ELISA方法研究多糖与人干扰素-γ相互作用

【作者】 冯伟云；

【导师】 赵鲁杭；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 [目的] 肝素是由N-乙酰葡萄糖胺与D-葡萄糖醛酸以1→4糖苷键连接起来的重复二糖单位组成的线性多糖，葡萄糖胺可带有N-硫酸基团，可在C6上形成0-硫酸酯，带有硫酸基团的独特结构参与了肝素与其他分子的相互作用。肝素能够与许多蛋白质结合并调节蛋白活性，包括细胞因子、生长因子、趋化因子、抗血管生成因子、粘附分子和补体成分等，肝素通过与蛋白相互作用发挥其生物学活性。肝素作为抗凝血试剂广泛地用于临床各种疾病并发的弥散性血管内凝血、心脏手术的体外循环、血栓形成或栓塞的防治。除了抗凝血活性外，肝素还具有调节免疫功能、抑制炎症反应、抑制肿瘤细胞的生成和转移及抗病毒等功能，但是肝素的抗凝血活性及出血性并发症限制了肝素抗炎症和抗肿瘤等其他生物学活性的临床应用。因此，我们试图寻找一些硫酸多糖来替代肝素的一些生物学活性而避免肝素的副作用。 以往主要是通过Sepharose-heparin亲和层析来研究肝素与蛋白的相互作用；20世纪90年代又发展了affinity-coelectrophoresis(ACE)技术研究蛋白质与肝素的相互作用。但亲和层析柱易被污染失活，ACE技术不能提供生理pH范围和离子强度及ACE技术必需的标记肝素的放射性物质对人和环境具有很大危害。为了避免这些不足及更加直观地反映肝素与蛋白的相互作用，本课题组通过化学方法合成肝素-牛血清白蛋白复合物(HBC)，通过牛血清白蛋白将肝素包被到酶标板上，建立了一种酶联免疫吸附实验(ELISA)技术来体外检测肝素与IFN-γ的结合，为进一步研究其他多糖与浙江大学硕士学位论文蛋白质的结合特性提供简单、灵敏的方法。〔方法〕 与蛋白多肤不同，糖链很难吸附在酶标板上，为了用ELI SA方法研究肝素与于扰素一Y的相互作用，肝素和牛血清白蛋白(日SA)在硼氢氰化钠作用下合成肝素一牛血清白蛋白复合物(lIBC)。反应产物经Sephorose4B凝胶过滤层析系统分离纯化日BC，除去反应产物中游离的肝素和l，SA。纯化的HBC经SDS一队GE鉴定，选择纯度最高的HBC组分，通过I3CA蛋白定量试剂盒测定HBC中的蛋白含量。 将HBC(相当于0.5拜g蛋白含量)包被在酶标板上，ELISA方法检测肝素与IFN一Y的相互作用，用相同蛋白含量的BSA一对照作为对照。为了进一步研究游离肝素(heparin)、低分子量肝素(LMw素A(CS一A)、硫酸软骨素C(CS一C)、(HA)、hepa五n)、硫酸软骨硫酸葡聚糖(DxS)、葡聚糖(Dx)、(Carrageenans)多聚半乳糖胺、等多糖对肝素与 透明质酸岩藻聚糖(fueoidan)以及卡拉胶IFN一Y结合的影响，进行了竞争性抑制ELISA实验。分析了多糖结构和硫酸基团在多糖与正N一Y相互作用中的作用。〔结果」 凝胶过滤层析获得了高纯度的HBC，通过HBC将肝素包被在酶标板上，EllSA实验表明，IFN一Y主要与HBC中的肝素结合(p(O，01)。IFN一Y与肝素的结合呈IFN一Y剂量依赖性，Zng左右达到饱和。游离肝素和低分子量肝素以及其他多糖均不同程度地抑制了IFN一Y与肝素的结合，各种多糖竞争性抑制的Ics。分别为游离肝素(2 .4拼留ml)，低分子量肝素(18.6环岁ml)，es一A(>500拜g/ml)，CS一e(巧00拜g/ml)，HA(247.13环g/ml)，岩藻聚糖(0.0177拜g/ml)，卡拉胶一K(8.33林留ml)，卡拉胶爪(0.17井g/ml)，卡拉胶一(6 .16拜g/ml)。〔结论}浙江大学硕士学位论文 多糖与工FN一Y的结合是一个复杂的过程，EL工SA是一个简单、灵敏的体外研究蛋白与多糖相互作用的方法，能够在纳克水平检测到工FN一Y与肝素的结合。工FN一Y与肝素、低分子量肝素、DxS、岩藻聚糖及卡拉胶高亲和力结合，而与CS一A、CS一C、HA、Dx结合力极弱。更多还原

【Abstract】 [Objective]Heparin is linear polysaccharides consisting of repeating disaccharide unit backbones of N-acetyl-D-glucosamine that is linked to D-glucuronic acid. The disaccharide repeat unit can be modified to include N-and O-sulfation (6-O and 3-O sulfation of the glucosamine and 2-O sulfation of the uronic acid). These specific sulfate patterns are involved in the interaction of heparin with other molecules. Heparin is known to bind and modulate the activity of various proteins, including cell growth factors, cytokines, angiogenic factors, complement components, adhesion molecules, heparin exerts its activities via interaction with some proteins. Potent anticoagulant activity of heparin by the interaction with antithrombin III is extensively used in medicine. In addition to its traditional anticoagulant activity, heparin has been shown to inhibit various aspects of the inflammatory response such as lymphocyte trafficking and neutrophil chemotaxis, modulate various aspects of immune function via multiple mechanisms and inhibit the metastasis of tumour cells via inhibiting the activities of endoglycosidase heparanase and angiogenesis. In addition, heparin has been shown to potently inhibit HIV -1 replication and affect cell growth, gene expression, modulate clinically relevant event such as diabetes and atherosclerosis. However, the clinical application of heparin for the treatment of both cancer and various inflammatory is limited by its anticoagulant activity. So we want to know whether LMW heparin and some polysaccharides from algae substitute heparin at some functions avoiding its side effects.A Sepharose-heparin column (a affinity chromatoraphic column) was used to study the binding of heparin to protein previously. In the 1990’s, a sensitive technique developed for the study of the potential interaction of cytokine with heparin and related polysaccharides is affinity coelectrophoresis (ACE). Butthese techniques can not provide physiological pH and ionic strength, and radio-iodination of heparin required for ACE endangers health of human and environment. For avoiding the radiological hazards and reflecting directly the binding characteristic of heparin to protein. Synthesis of heparin-BSA complex used a chemistry method, established an ELISA technique to detect the interaction of heparin with protein in vitro. ELISA is a simple, effective method to study the interaction of polysaccharides and proteins.[Methods]Consideration of free heparin, unlike polypeptides, adsorb poorly onto plastic surfaces. In order to study the interaction of heparin with IFN- Y using ELISA assay, heparin-bovine complex (HBC) was synthesized by conjugating heparin to BSA in the presence of sodium cyanoborohydride. For removing the free heparin and free BSA, The reaction products were resolved by a 1 X 90 cm gel filtration column on Sepharose 4B, the elution of high Mr HBC was identified by SDS-PAGE, stained for heparin with Azure A and for protein with Coomassie blue. HBC was quantified for protein using the bicinchoninic acid assay (BCA reagent Kit).ELISA plate wells were coated either with HBC (0.5 g BSA equivalent )or with the same amount of mock-treated BSA, detected the binding of HBC to IFN- . For further observation the influence of some polysaccharides including free heparin, LMW heparin, CS-A, CS-C, HA, DxS, fucoidan and carrageenan family on the interaction of heparin with IFN-, 1.5 ng IFN- y waspreincubated at 37 for 30 mins in the presence and absence of different concentration of these polysaccharides before addition to coated well.[Results]We got HBC with high purity by Sepharose-4B gel filtration chromatography, and confirmed the important role of heparin in the interaction of HBC withIFN- γ with ELISA assay. The binding of IFN-γ to HBC is readily detectable at as little as 0.25 ng IFN- γ /well, increases in a cytokine dose-dependent manner, and saturates at around 2ng/well. Free heparin, LMW heparin and other polysaccharides inhibit the binding of HBC to IFN-γ with significant更多还原