中脂肪酸组成、摄取及血液脂质指标的影响.选用6头经产的荷斯坦奶牛(BW=(576.33±36.67)kg,DIM=(185.67±30.11)d),随机分成2组.采用交叉试验设计,每期试验7d,间隔期14d;试验组阴外动脉灌注LPS(Escherichia coli 0...     

培养时间对柱状嗜纤维菌脂多糖蛋白质成分和免疫原性的影响

从培养不同时间后的柱状嗜纤维菌中提取菌体脂多糖 (LPS) ,测定了其中蛋白质含量及成分的变化 ,并分别作为免疫原接种翘嘴鳜对其免疫原性进行了比较。结果表明 ,培养时间对菌体LPS中蛋白质含量没有明显影响 ,对其蛋白质成分则有一定影响 ,而菌体LPS中蛋白质成分与其免疫原性有关 ,从培养 36h的菌体中提取的菌体LPS的免疫原性最强     

The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4

The inflammatory toxicity of lipopolysaccharide (LPS), a component of bacterial cell walls, is driven by the adaptor proteins myeloid differentiation factor 88 (MyD88) and Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta (TRIF), which together mediate signaling by the endotoxin receptor Toll-like receptor 4 (TLR4). Monophosphoryl lipid A (MPLA) is a low-toxicity derivative of LPS with useful immunostimulatory properties, which is nearing regulatory approval for use as a human vaccine adjuvant. We report here that, in mice, the low toxicity of MPLA's adjuvant function is associated with a bias toward TRIF signaling, which we suggest is likely caused by the active suppression, rather than passive loss, of proinflammatory activity of this LPS derivative. This finding may have important implications for the development of future vaccine adjuvants.     

不同血清型柱状嗜纤维菌脂多糖蛋白质成分及其对鳜的免疫原性影响的研究

从３种不同血清型的柱状嗜纤维菌中提取菌体脂多糖（ＬＰＳ）,测定了其中蛋白质含量及其成分的变化,并分别作为免疫原接种翘嘴鳜,对ＬＰＳ的免疫原性进行了比较。结果表明,不同血清型的柱状嗜纤维菌菌体ＬＰＳ中蛋白质含量没有明显差别（ｔ检测,Ｐ〉０．０５）,而其中蛋白质成分则有明显的血清型特异性。不同血清型的菌体ＬＰＳ对翘嘴鳜的免疫原性没有明显差别（ｔ测验,Ｐ〉０．０５）。     

基于LBP纹理和改进Camshift算子的车辆检测与跟踪

提出了利用背景图像LBP（局部二值模式）纹理和当前帧图像LBP纹理的相似度分析提取前景的方法,克服了车辆检测中常用的帧差法、背景差分法对光照比较敏感的缺点.同时基于H,S,V分量及改进的LBP纹理的联合直方图与金字塔L-K光流法中心跟踪相结合的Camshift跟踪算法,有效地解决了背景目标颜色相近可能会导致跟踪的目标区域加入背景后变大、处理较大帧间位移的视频跟踪上搜索窗口的位置准确度较低的问题.实验证明,该方法具有良好的检测和追踪效果.     

Effects of yeast and yeast cell wall polysaccharides supplementation on beef cattle growth performance,rumen microbial populations and lipopolysaccharides production

This experiment was conducted to investigate the effects of live yeast and yeast cell wall polysaccharides on growth performance, rumen function and plasma lipopolysaccharides(LPS) content and immunity parameters of beef cattle. Forty Qinchuan cattle were randomly assigned to one of four treatments with 10 replicates in each treatment. The dietary treatments were: control diet(CTR), CTR supplemented with 1 g live yeast(2×1010 live cell g–1 per cattle per day(YST1), CTR supplemented with 2 g live yeast per cattle per day(YST2) and CTR supplemented with 20 g of yeast cell wall polysaccharides(30.0%≤β-glucan≤35.0%, and 28.0%≤mannanoligosaccharide≤32.0%) per cattle per day(YCW). The average daily gain was higher(P=0.023) and feed conversion ratio was lower(P=0.042) for the YST2 than the CTR. The digestibility of neutral detergent fiber(P=0.039) and acid detergent fiber(P=0.016) were higher in yeast supplemented groups. The acetic acid:propionic acid of the YST2 was lower compared with the CTR(P=0.033). Plasma LPS(P=0.032), acute phase protein haptoglobin(P=0.033), plasma amyloid A(P=0.015) and histamine(P=0.038) were lower in the YST2 compared with the CTR. The copies of fibrolytic microbial populations such as Fibrobacter succinogenes S85, Ruminococcus albus 7 and Ruminococcus flavefaciens FD-1 of the YST2 were higher(P0.001), while the copies of typical lactate producing bacteria Streptococcus bovis JB1 was lower(P0.001) compared with the CTR. Little differences were observed between the CTR, YST1 and YCW in growth performance, ruminal fermentation characteristics, microbial populations, immunity indices and total tract nutrient digestibility. It is concluded that the YST2 could promote fibrolytic microbial populations, decrease starch-utilizing bacteria, reduce LPS production in the rumen and LPS absorption into plasma and decrease inflammatory parameters, which can lead to an improvement in growth performance in beef cattle.     

原花青素对脂多糖诱导的环氧合酶-2表达的抑制作用

以脂多糖(LPS)诱导的RAW264.7细胞为模型,研究原花青素对LPS诱导的小鼠RAW264.7细胞中环氧合酶-2(COX-2)mRNA转录的抑制机制。采用RT-PCR法测定原花青素对LPS诱导的RAW264.7细胞中COX-2mRNA转录的影响,采用Western blot和免疫组化法考察原花青素对LPS诱导的RAW264.7细胞核转录因子κB(NF-κB)亚基p65(NF-κB/p65)及NF-κB结合蛋白(I-κB)表达的影响。结果发现LPS处理RAW264.7细胞可以明显上调COX-2 mRNA的表达,同时降低胞质蛋白I-κB的表达水平,增加NF-κB的水平。原花青素对RAW264.7细胞中COX-2 mRNA的转录有较强抑制作用,抑制NF-κB/p65的表达及I-κB的降解,原花青素可能是通过抑制NF-κB/p65的表达及I-κB/p65的降解而抑制COX-2的表达。     

Drug-induced change in the distribution of sulfonamides in the mother rat and its fetus

The distribution of a highly bound antibacterial sulfonamide was markedly altered in both the mother rat and its fetus by interfering with the binding of this drug to plasma protein in the mother. This effect was due to binding displacement, since the displacing agent had little or no effect on the distribution of another sulfonamide with very low binding to plasma protein.     

The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.