CN1890264A - Gag结合蛋白 - Google Patents

Gag结合蛋白 Download PDF

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CN1890264A
CN1890264A CNA2004800361606A CN200480036160A CN1890264A CN 1890264 A CN1890264 A CN 1890264A CN A2004800361606 A CNA2004800361606 A CN A2004800361606A CN 200480036160 A CN200480036160 A CN 200480036160A CN 1890264 A CN1890264 A CN 1890264A
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安德烈亚斯·J·孔格尔
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Abstract

本发明提供了一种将GAG结合位点导入蛋白的方法，包括步骤：－鉴定出蛋白中对于维持结构而言非必需的区；－将至少一个碱性氨基酸导入所述位点和/或在所述位点中删除至少一个体积大的和/或酸性的氨基酸，其中所述GAG结合位点的GAG结合亲和力为Kd≤10μM，优选≤1μM，更优选≤0.1μM，本发明还涉及修饰的GAG结合蛋白。

Description

GAG结合蛋白
本发明涉及抑制趋化因子与它们在白细胞上的高亲和力受体结合的方法和工具(tool)，以及治愈性治疗(therapeutic treatment)炎症疾病的方法。
趋化因子最初衍生自化学吸引细胞因子(chem.oattractant cytokine)，实际上包括的成员多于50种，代表低分子量(单体形式为6-12kDa)、可诱导型、分泌型小蛋白家族，这些蛋白在免疫监视和炎症过程中起决定性作用。根据它们在免疫和炎症中的作用，可将它们分成两类。炎性趋化因子由许多不同的组织细胞产生，也可以由白细胞应答细菌毒素和炎性细胞因子如IL-1、TNF和干扰素而发生迁移时产生。它们的主要功能是，为了宿主防御的目的募集白细胞以及在炎症过程中募集白细胞。另一方面，归巢趋化因子(homing chemokine)在淋巴组织的限定区域内组成性表达。它们指导淋巴细胞和树突细胞在免疫系统内运输和归巢。这些趋化因子，例如BCA-1，SDF-1或SLC，控制淋巴细胞在成熟、分化、活化时的再定位和再循环，确保它们在二级淋巴器官内正确归巢。
趋化因子有众多的代表，它们的序列同源性为20-70％不等，但它们显示有明显相似的结构性折叠。趋化因子由约70-130个氨基酸构成，有四个保守的半胱氨酸残基。这些半胱氨酸形成两个二硫键(Cys1→Cys3，Cys2→Cys4)，它们负责其特有的三维结构。趋化性细胞因子的构成如下：短氨基末端域(3-10个氨基酸)，之后是第一个半胱氨酸残基，位于第二和第四个半胱氨酸残基之间、由β链和连接环构成的核心，以及，20-60个氨基酸的羧基末端α螺旋。该蛋白核心具有非常规则的结构，但N-和C-末端部分不规则。由于是分泌蛋白，因此它们在合成时含有20-25个氨基酸的前导序列，该前导序列在释放前被切除。
趋化因子基于其半胱氨酸残基在成熟蛋白中的相对位置可细分为4个家族。在α-趋化因子亚家族中，四个半胱氨酸中的前两个半胱氨酸被一个氨基酸(CXC)隔开，而在β-趋化因子中，相应的半胱氨酸彼此相邻(CC)。α-趋化因子可进一步分为：在N末端含有ELR序列、从而可以趋向中性粒细胞的α-趋化因子(例如IL-8)，和缺少ELR基序并作用于淋巴细胞的α-趋化因子(例如I-TAC)。从结构上看，β-趋化因子可以细分为两个家族：单核细胞-趋化蛋白eotaxin家族和其它β-趋化因子，前者包括五种单核细胞-趋化蛋白(MCP)和eotaxin(它们彼此有近65％相同)。在CXX-家族，CC-残基之前的N末端氨基酸对这些趋化因子的生物活性和白细胞选择性而言是关键成分。通常，β-趋化因子不对嗜酸性粒细胞起作用，而是以不同选择性吸引单核细胞、嗜酸性粒细胞、嗜碱性粒细胞和淋巴细胞。
仅有少数趋化因子不适合归类为CC-或CXC-家族。Lymphotactin是迄今仅有的在其一级结构中只有两个而非四个特征性半胱氨酸的趋化因子，它因此被归为γ-或C-趋化因子。另一方面，通过总结此分类法，fractalkine被认为是δ-或CXXXC-亚家族的唯一代表，其在前两个半胱氨酸之间有三个氨基酸。Lymphotaxin和fractalkine都诱导T-细胞和自然杀伤细胞的趋化性。
趋化因子通过与特异性细胞表面即靶细胞表面7个跨膜(7TM)G蛋白偶联受体结合而诱导细胞迁移和活化。迄今克隆的18种趋化因子受体包括6种CXC，10种CC，1种CX3C和1种XC受体。趋化因子受体在不同类型的白细胞上表达，其中一些受体限于一些细胞(例如，CXCR1限于中性粒细胞)，而其它受体则更广泛地表达(例如，CCR2在单核细胞、T细胞、自然杀伤细胞和嗜碱粒细胞上表达)。与趋化因子相似，受体可以在一些细胞上组成性表达，另一些为可诱导型。还有一些甚至可以被下调，从而使细胞不应答某个趋化因子但能应答其它趋化因子。大多数受体识别不止一种趋化因子，反之亦然，但识别限于相应亚家族的趋化因子(参见表1)。
                                           表1
         趋化因子   受体  趋向于   炎症疾病
CXC-趋化因子(+ELF-基序) IL-8 CXCR1CXCR2 中性粒细胞   急性呼吸窘迫综合症[71]；细菌性肺炎[72]；类风湿性关节炎[73]；炎症性肠病[74]；牛皮癣[75]；细菌性脑膜炎[76]
CC-趋化因子 MCP-1 CCR2 嗜碱性粒细胞；单核细胞；活化的T细胞；树突细胞；自然杀伤细胞   哮喘[77]；肾小球性肾炎[78]；动脉硬化症[79]；炎症性肠病[80]；牛皮癣[81]；细菌性和病毒性脑膜炎[82，83]
  RANTES CCR1  嗜酸性粒细胞；单核细胞；活化的T细胞；树突细胞   哮喘[84]；肾小球性肾炎[85]
  CCR3  嗜酸性粒细胞；嗜碱性粒细胞；树突细胞
CCR5  单核细胞；活化的T细胞；树突细胞；自然杀伤细胞
趋化因子有两个主要的与受体相互作用的位点，一个位点在氨基末端域内，另一个位点在第二和第三个半胱氨酸残基间扩展的骨架的暴露环内。这两个位点通过二硫键保持密切接近。受体首先识别环区域内的结合位点，该位点看来作为停靠区(docking domain)起作用。此相互作用限制了趋化因子的活动性，从而便于氨基末端结构域的正确取向。有人对那些仍然有效结合其受体但并不传导信号的突变趋化因子进行了研究。这些突变通过在例如IL-8、RANTES和MCP-1的N-末端内进行氨基酸删除或修饰来获得。
多种细胞内信号传导途径在受体激活后因趋化因子的结合而出现。趋化因子还与两种类型的非信号传导分子相互作用。一种是DARC受体，其在红细胞上和内皮细胞上表达，并结合CC-和CXC-趋化因子，防止它们循环。第二种类型是硫酸类肝素糖胺聚糖(GAGs)，其是蛋白聚糖的一部分，可作为趋化因子的辅助受体(co-receptor)。它们在归巢组织(如内皮细胞)表面上捕获和呈递趋化因子，以建立局部浓度梯度。在炎症反应(如类风湿性关节炎)中，在选择素介导的(selectin-mediated)过程中，滚动在内皮上的白细胞开始与细胞表面蛋白聚糖呈递的趋化因子相接触。这样，就激活了白细胞整联蛋白，促使紧密粘连和外渗。募集的白细胞被局部炎症细胞因子激活，并且由于趋化因子的局部高浓度而对进一步的趋化因子信号传导变得不敏感。为了保持组织血流趋化因子梯度，DARC受体起到多余趋化因子的接收器的作用。
硫酸类肝素(heparin sulphate，HS)蛋白聚糖(proteoglycan)由核心蛋白以及共价结合的糖胺聚糖(glycosaminoglycan，GAG)侧链组成，可见于大多数哺乳动物细胞和组织。所述蛋白组分决定该蛋白聚糖在细胞膜或胞外基质中的定位，而糖胺聚糖组分介导与各种胞外配体(如生长因子、趋化因子和粘附分子)的相互作用。蛋白聚糖的生物合成在此之前已有大量综述。细胞表面蛋白聚糖的主要基团是跨膜蛋白的多配体聚糖(syndecan)家族(哺乳动物中的四个成员)和通过糖基磷脂酰肌醇(GPI)尾粘附到细胞膜上的蛋白的磷脂酰肌醇聚糖(glypican)家族(哺乳动物中的六个成员)。磷脂酰肌醇聚糖在神经系统、肾脏中广泛表达，在骨骼肌和平滑肌中表达相对较少，syndecan-1是上皮细胞中的主要HSPG，syndecan-2在成纤维细胞和内皮细胞中占主导，syndecan-3在神经元细胞中大量存在，syndecan-4则广泛表达。通过四糖键合区域添加到syndecan核心蛋白的特定丝氨酸上的GAG链大多是HS链。具体类型syndecan的胞外区氨基酸序列在不同物种之间并不保守，GAG链的数量和位置却是高度保守的，这与跨膜区和胞浆区相反。但是，GAG的结构具有物种特异性，而且由表达HSPG的组织的天性决定。
硫酸类肝素(HS)是线性多糖的糖胺聚糖(GAG)家族的最丰富成员，该家族还包括肝素、硫酸软骨素、硫酸皮肤素和硫酸角质素。天然存在的HS的特点是由N-乙酰基-D-葡糖胺(GlcNAc)和D-葡糖醛酸(GlcA)组成的20-100个二糖单元的线性链，其可以被修饰为包括N-和O-硫酸化(葡糖胺的6-O和3-O硫酸化以及糖醛酸的2-O硫酸化)，以及β-D-葡糖醛酸到α-L-艾杜糖醛酸(IdoA)的表异构化。
由低硫酸化区域隔开的N-和O-硫酸化糖残基簇被认为主要负责HS的多种蛋白结合和调节特性。除了HS硫酸根基团与碱性氨基酸的静电相互作用之外，范德华力和疏水相互作用也被认为参与了蛋白结合。另外，构象可变的艾杜糖醛酸残基的存在看来有利于GAG与蛋白结合。其它因素如蛋白结合位点间的间隔对蛋白-GAG结合相互作用也起了关键作用。例如，由HS诱导的γ-干扰素二聚化需要具有两个蛋白结合序列的GAG链，所述蛋白结合序列被低硫酸化的7kDa区隔开。有时，一些配体的完整生物活性需要额外的序列：为了支持FGF-2信号传导，HS必须具有最小的结合序列以及可以与FGF受体相互作用的额外残基。
肝素结合蛋白通常包括由碱性氨基酸残基簇组成的共有序列。赖氨酸、精氨酸、天冬酰胺、组氨酸和谷氨酰胺经常参与和GAG上的硫酸和羧基基团的静电接触。碱性氨基酸之间的间隔有时通过蛋白三维结构来确定，它们被认为控制GAG结合特异性和亲和力。配体的生物活性还可以受HS-蛋白相互作用动力学的影响。降低生长因子扩散范围被视为HSPG的功能之一，GAG链的长重复特性以及其相对快的蛋白结合开关率非常适于该功能。在一些情况下，动力学比热力学更能驱动HS-蛋白结合的生理功能。大部分HS配体需要明确长度和结构的GAG序列。由肥大细胞产生的肝素，结构上与硫酸类肝素非常相似，但是它的特点是高水平的聚合后修饰，导致较均一的高度硫酸化而结构多样程度相对低。因此，硫酸类肝素中的高度修饰模块有时被称为“肝素样”。因此，肝素可以作为完美HS类似物，象HS一样，用于蛋白生物物理学研究，而其可获得的量更大因而比HS更便宜。不同的细胞类型显示出可以合成具有不同葡糖胺聚糖结构的蛋白聚糖，所述结构在发病期间、发育期间或者应答胞外信号如生长因子时发生变化。HSPG的这种结构多样性导致较高的结合通用性，从而突出蛋白聚糖的巨大重要性。
由于已证实硫酸类肝素蛋白聚糖对于FGF信号传导非常重要，多项研究都显示了趋化因子-GAG结合对促进趋化因子活性的重要性。首先，迄今为止研究的几乎所有趋化因子都显示在体外结合HS，说明这是这些蛋白的基本特性。第二，发现T淋巴细胞在体内以与葡糖胺聚糖形成的复合体的形式分泌CC-趋化因子，说明这种相互作用形式是与生理有关的。另外，已经知道，趋化因子结合HS可以帮助稳定横跨内皮表面的浓度梯度，从而为迁移白细胞提供指导信息。还认为HS可以防止趋化因子的蛋白水解降解，并诱导其寡聚化，从而提高在G-偶联信号传导受体附近的局部高浓度。然而，虽然所有趋化因子具有适合多聚化的明确结构基础，寡聚化的功能相关性仍然存在争议。在CXC-家族中所有趋化因子观察到了通过β-折叠结构的结合而形成的二聚化(如IL-8)，与此相反，CC-趋化因子家族的大多数成员(如RANTES)通过它们的N-末端链而二聚化。
关于利用低分子量化合物抑制趋化因子与它们在白细胞上的高亲和力受体之间的相互作用已经积累了大量数据。然而，通过该途径对炎症疾病进行治愈性治疗仍未获得突破。
白细胞介素-8(IL-8)是在慢性和急性炎症过程中参与吸引中性粒细胞的一个关键分子。迄今为止，已采取了多种方法来阻断IL-8的作用，首先通过如糖肾上腺皮质激素，维生素D3，环孢素A，转化生长因子β，干扰素等在IL-8mRNA生成水平上抑制IL-8活性的物质抑制IL-8的生成。先前使用的另一方法是，利用抗白细胞受体或抗IL-8本身的特异性抗体作为特异性拮抗剂抑制IL-8与其受体的结合，从而抑制IL-8。
因此，本发明的目的是提供抑制或干扰趋化因子/白细胞表面受体相互作用的可选策略。特别是，该策略应该靶定IL-8、RANTES或MCP-1的作用。
因此，本发明的主题是，制备新GAG结合蛋白以及可选的GAG结合蛋白的方法，其中所述蛋白(比野生型)表现出对GAG辅助受体的高(更高)亲和力。该修饰的GAG结合蛋白可以作为野生型GAG结合蛋白的竞争对手，能够抑制或下调野生型GAG结合蛋白的活性，而不导致现阶段所用的已知重组蛋白所发生的副反应。本发明所述的分子不具备上述缺点。本发明修饰的GAG结合蛋白可以在各种治疗用途的药物中使用，尤其是-在趋化因子情况下-用于治疗炎症疾病，而不会发生在现阶段已知重组趋化因子中所出现的已知危害。本发明对GAG结合位点的修饰可以广泛应用于所有基于与该位点的结合而具有活性的蛋白，尤其是具有GAG位点的趋化因子。本发明优选的具有高GAG结合亲和力的分子被证实具有良好的生物效应，尤其通过与野生型分子竞争GAG位点而具有良好的抗炎症活性。
因此，本发明提供了将GAG结合位点导入蛋白的方法，其特征是包括以下步骤：
-鉴定出蛋白中对于维持结构而言非必需的区，
-将至少一个碱性氨基酸导入所述位点和/或在所述位点中删除至少一个体积大的和/或酸性的氨基酸，
其中所述GAG结合位点具有Kd≤10μM，优选≤1μM，更优选≤0.1μM的GAG结合亲和力。通过在所述区域中导入至少一个碱性氨基酸和/或删除至少一个体积大的和/或酸性的氨基酸，可以将新的改进的“人工”GAG结合位点导入所述蛋白。这包括在被修饰前不表现GAG结合活性的蛋白内完整导入新GAG结合位点。这还包括在已经表现GAG结合活性的蛋白内导入GAG结合位点。可将新GAG结合位点导入不表现GAG亲和力的蛋白区域以及表现GAG结合亲和力的区域内。然而，本发明最优选的实施方式提供了对给定GAG结合蛋白的GAG结合亲和力的修饰，所述修饰蛋白比野生型蛋白提高了GAG结合能力。本发明涉及将GAG结合位点导入蛋白的方法、修饰的GAG结合蛋白以及分离的DNA分子、载体、重组细胞、药物组合物，以及所述修饰的蛋白的用途。
术语“导入至少一个碱性氨基酸”涉及导入额外氨基酸以及取代氨基酸。主要目的是提高所述位点中碱性氨基酸(优选Arg，Lys，His，Asn和/或Gln)占氨基酸总量的相对量，因此所得GAG结合位点应优选包括至少3个碱性氨基酸，更优选4个，最优选5个氨基酸。
GAG结合位点优选在溶剂暴露位置，例如环上。这可保证有效修饰。
蛋白区域是否对结构保持是必需的可以如通过本领域技术人员已知的含特殊程序的计算机方法来进行检测。在蛋白修饰后，优选在计算机上(insilico)检测构象稳定性。
术语“体积大的氨基酸”是指含长侧链或空间干扰侧链的氨基酸；具体是Trp，Ile，Leu，Phe，Tyr。具体的酸性氨基酸是Glu和Asp。优选，所得GAG结合位点不含体积大的和酸性的氨基酸，意思是去除了所有体积大的和酸性氨基酸。
对于本申请的保护范围而言，GAG结合亲和力由解离常数Kd决定。一种可能是通过配体结合中的结构变化，来确定任何给定蛋白的解离常数(Kd)。本领域技术人员熟知各种技术，例如等温荧光滴定法、如等温滴定量热法、表面质子共振(surface plasmon resonance)，凝胶迁移分析(gel mobilityassay)，和用放射性标记的GAG配体进行竞争试验来间接测定的方法。另外还可以用本领域已知的计算机方法，使用多种程序进行计算，来预测结合区。
例如，将GAG结合位点导入蛋白的方案如下：
-鉴定对维持整体结构并非必需且可能适合GAG结合的蛋白区域
-通过在任何位置导入(取代或插入)碱性Arg、Lys、His、Asp和Gln残基，或通过删除干扰GAG结合的氨基酸来设计新的GAG结合位点
-在计算机上(in silico)检查所得突变蛋白的构象稳定性
-克隆野生型蛋白cDNA(或者购买该cDNA)
-利用该cDNA作为PCR-辅助诱变的模板，将上面提及的变化导入氨基酸序列内
-将突变基因亚克隆入适合的表达系统(根据生物必需的翻译后修饰决定原核或者真核)
-体外表达，纯化和鉴定突变蛋白
-导入的GAG结合亲和力的标准：Kd GAG(突变体)＜10μM。
具有新GAG结合位点的所述工程化蛋白实例是，例如，如下述修饰的IgG Fc部分以及补体因子C3和C4：
Fc：(439)KSLSLS(444)->KSKKLS
C3：(1297)WIASHT(1302)->WKAKHK
C4：(1)MLDAERLK(8)->MKKAKRLK
本发明另一方面是可通过上述发明方法获得的蛋白。因此，该发明蛋白包括上述相对于野生型蛋白而言新的GAG结合位点，从而可以作为天然GAG结合蛋白的竞争对手，特别是由于该发明蛋白的GAG结合亲和力非常高，例如Kd≤10μM。
本发明另一方面是修饰的GAG结合蛋白，其中所述蛋白的GAG结合区通过取代，插入，和/或删除至少一个氨基酸而进行了修饰，从而提高所述GAG结合区内的碱性氨基酸相对量，和/或降低所述GAG结合区内的体积大的和/或酸性的氨基酸的量，优选在溶剂暴露位置上进行修饰，所述蛋白的GAG结合亲和力比相应野生型蛋白的GAG结合亲和力提高。
令人吃惊地是，通过增加GAG结合区内的碱性氨基酸(尤其是Arg，Lys，His，Asn和Gln)的相对量，修饰的GAG结合蛋白相比于野生型蛋白的GAG结合亲和力增加，尤其是在溶剂暴露位置上的碱性氨基酸相对量增加时，因为蛋白表面的正电荷区显示增强结合亲和力。优选在GAG结合区中存在至少3，更优选4，最优选5个碱性氨基酸。
术语“GAG结合蛋白”涉及结合GAG辅助受体的任何蛋白。蛋白是否结合GAG辅助受体可以在上述已知方案的帮助下进行测定。Hileman等(BioEssays 20(1998)，156-167)公开了葡糖胺聚糖结合蛋白中的共有位点。在该文章中公开的信息作为本发明的起始信息也是有用的。术语“蛋白”解释为本发明提供的分子长度至少为80个氨基酸。这是它们作为本发明抗炎症策略的适合候选物所必需的。与GAG结合位点相互作用且因所述相互作用而具有生理或病理相关性的更小分子还未知，因此与本发明无关。优选，本发明的分子由至少90，至少100，至少120，至少150，至少200，至少300，至少400或至少500个氨基酸残基组成。
在本申请的范围内，术语“GAG结合区”定义为，以小于100μM，优选小于50μM，更优选小于20μM的解离常数(Kd-值)与GAG结合的区，所述常数通过等温荧光滴定法测定(参见下文实施例)。
本发明提及的任何修饰可以通过生物化学方法，例如，定点诱变来实现。还应指出，GAG结合位点的分子克隆显然是现有技术(参见例如WO96/34965 A，WO 92/07935 A，Jayaraman等(FEBS Letters 482(2000)，154-158)，WO02/20715 A，Yang等(J.Cell.Biochem.56(1994)，455-468)，其中描述了GAG区域的分子改组或重头合成(de novo)；Butcher等(FEBSLetters 4009(1997)，183-187)(与人工肽而非蛋白有关)；Jinno-Oue等(J.Virol.75(2001)，12439-12445)重头合成))。
可通过取代，插入和/或删除来对GAG结合区进行修饰。这意味着非碱性氨基酸可以被碱性氨基酸取代，碱性氨基酸可以插入到GAG结合区或者可以删除非碱性氨基酸。另外，删除干扰GAG结合的氨基酸，优选所有干扰氨基酸结合的氨基酸。尤其，所述氨基酸是上述体积大的氨基酸，以及酸性氨基酸如Glu和Asp。氨基酸是否干扰GAG结合可以通过例如数学方法或计算机方法来进行检测。所述任一修饰的结果都是在所述GAG结合区中的碱性氨基酸相对量提高，其中“相对”是指所述GAG结合区内碱性氨基酸的量相对于所述GAG结合区内全部氨基酸的数。另外，删除了空间或静电干扰GAG结合的氨基酸。
氨基酸是否存在于溶剂暴露位置，可以在例如蛋白的已知三维结构的帮助下，或在上述计算机方法的帮助下来进行测定。
所述修饰蛋白的GAG结合亲和力是否比相应野生型蛋白的GAG结合亲和力提高，可以借助例如测定解离常数的荧光滴定实验如上所述进行测定。GAG结合亲和力提高的标准是Kd(突变体)＜Kd(野生型)，优选相差至少100％。尤其是，改进型修饰的蛋白的GAG结合亲和力比野生型Kd高至少5倍，优选至少10倍，更优选至少100倍。因此，增高的GAG结合亲和力显示的Kd在10μM之下，优选在1μM之下，更优选在0.1μM之下。
通过增加GAG结合亲和力，修饰的蛋白将可以起特异性拮抗剂的作用，并且可以与野生型GAG结合蛋白竞争结合GAG。
优选，将选自Arg，Lys和His的至少一种碱性氨基酸插入所述GAG结合区。这些氨基酸可以很容易地插入到所述GAG结合区，而术语“插入”是指插入以及用精氨酸、赖氨酸或组氨酸取代任何非碱性氨基酸。当然，可以插入不止一个碱性氨基酸，其中所插入的可以是相同的碱性氨基酸或者可以是两种或三种上述氨基酸。
更优选，所述蛋白是趋化因子，优选IL-8，RANTES或MCP-1。已知趋化因子具有与辅助受体GAG相互作用的位点，而趋化因子的这种结合常常是上述进一步受体激活的条件。由于趋化因子经常出现在炎症疾病中，因此主要兴趣就是阻断趋化因子受体激活。这类趋化因子优选是IL-8，RANTES或MCP-1，它们都是性质较明确的分子，且它们的GAG结合区是已知的(参见，例如Lortat-Jacob等，PNAS 99(3)(2002)，1229-1234)。通过增加这些趋化因子GAG结合区内的碱性氨基酸量，可以提高其结合亲和力，使野生型趋化因子以较小频率结合或根本不结合，这取决于修饰蛋白相对于野生型蛋白的浓度。
根据有利方面，所述GAG结合区是C末端α-螺旋。典型的化学单体围绕被C末端α-螺旋覆盖的三股反向-平行β-折叠来排列。有迹象表明，趋化因子中的这种C末端α-螺旋是参与GAG结合的主要部分，因此为增加碱性氨基酸量而对该C末端α-螺旋进行修饰，可使修饰的趋化因子的GAG结合亲和力增强。
有利地是，将IL-8中位置17，21，70和/或71用Arg，Lys，His，Asn和/或Gln取代。可以仅对其中一个位点进行修饰。也可以对不止一个所述位点进行修饰以及对所有位点进行修饰，其中所有修饰可以为Arg或Lys或His或Asn或Gln或其混和。在IL-8中，这些位置显示高度促进IL-8的GAG结合亲和力，因此尤其适合对这些位置进行修饰。
优选，增加的结合亲和力是增加了对硫酸类肝素和/或肝素的结合亲和力。硫酸类肝素是线性多糖GAG家族的最丰富成员，该家族还包括肝素。肝素在结构上与硫酸类肝素非常相似，其特点是聚合后修饰水平更高，导致较均一的高度硫酸化而结构多样程度相对低。因此，硫酸类肝素中的高度修饰模块有时被称为肝素样，肝素可以作为完美的硫酸类肝素类似物用于蛋白生物物理学研究。在任何情况中，硫酸类肝素和肝素都特别适合。
更优选，对另外的生物活性区进行修饰，从而抑制或下调所述蛋白的另外的生物活性。此另外的生物活性对于多数GAG结合蛋白，例如趋化因子来说是已知的。它可以是受体(例如7TM受体)的结合区。术语“另外的”用于定义一种生物活性区，它并非GAG结合区，但它结合其它分子、细胞或受体和/或激活它们。通过对该另外的生物活性区进行修饰来抑制或下调该蛋白的另外的生物活性，从而提供一种修饰的蛋白作为野生型蛋白的强拮抗剂。这就意味着，一方面GAG结合亲和力比野生型GAG结合蛋白高，从而该修饰的蛋白而不是野生型蛋白可以较多地结合GAG。另一方面，野生型蛋白中主要在蛋白结合GAG时出现的另外的活性被抑制或下调，因为修饰的蛋白将不执行此特异性活性或以较小程度执行此活性。有了这种修饰的蛋白，可以提供野生型GAG结合蛋白的有效拮抗剂，它不具备现有技术所述其它重组蛋白的已知副作用。此另外的生物活性区可以例如通过体外受体竞争试验(利用荧光标记的野生型趋化因子，钙流和细胞迁移(在天然白细胞或7TM稳定转染的细胞系上进行))来测定。除了另外的受体结合位点(如在生长因子家族的情况中)之外，所述另外的生物活性区的实例是酶位点(如在水解酶，裂解酶，磺基转移酶，N-脱乙酰基酶和共聚酶(copolymerase)的情况中)，蛋白相互作用位点(如在抗凝血酶III的情况中)，和膜结合域(如在单纯疱疹病毒gD蛋白的情况中)。有了双修饰蛋白的优选实施方式，可以提供显性(对于GAG结合)阴性(对于受体)突变体，它们对本发明的目的特别有利。
更优选，所述另外的生物活性区是通过删除，插入和/或取代进行修饰，优选用丙氨酸、空间相似残基和/或静电相似残基来进行修饰。当然，在所述另外的生物活性区中可以删除或插入或取代至少一个氨基酸。然而，也可以提供至少两种或所有三种上述修饰。通过用丙氨酸或空间/静电相似残基取代指定氨基酸-“相似”的意思是与被取代的氨基酸相似-而修饰的蛋白在空间/静电上未改变或者仅有很小程度的改变。此点尤其有利，因为修饰的蛋白的其它活性，尤其是对GAG结合区的亲和力不变。
有利地，所述蛋白是趋化因子且所述另外的生物活性是白细胞活化。如上所述，在慢性和急性发炎时，趋化因子参与吸引白细胞。因此，通过抑制或下调白细胞活化，可降低或抑制炎症，而使得这种具体的修饰蛋白成为研究，诊断和治疗炎症疾病的重要工具。
根据有利的方面，所述蛋白是IL-8且所述另外的生物活性区位于前10个N-末端氨基酸内。前面的N-末端氨基酸参与白细胞活化，尤其是经鉴定Glu-4，Leu-5和Arg-6对于受体结合和活化是必需的。因此，可以对这3个或甚至所有的前10个N-末端氨基酸进行取代或删除，从而抑制或下调受体结合和活化。
另外的有利蛋白是删除了前6个N-末端氨基酸的IL-8突变体。如上所述，此变体将不能或能但以很小程度结合和活化白细胞，因此它对于研究，诊断和治疗炎症疾病是特别适合的。
优选，所述蛋白是选自下组的IL-8突变体：de16F17RE70KN71R，de16F17RE70RN71K和de16E70KN71K。这些突变体尤其有利，因为删除前6个N-末端氨基酸可抑制或下调受体结合和活化。另外，发现位置17和21的两个苯丙氨酸首先与受体N-末端胞外区接触，从而便于受体的随后激活。为了防止任何中性粒细胞接触，将这两个氨基酸17和21调换为碱性氨基酸，因为从蛋白的三维模型可看出，这两个氨基酸非常接近C-末端α-螺旋的GAG结合基序。通过将位置17和/或21调换为精氨酸或赖氨酸，可使GAG结合亲和力增强。
本发明另一方面是分离的多核酸分子，该分子编码如上所述的发明蛋白。多核酸可以是DNA或RNA。因此，可以在DNA或RNA水平进行导致本发明修饰蛋白的修饰。本发明分离的多核酸分子适合诊断方法以及基因治疗和大量制备本发明的修饰蛋白。
更优选，分离的多核酸分子与上述定义的本发明多核酸分子在严谨条件下杂交。根据不同杂交条件，两DNA或RNA分子间可通过完全匹配形成互补双链，或者也可以包括错配碱基(参见Sambrook等，分子克隆：实验室手册，第二版，冷泉港，N.Y.1989)。长度大于约50个核苷酸的探针可接纳多达25-30％的错配碱基。较小的探针接纳的错配较少。靶和探针形成含错配碱基对的双链的趋势是由杂交条件的严谨性控制的，所述杂交条件本身是杂交缓冲液中盐浓度或甲酰胺浓度、杂交温度和杂交后洗涤条件等因素的函数。通过利用众所周知的形成杂交双链时的原则，本领域技术人员可以从各种杂交缓冲液、温度和洗涤条件中选出具有所需严谨性的条件。因此，所选择的条件可以是允许检测完全匹配或部分错配的杂交双链。可用双链的解链温度(Tm)选择合适的杂交条件。长度超过200个核苷酸的多核苷酸分子的严谨杂交条件通常包括：在比预期双链的解链温度低15-25℃的温度杂交。超过30个核苷酸的寡核苷酸探针形成的双链稳定性低于更长探针，其严谨杂交通常在比Tm低5-10℃时进行杂交而获得。核酸双链的Tm可利用基于核酸中所含G+C百分数并考虑了链长的公式进行计算，如公式Tm＝81.5-16.6(log[Na+)])+0.41(％G+C)-(600/N)，其中N＝链长。
本发明另一方面涉及含有上述本发明的分离的DNA分子的载体。该载体包括蛋白有效转染以及有效表达所需的所有调节元件。这种载体是本领域所熟知的，可以选择任何适合的载体用于该目的。
本发明另一方面涉及稳定转染上述发明载体的重组细胞。这种重组细胞及其任何后代细胞包含该载体。因此，本发明提供了细胞系，根据载体的不同，所述细胞系可以组成型表达或通过活化而表达经修饰的蛋白。
本发明另一方面涉及包括本发明所述蛋白，多核酸或载体和药学上可接受载体的药物组合物。当然，该药物组合物可进一步包括常规存在于药物组合物中的其它物质，如盐，缓冲液，乳化剂，着色剂等。
本发明另一方面涉及本发明的修饰的蛋白，多核酸或载体在抑制或遏制相应野生型蛋白的生物活性的方法中的用途。如上所述，修饰的蛋白可以作为拮抗剂，用本发明的修饰蛋白将不会发生用已知重组蛋白发生的副作用。在趋化因子的情况下，这特别就是参与炎症反应的生物活性。
因此，本发明的修饰蛋白，多核酸或载体的另外的用途是在制备治疗炎症病症的药物的方法中的用途。特别是，当修饰的蛋白是趋化因子时，可以作为无副作用的拮抗剂并且尤其适于治疗炎症。因此，本申请另一方面还涉及治疗炎症的方法，其中将本发明的修饰的蛋白、本发明的分离的多核酸分子或载体、或本发明的药物组合物施给患者。
优选，炎症病症选自下组：类风湿性关节炎，牛皮癣，骨关节炎，哮喘，阿尔茨海默氏症(Alzheimer’s disease)，和多发性硬化。由于通过趋化因子进行的激活可以用本发明的修饰的蛋白来抑制，因此可以抑制或下调炎症反应，从而可以预防或治疗上述炎症病症。
在下述实施例和附图的帮助下，将对本发明进行更详细的描述，但是本发明并不限于此，其中图1是CD光谱；图2显示各种突变体的二级结构；图3和4显示各种突变体的荧光各向异性试验结果图；图5显示等温荧光滴定结果图；图6显示各种突变体的解折叠实验结果图；图7显示IL-8突变体的趋化性指数；图8显示RANTES趋化性试验结果。
实施例
实施例1：产生重组IL-8基因和克隆突变体
通过利用有义和反义诱变引物将突变导入，使用聚合酶链式反应(PCR)技术产生所需突变体的cDNA。用含wtIL-8 cDNA的合成质粒作模板，Clontech Advantage2 Polymerase Mix作DNA聚合酶，用MastergradientCycler of Eppendorf进行PCR反应。使用的诱变引物在下表中概括，正向序列为(5′到3′)：
·CACC ATG TGT CAG TGT ATA AAG ACA TAC TCC(突变Δ6的引物)
·CACC ATG TGT CAG TGT ATA AAG ACA TAC TCC AAA CCT AGGCAC CCC AAA AGG ATA(突变Δ6 F17R F21R的引物)
反向序列是(5′到3，)：
·TTA TGA ATT CCT AGC CCT CTT(突变E70R的引物)
·TTA TGA ATT CTT AGC CCT CTT(突变E70K的引物)
·TTA TGA CTT CTC AGC CCT CTT(突变N71K的引物)
·TTA TGA CTT CTT AGC CCT CTT(突变E70K N71K的引物)
·TTA TGA CTT CCT AGC CCT CTT(突变E70R N71K的引物)
·TTA TGA CCT CTT AGC CCT CTT(突变E70K N71R的引物)
·TTA TGA CCT CCT AGC CCT CTT(突变E70R N71R的引物)
将PCR产物纯化，克隆入pCRT7/NT-TOPOTA(Invitrogen)载体，并转化入TOP10F感受态大肠杆菌(Invitrogen)。接着利用ABI PRISM CE1测序仪，通过双链DNA定序进行序列确认。
实施例2：重组蛋白的表达和纯化
一旦确认序列后，将构建体转化入用于表达的钙-感受态BL21(DE3)大肠杆菌。在37℃，将细胞在含100μg/ml氨苄青霉素的1升Lennox Broth(Sigma)中震荡培养至OD600达到约0.8。通过添加异丙基β-D-硫代半乳糖苷(IPTG)至终浓度为1mM，来诱导蛋白表达。4小时后，细胞在6000g离心20分钟进行收集。然后细胞沉淀物在含20mM TRIS/HCl，50mM NaCl，pH8的缓冲液中重悬，在100瓦超声处理5×20s，最后再于10,000g离心20分钟。由于发现重组IL-8蛋白的主要级分是在包涵体中，选择变性条件进行进一步纯化。因此，细胞沉淀物在6M Gua/HCl和50mM MES，pH 6.5的缓冲液中重新悬浮。然后，悬浮液在4℃搅拌4小时，接着对50mM MES，pH 6.5透析。然后对所得悬浮液进行离心，过滤，加样到强阳离子交换柱(Pharmacia Biotech的SP Sepharose)上。在60分钟内，用50mM MES缓冲液中的0M-1M NaCl的线性梯度完成洗脱。在冻干含所需蛋白的级分后，采用C18柱，通过反相HPLC进行二次纯化。在此情况下，选择10％-90％乙腈的非线性梯度洗脱所需蛋白。在如上所述相同的条件下，用相同的阳离子交换柱最终实现变性蛋白的重折叠。
然后，检测蛋白纯度和同一性，所用方法可以是银染色，也可以是利用抗wtIL-8的特异性单克隆抗体进行Western印迹。蛋白的重折叠也可以通过圆二色性(CD)测量法进行证实。
实施例3：突变体的生物物理学鉴定
3.1圆二色性测量和分析
使用圆二色谱研究存在和不存在硫酸类肝素(HS)时的突变蛋白的二级结构变化。在Jasco J-710旋光分光计上，记录在覆盖195-250nm范围的测量，使用径长(path length)0.1cm的细胞。记录浓度5μM的蛋白溶液光谱，反应时间1s，步进分辨率(step resolution)0.2nm，速度50nm/min，频带宽度1nm，灵敏度20mdeg。将三次扫描平均获得平滑光谱。然后，对蛋白光谱相对缓冲液本身或缓冲液/HS的CD-信号进行背景校正。利用程序SELCON最终获得存在和缺少HS时的蛋白二级结构分析。
由于许多新组合物中的多数氨基酸都发生了变化，试图通过圆二色性法揭开获得的二维结构。
获得了依赖于所导入突变的不同结构。除了一个表达突变体(Δ6 F17RF21R E70K N71R)未显示任何结构之外，所有突变体都表现了可测的α-螺旋，β-折叠和环。相比于IL-8wt，仅有一个突变体(Δ6 E70R)显示了几乎相同的结构，而其它突变体主要在它们的α-螺旋上有区别，范围从全部结构的17.2％到45.2％。然而，此事实说明尽管在蛋白序列中有许多改变，但仍保持着IL-8wt的整体结构。这一点是先前无法预料的。已发现仅硫酸类肝素寡糖，而非肝素，能够影响IL-8wt二级结构，注意力集中在由普通(unfractionated)硫酸类肝素诱导的效应。所有的检测突变体在HS结合时都显示结构变化，这可以看做是复合物形成的证据。
为了证明在导入突变和硫酸类肝素添加物时的结构变化，获得的一些数据在上面和下面的图表进行了概括。
3.2荧光测定
为了研究浓度和配体依赖性四级结构变化，进行荧光光谱。由于敏感性很高，仅需要纳克量的蛋白，荧光技术是进行所需研究的最佳方法。使用Perkin-Elmer(Beaconsfield，England)LS50B荧光仪进行测定。
3.3荧光各向异性
通过记录从外在发色团bisANS获得的趋化因子的浓度依赖性荧光各向异性，旨在发现突变体的二聚化常数。在以高浓度(高至4μM蛋白)起始，随后分步稀释的PBS中进行测定。对各数据点，60秒内，507nm记录的各向异性信号(r)进行平均。
已经报道了IL-8寡聚化会相应地影响蛋白GAG结合特性。设定于单体浓度，由寡糖定义的IL-8束缚尺寸比二聚浓度下的紧1000倍。因此，采用荧光各向异性研究IL-8突变体的寡聚特性。由于IL-8内在荧光团(Trp57)并不是对所有的突变体都敏感，因此使用外在荧光团bis-ANS进行测量。此外，由于已注意二级结构，突变体Δ6 E70R显示的的寡聚常数(koligo＝350nM)与IL-8wt(koligo＝379nM)非常相似。koligo最高的突变体(koligo＝460nM)，也就是二聚化最差，是Δ6 F17RF21R E70RN71K。以前的研究发现β-折叠主要参与二聚化过程，事实上，它与这个突变体的二级结构结果有关系，而且二聚化过程中的β-折叠份额非常低，仅为11.4％。具有最低koligo的突变体(koligo＝147nM)是Δ6 F17RF21R E70K，它又具有所有检测突变体的最高β-折叠结构份额(29.8％)。同时还观察到添加硫酸类肝素的影响。对于IL-8wt，在IL-8wt中硫酸类肝素导致寡聚常数移到更高水平(koligo＝1.075μM)，这也在研究的IL-8突变体中发现。Δ6 F17RF21R E70K从0.147μM变到1.162μM，Δ6 E70R从0.350μM变到存在硫酸类肝素时的1.505μM。获得的一些结果显示在图3和图4中，其中图3显示在趋化因子浓度下的PBS中，IL-8突变体的荧光各向异性依赖性，图4显示在趋化因子浓度下的PBS中，存在(超过10倍)和缺少HS((pc＝超过10xy)蛋白浓度).时，Δ6 F17RF21R E70K的荧光各向异性依赖性。
3.4等温荧光滴定(IFT)实验
解离常数(Kd值)是配体与蛋白结合亲和力单位，因此，当结合配体时，使用蛋白荧光发射特性中的荧光浓度依赖性改变(荧光淬灭)来测定Kd。由于这些突变体包括内在色氨酸发色团，该发色团位于或靠近建议的GAG结合位点，所以应该对配体结合时的荧光结构变化敏感，IFT实验似乎适合此类研究。在PBS中，利用700nM的蛋白浓度进行荧光强度滴定。记录下在300-400nm范围内，282nm激发的蛋白溶液发射波长，接着添加各自的GAG配体等分试样，平衡周期60秒。
观察与普通肝素(unfractionated heparin)和硫酸类肝素的结合。将突变体设置为二聚浓度以保证足够的敏感性。结合GAG时，登记的Trp57荧光淬灭强度的在25-35％范围内。观察到了配体结合的显著改善，尤其是肝素结合。相比于IL-8wt(Kd＝37μM)，Δ6 F17RN71R E70K(Kd＝14nM)和Δ6F17RF21R N71K(Kd＝14.6nM)的结合强2600倍，Δ6 E70K N71K(Kd＝74nM)的结合强1760倍。在硫酸类肝素结合中也获得了较好结果。Δ6 F17RN71RE70K的Kd为107nM，Δ6 F17RF21R N71K的Kd为95nM，突变体Δ6 E70KN71K的Kd为34nM。由于IL-8wt结合的Kd为4.2μM，发现的突变体的Kds意味着在结合方面惊人地提高，参见图5。
3.5解折叠实验
为了获得蛋白稳定性以及此稳定性是否会在GAG配体结合时发生变化的信息，进行解折叠实验。如上所述，荧光技术对于观察四级结构变化是非常敏感的，而且也是研究蛋白热结构变化的最佳方法。如IFT所述进行测量，其中不是配体浓度改变而是温度改变。在温度15-85℃，缺少和存在超过10倍硫酸类肝素或肝素时，浓度0.7μM下观察蛋白结构。
蛋白的最大发射波长(emission maximum)范围从340nm到357nm，该值是溶剂暴露的色氨酸残基所特有的。于15℃下开始解折叠实验，突变体的最大发射波长在340nm-351nm间变化。与IL-8wt相比，其观察的最大发射波长在340nm，说明该值略微偏高。一旦温度增加，最大发射波长强度就降低，同时最大发射波长向更长或更短波长移动。Δ6 E70R和Δ6 E70K N71K的最大发射波长从352.5nm移到357nm和从343nm移到345nm，这是随着温度升高，Trp57残基进一步暴露给溶剂所特有的，但有趣地是突变体Δ6F17RN71R E70K和Δ6 F17RF21R E70R N71K表现为蓝移(blue shift)，范围从350nm到343nm，和更不明显地从350nm到348nm(参见图6)。观察波长移动和强度变化，通过缓慢降低温度，解折叠过程是部分可逆的。加入超过5倍的硫酸类肝素导致蛋白稳定性增加，可能是通过复合物形成所致。一方面，这可以通过熔点移至更高的温度，另一方面，一旦温度升高，通过最大发射波长显著地不明显移位而观察到。
实施例4：基于细胞分析显性-阴性IL-8突变体的受体-“阴性”功能
为了测定IL-8突变体在吸引中性粒细胞方面的受体功能损伤情况，在配备有5μm不含PVP的聚碳酸酯膜的微量趋化小室中分析中性粒细胞应答IL-8突变体时基于越滤膜(transfilter)的趋化性。
细胞制备：
简单地说，中性粒细胞级分从新鲜采集的人血液制备。这通过将6％葡聚糖溶液加到用肝素处理的血液(1∶2)中，然后静置沉淀45分钟来完成。收集上层透明细胞溶液，用HBSS w/o Ca和Mg洗涤两次。对细胞进行计数，最后用HBSS稀释成2Mio/ml细胞悬液，计算得出仅有60％的被计数的细胞是中性粒细胞。
趋化性分析：
将IL-8突变体稀释至10μg/ml，1μg/ml和0.1μg/ml，一式三份置于趋化小室(chamber)的下层间格内(26μl每孔)。将新鲜制备的中性粒细胞接种在上层趋化小室内(50μl每孔)，于37℃、5％CO2湿润培养箱中孵育30分钟。孵育后，拆开趋化小室，清洗滤膜上侧并擦净，将附着在下侧的细胞用甲醇固定，并用Hemacolor溶液(Merck)染色。然后，每孔随机选4个显微镜视野，于400倍放大倍数对细胞计数。最后，取三个独立实验的平均值对趋化因子浓度进行作图。在图7中，显示了各种IL-8突变体的趋化指数。正如所料，所有突变体都显示了显著降低的受体结合活性。
实施例5：重组RANTES基因的产生，突变体的表达、生物物理特征和活性特征
对RANTES应用显性-阴性“GAG-隐蔽(masking)”趋化因子突变体概念。所述RANTES是参与IV型超敏反应(例如移植物排斥、特应性皮炎以及诸如关节炎、进行性肾小球性肾炎和炎症性肺疾病等炎症)的一种趋化因子。
通过向野生型蛋白导入一个起始蛋氨酸残基而使受体结合能力受损。野生型RANTES(wt RANTES)在大肠杆菌中的表达导致该蛋氨酸残基滞留，它使野生型RANTES成为单核细胞迁移的强效抑制剂，即Met-RANTES。能提高GAG结合亲和力的多种不同突变通过基于PCR的定点诱变法导入。
到目前为止，通过这些方式克隆、表达并纯化了9种RANTES突变体：Met-RANTES A22K，Met-RANTES H23K，Met-RANTES T43K，Met-RANTESN46R，Met-RANTES N46K，Met-RANTES Q48K，Met-RANTES A22K/N46R，Met-RANTES V49R/E66S和Met-RANTES15LSLA18V49R/E66S。
进行等温荧光滴定实验来测量RANTES突变体对大小确定的肝素的相对亲和力常数(Kd值)。如表中所见，所有RANTES突变蛋白都显示了对该肝素的更高亲和性，其中Met-RANTES A22K，Met-RANTES H23K，Met-RANTES T43K和Met-RANTES A22K/N46R显示了最有前景的结果。
                                  Kd(nM)
wt Rantes                         456.2±8.5
Met-Rantes V49R/E66S              345.5±21.7
Rantes 15LSLA18 V49R/66S          297.3±14.1
Rantes N46R                       367.7±11.7
Rantes N46K                       257.4±10.2
Rantes H23K                       202.5±12.8
Rantes Q48K                       383.4±39.6
Rantes T43K                       139.2±30.1
Rantes A22K                       202.1±9.8
Rantes A22K/N46R                  164.0±16.6
RANTES趋化性分析
利用配备有5μm被PVP-包被的聚碳酸酯膜的48-孔Boyden趋化小室系统对指导细胞迁移的RANTES突变体进行研究。将稀释在含20mM HEPESpH 7.3和1mg/ml BSA的RPMI 1640中的RANTES和RANTES突变体一式三份置于趋化小室的下层孔内。将50μl悬浮在相同培养基中的THP-1细胞悬液(从欧洲细胞培养物保藏中心(European collection of cell cultures)获得的前单核细胞系)按2×106个细胞/ml置于上层孔中。在37℃，5％CO2中孵育2小时后，用HBSS溶液清洗滤膜上表面。将迁移的细胞固定在甲醇中，用Hemacolor溶液(Merck)染色。每孔取5处按400倍放大倍数计数，将三个独立实验的平均值对趋化因子浓度作图，见图8。误差栏代表三个实验的平均值的标准误差。与IL-8突变体的情况相同，所有RANTES都显示了显著降低的受体结合活性。
实施例6：含GAG结合区的蛋白
利用生物信息学和蛋白质组分析手段，对GAG结合蛋白及其GAG结合区进行鉴定。下表2和3中显示了趋化因子及其GAG结合区(表2)、其它蛋白实例及其其GAG结合区(表3)。
表2：
趋化因子及其GAG结合区
                              CXC-趋化因子
IL-8：18HPK20，(R47)60RVVEKFLKR68
SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS
MGSA/GRO：19HPK21，45KNGR48，60KKIIEK66
ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN
MIP-2/GRO：19HLK21，K45，60KKIIEKMLK68
APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSN
NAP-2：15HPK18，42KDGR45，57KKIVQK62
AELRCLCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDESAD
PF-4：20RPRH23，46KNGR49，61KKIIKK66
EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES
SDF-1：K1，24KHLK27，41RLK43
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN
                              CXC-趋化因子
RANTES：(17RPLPRAH23)44RKNR47
SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS
MCP-2：18RKIPIQR24，46KRGK49
QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP
MCP-3：22KQR24，47KLDK50，66KHLDKK71
QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL
MIP-1：R17，44KRSR47
SLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYVSDLELSA
MlP-1：R18，45KRSK48
APMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN
MPIF-1：R18，45KKGR48
MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKTRKN
MIP-5/HCC-2：40KKGR43
HFAADCCTSYISQSIPCSLMKSYFETSSECSKPGVIFLTKKGRQVCAKPSGPGVQDCMKKLKPYSI
表3
过氧化物酶体生物发生因子1(Peroxisome biogenesis factor 1) 181 TRRAKE 186
                                                          367 QKKIRS 372
                                                          1263 PKRRKN 1268
                                                          181 TRRAKE 186
                                                          367 QKKIRS 372
                                                          1263 PKRRKN 1268
MLTK-β                                                   415 SKRRGKKV 422
                                                          312 ERRLKM 317
                                                          416 KRRGKK 421
                                                          312 ERRLKM 317
                                                          416 KRRGKK 421
BHLH因子Hes4                                              43 EKRRRARI 50
                                                          43 EKRRRA 48
                                                          43 EKRRRA 48
原钙黏着蛋白(Protocadherin)11                             867 MKKKKKKK 874
                                                          867 MKKKKK 872
                                                          867 MKKKKK 872
                                                          899 MKKKKKKK 906
                                                          899 MKKKKK 904
                                                          899 MKKKKK 904
联蛋白(钙粘着蛋白-相关蛋白)，δ1                          315 RRRLRS 320
                                                          404 VRKLKG 409
                                                          460 LRKARD 465
                                                          545 RRKLRE 550
                                                          621 AKKGKG 626
                                                          787 AKKLRE 792
                                                          315 RRRLRS 320
                                                          404 VRKLKG 409
                                                          460 LRKARD 465
                                                          545 RRKLRE 550
                                                          621 AKKGKG 626
                                                          787 AKKLRE 792
蝇蕈碱性乙酰胆碱受体M5                                    221 EKRTKD 226
                                                          427 TKRKRV 432
                                                          514 WKKKKV 519
                                                          221 EKRTKD 226
                                                          427 TKRKRV 432
                                                          514 WKKKKV 519
α-2A肾上腺素能受体                                       147 PRRIKA 152
                                                          224 KRRTRV 229
                                                          147 PRRIKA 152
                                                          224 KRRTRV 229
IL-5启动子REII-区-结合蛋白                                440 TKKKTRRR 447
                                                          569 GKRRRRRG 576
                                                          38 ARKGKR 43
                                                          437 GKKTKK 442
                                                          444 TRRRRA 449
                                                          569 GKRRRR 574
                                                          38 ARKGKR 43
                                                          437 GKKTKK 442
                                                          444 TRRRRA 449
                                                          569 GKRRRR 574
Mito fusin 1                                              291 ARKQKA 296
                                                          395 KKKIKE 400
                                                          291 ARKQKA 296
                                                          395 KKKIKE 400
N-cym蛋白                                                 71 VRRCKI 76
                                                          71 VRRCKI 76
Smad泛素化调节因子1                                       672 ERRARL 677
                                                          672 ERRARL 677
CUG-BP和ETR-3样因子5                                      468 MKRLKV 473
                                                          475 LKRPKD 480
                                                          468 MKRLKV 473
                                                          475 LKRPKD 480
尤因肉瘤(Ewings sarcoma)EWS-Flil                          347 QRKSKP 352
                                                          347 QRKSKP 352
NUF2R                                                     455 LKRKMFKM 462
                                                          331 LKKLKT 336
                                                          347 VKKEKL 352
                                                          331 LKKLKT 336
                                                          347 VKKEKL 352
枯否(Kruppel)-样锌指蛋白GLIS2                             22 EKRERT 27
                                                          22 EKRERT 27
FKSG32                                                    15 LKRVRE 20
                                                          431 VRRGRI 436
                                                          15 LKRVRE 20
                                                          431 VRRGRI 436
BARH-样1蛋白                                              175LKKPRK 180
                                                          228 NRRTKW 233
                                                          175 LKKPRK 180
                                                          228 NRRTKW 233
核仁GTP-结合蛋白1                                         393 SRKKRERD 400
                                                          624 GKRKAGKK 631
                                                          48 MRKVKF 53
                                                          141 IKRQKQ 146
                                                          383 ARRKRM 388
                                                          393 SRKKRE 398
                                                          490 KKKLKI 495
                                                          543 ARRSRS 548
                                                          550 TRKRKR 555
                                                          586 VKKAKT 591
                                                          629 GKKDRR 634
                                                          48 MRKVKF 53
                                                          141 IKRQKQ 146
                                                          383 ARRKRM 388
                                                          393 SRKKRE 398
                                                          490 KKKLKI 495
                                                          543 ARRSRS 548
                                                          550 TRKRKR 555
                                                          586 VKKAKT 591
                                                          629 GKKDRR 634
EVG1                                                      17 RRRPKT 22
                                                          138 ERKRKA 143
                                                          17 RRRPKT 22
                                                          138 ERKRKA 143
ASPL                                                      282 PKKSKS 287
                                                          282 PKKSKS 287
锌转运蛋白1                                               477 EKKPRR 482
                                                          477 EKKPRR 482
Uveal自身抗原                                             603 EKKGRK 608
                                                             995 ERKFKA 1000
                                                             1023 VKKNKQ 1028
                                                             603 EKKGRK 608
                                                             995 ERKFKA 1000
                                                             1023 VKKNKQ 1028
RAB39                                                        7 VRRDRV 12
                                                             7 VRRDRV 12
唐氏综合征细胞粘附分子(Down syndrome cell adhesion molecule) 320 PRKVKS 325
                                                             387 VRKDKL 392
                                                             320 PRKVKS 325
                                                             387 VRKDKL 392
蛋白-酪氨酸磷酸酶，12型非受体                                139 GRKKCERY 146
                                                             59 VKKNRY 64
                                                             59 VKKNRY 64
WD-重复蛋白11                                                752 VRKIRF 757
                                                             752 VRKIRF 757
胃癌相关蛋白VRG107                                           20 SRKRQTRR 27
                                                             25 TRRRRN 30
                                                             25 TRRRRN 30
早期生长应答蛋白4                                            356 ARRKGRRG 363
                                                             452 EKKRHSKV 459
                                                             357 RRKGRR 362
                                                             357 RRKGRR 362
小泡转运相关蛋白(Vesicle transport-related protein)          309 PKRKNKKS 316
                                                             226 DKKLRE 231
                                                             310 KRKNKK 315
                                                             355 VKRLKS 360
                                                             226 DKKLRE 231
                                                             310 KRKNKK 315
                                                             355 VKRLKS 360
UPF3X                                                        140 AKKKTKKR 147
                                                             141 KKKTKK 146
                                                             217 ERRRRE 222
                                                             225 RKRQRE 230
                                                             233 RRKWKE 238
                                                             240 EKRKRK 245
                                                             296 DKREKA 301
                                                             373 RRRQKE 378
                                                             393 MKKEKD 398
                                                             426 VKRDRI 431
                                                          140 AKKKTKKRD 148
                                                          141 KKKTKK 146
                                                          217 ERRRRE 222
                                                          225 RKRQRE 230
                                                          233 RRKWKE 238
                                                          240 EKRKRK 245
                                                          296 DKREKA 301
                                                          373 RRRQKE 378
                                                          393 MKKEKD 398
                                                          426 VKRDRI 431
CGI-201蛋白，IV型                                         49 ARRTRS 54
                                                          49 ARRTRS 54
环指蛋白23                                                98 KRKIRD 103
                                                          98 KRKIRD 103
FKSG17                                                    72 EKKARK 77
                                                          95 IRKSKN 100
                                                          72 EKKARK 77
                                                          95 IRKSKN 100
P83                                                       681 ARKERE 686
                                                          681 ARKERE 686
卵巢癌相关蛋白1                                           62 LKRDRF 67
                                                          62 LKRDRF 67
MHC II类反式激活因子CIITA                                 407 HRRPRE 412
                                                          741 PRKKRP 746
                                                          783 DRKQKV 788
                                                          407 HRRPRE 412
                                                          741 PRKKRP 746
                                                          783 DRKQKV 788
血小板糖蛋白VI-2                                          275 SRRKRLRH 282
                                                          275 SRRKRL 280
                                                          275 SRRKRL 280
泛素样蛋白5                                               11 GKKVRV 16
                                                          11 GKKVRV 16
蛋白激酶D2                                                191 ARKRRL 196
                                                          191 ARKRRL 196
同源框(homeobox)蛋白GSH-2                                 202 GKRMRT 207
                                                          252 NRRVKH 257
                                                          202 GKRMRT 207
                                                          252 NRRVKH 257
ULBP3蛋白                                                 166 ARRMKE 171
                                                          201 HRKKRL 206
                                                          166 ARRMKE 171
                                                          201 HRKKRL 206
II型碘化甲状腺氨酸脱碘酶                                  87 SKKEKV 92
                                                          87 SKKEKV 92
                                                          299 SKRCKK 304
                                                          299 SKRCKK 304
精子抗原                                                  160 LKKYKE 165
                                                          478 IKRLKE 483
                                                          160 LKKYKEKRT 168
                                                          160 LKKYKE 165
                                                          478 IKRLKE 483
UDP-GalNAc：多肽N-乙酰半乳糖氨基转移酶                    4 ARKIRT 9
                                                          44 DRRVRS 49
                                                          138 PRKCRQ 143
                                                          4 ARKIRT 9
                                                          44 DRRVRS 49
                                                          138 PRKCRQ 143
NCBE                                                      62 HRRHRH 67
                                                          73 RKRDRE 78
                                                          1012 SKKKKL 1017
                                                          62 HRRHRH 67
                                                          73 RKRDRE 78
                                                          1012 SKKKKL 1017
WD重复蛋白                                                372 LKKKEERL 379
                                                          384 EKKQRR 389
                                                          400 AKKMRP 405
                                                          384 EKKQRR 389
                                                          400 AKKMRP 405
磷酸二酯酶11A                                             27 MRKGKQ 32
                                                          27 MRKGKQ 32
可能的转运阳离子的ATP酶2                                  891 ERRRRPRD 898
                                                          306 SRKWRP 311
                                                          891 ERRRRP 896
                                                          306 SRKWRP 311
                                                          891 ERRRRP 896
HMG-box转录因子TCF-3                                      420 GKKKKRKR 427
                                                          399 ARKERQ 404
                                                          420 GKKKKR 425
                                                          420 GKKKKRKRE 428
                                                          399 ARKERQ 404
                                                          420 GKKKKR 425
HVPS 11                                                   793 VRRYRE 798
                                                          793 VRRYRE 798
PIST                                                      165 NKKEKM 170
                                                          165 NKKEKM 170
FYN-结合蛋白                                              473 KKREKE 478
                                                          501 KKKFKL 506
                                                          682 LKKLKK 687
                                                          696 RKKFKY 701
                                                          473 KKREKE 478
                                                          501 KKKFKL 506
                                                          682 LKKLKK 687
                                                          696 RKKFKY 701
Clorf25                                                   620 GKKQKT 625
                                                          620 GKKQKT 625
Clorfl4                                                   441 LRRRKGKR 448
                                                          70 LRRWRR 75
                                                          441 LRRRKG 446
                                                          70 LRRWRR 75
                                                          441 LRRRKG 446
T-box转录因子TBX3                                         144 DKKAKY 149
                                                          309 GRREKR 314
                                                          144 DKKAKY 149
                                                          309 GRREKR 314
线粒体RNA 39S核糖体蛋白L47                                121 AKRQRL 126
                                                          216 EKRARI 221
                                                          230 RKKAKI 235
                                                          121 AKRQRL 126
                                                          216 EKRARI 221
                                                          230 RKKAKI 235
CGI-203                                                   33 VRRIRD 38
                                                          33 VRRIRD 38
Jagged1                                                   1093 LRKRRK 1098
                                                          1093 LRKRRK 1098
分泌型载体-相关膜蛋白1                                    102 DRRERE 107
                                                          102 DRRERE 107
维生素D受体相互作用蛋白复合物组分DRIP205                  673 KKKKSSRL 680
                                                          672 TKKKKS 677
                                                          954 QKRVKE 959
                                                          978 GKRSRT 983
                                                          995 PKRKKA 1000
                                                          1338 GKREKS 1343
                                                          1482 HKKHKK 1487
                                                          1489 KKKVKD 1494
                                                          672 TKKKKS 677
                                                          954 QKRVKE 959
                                                          978 GKRSRT 983
                                                          995 PKRKKA 1000
                                                          1338 GKREKS 1343
                                                          1482 HKKHKK 1487
                                                          1489 KKKVKD 1494
分泌型载体-相关膜蛋白2                                    100 ERKERE 105
                                                          100 ERKERE 105
Nogo受体                                                  420 SRKNRT 425
                                                          420 SRKNRT 425
FLAMINGO1                                                 169 GRRKRN 174
                                                          2231 ARRQRR 2236
                                                          169 GRRKRN 174
                                                          2231 ARRQRR 2236
CC-趋化因子受体                                           58 CKRLKS 63
                                                          58 CKRLKS 63
促乳激素调节元件-结合蛋白                                 271 HKRLRQ 276
                                                          271 HKRLRQ 276
Kappa B和V(D)J重组信号序列结合蛋白                        17 PRKRLTKG 24
                                                          713 RKRRKEKS 720
                                                          903 PKKKRLRL 910
                                                          180 HKKERK 185
                                                          629 TKKTKK 634
                                                          712 LRKRRK 717
                                                          903 PKKKRL 908
                                                          1447 QKRVKE 1452
                                                          1680 SRKPRM 1685
                                                          180 HKKERK 185
                                                          629 TKKTKK 634
                                                          712 LRKRRK 717
                                                          903 PKKKRL 908
                                                          1447 QKRVKE 1452
                                                          1680 SRKPRM 1685
乳腺癌转移-抑制因子1                                      200 SKRKKA 205
                                                          229 IKKARA 234
                                                          200 SKRKKA 205
                                                          229 IKKARA 234
Forkhead box蛋白P3                                        414 RKKRSQRP 421
                                                          413 FRKKRS 418
                                                          413 FRKKRS 418
FAS结合蛋白                                               228 LKRKLIRL 235
                                                          391 RKKRRARL 398
                                                          358 ARRLRE 363
                                                          390 ERKKRR 395
                                                          629 CKKSRK 634
                                                          358 ARRLRE 363
                                                          390 ERKKRR 395
                                                          629 CKKSRK 634
泛素羧基末端水解酶12                                      228 HKRMKV 233
                                                          244 LKRFKY 249
                                                          228 HKRMKV 233
                                                          244 LKRFKY 249
KIAA0472蛋白                                              110 HRKPKL 115
                                                          110 HRKPKL 115
PNAS-10                                                   168 LKRSRP 73
                                                          106 PRKSRR 111
                                                          68 LKRSRP 73
                                                          106 PRKSRR 111
PNAS-26                                                   118 DRRTRL 123
                                                          118 DRRTRL 123
髓磷脂转录因子2                                           176 GRRKSERQ 183
钠/钾-转运ATP酶γ链                                       47 SRRFRC 52
                                                          55 NKKRRQ 60
                                                          47 SRRFRC 52
                                                          55 NKKRRQ 60
Mdm4蛋白                                                  441 EKRPRD 446
                                                          464 ARRLKK 469
                                                          441 EKRPRD 446
                                                          464 ARRLKK 469
G抗原家族D2蛋白                                           87 QKKIRI 92
                                                          87 QKKIRI 92
NipSnap2蛋白                                              153 FRKARS 158
                                                          153 FRKARS 158
Stannin                                                   73 ERKAKL 78
                                                          73 ERKAKL 78
碳酸氢钠协同转运蛋白                                      973 EKKKKKKK 980
                                                          165 LRKHRH 170
                                                          666 LKKFKT 671
                                                          966 DKKKKE 971
                                                          973 EKKKKK 978
                                                          165 LRKHRH 170
                                                          666 LKKFKT 671
                                                          966 DKKKKE 971
                                                          973 EKKKKK 978
肌球蛋白(Myosin)X                                         683 YKRYKV 688
                                                          828 EKKKRE 833
                                                          1653 LKRIRE 1658
                                                          1676 LKKTKC 1681
                                                          683 YKRYKV 688
                                                          828 EKKKRE 833
                                                          1653 LKRIRE 1658
                                                          1676 LKKTKC 1681
PNAS-20                                                   21 RKRKSVRG 28
                                                          20 ERKRKS 25
                                                          20 ERKRKS 25
Pellino                                                   36 RRKSRF 41
                                                          44 FKRPKA 49
                                                          36 RRKSRF 41
                                                          44 FKRPKA 49
乙酰透明质酸介导的迁移受体                                66 ARKVKS 71
                                                          66 ARKVKS 71
短瞬时受体潜在通道7                                       753 FKKTRY 758
                                                          753 FKKTRY 758
Liprin-alpha2                                             825 PKKKGIKS 832
                                                          575 IRRPRR 580
                                                          748 LRKHRR 753
                                                          839 GKKEKA 844
                                                          875 DRRLKK 880
                                                          575 IRRPRR 580
                                                          748 LRKHRR 753
                                                          839 GKKEKA 844
                                                          875 DRRLKK 880
转录中间因子1-alpha                                       904 DKRKCERL 911
                                                          1035 PRKKRLKS 1042
                                                          321 NKKGKA 326
                                                          1035 PRKKRL 1040
                                                          321 NKKGKA 326
                                                          1035 PRKKRL 1040
软骨中间层蛋白                                            719 QRRNKR 724
                                                          719 QRRNKR 724
含UBX结构域的蛋白1                                        194 YRKIKL 199
                                                          194 YRKIKL 199
花生四烯酸-12-脂加氧酶，12R型                             166 VRRHRN 171
                                                          233 WKRLKD 238
                                                          166 VRRHRN 171
                                                          233 WKRLKD 238
造血性PBX-相互作用蛋白                                    159 LRRRRGRE 166
                                                          698 LKKRSGKK 705
                                                          159 LRRRRG 164
                                                          703 GKKDKH 708
                                                          159 LRRRRG 164
                                                          703 GKKDKH 708
NAG18                                                     28 LKKKKK 33
                                                          28 LKKKKK 33
POU5结构域蛋白                                            222 ARKRKR 227
                                                          222 ARKRKR 227
NRCAM蛋白                                                 2 PKKKRL 7
                                                          887 SKRNRR 892
                                                          1185 IRRNKG 1190
                                                          1273 GKKEKE 1278
                                                          2 PKKKRL 7
                                                          887 SKRNRR 892
                                                          1185 IRRNKG 1190
                                                          1273 GKKEKE 1278
原钙黏着蛋白(Protocadherin)γ簇                           11 TRRSRA 16
                                                          11 TRRSRA 16
SKD1蛋白                                                  288 IRRRFEKR 295
                                                          251 ARRIKT 256
                                                          362 FKKVRG 367
                                                          251 ARRIKT 256
                                                          362 FKKVRG 367
抗-死亡蛋白                                               58 HRKRSRRV 65
                                                          59 RKRSRR 64
                                                          59 RKRSRR 64
中心粒蛋白(Centrin)3                                      14 TKRKKRRE 21
                                                          14 TKRKKR 19
                                                          14 TKRKKR 19
胞外核苷三磷酸二磷酸水解酶3
(Ectonucleoside triphosphate diphosphohydrolase 3)        512 TRRKRH 517
                                                          512 TRRKRH 517
Homeobox protein prophet of PIT-1                         12 PKKGRV 17
                                                          69 RRRHRT 74
                                                          119 NRRAKQ 124
                                                          12 PKKGRV 17
                                                          69 RRRHRT 74
                                                          119 NRRAKQ 124
前列腺素EP3受体                                           77 YRRRESKR 84
                                                          389 MRKRRLRE 396
                                                          82 SKRKKS 87
                                                          389 MRKRRL 394
                                                          82 SKRKKS 87
                                                          389 MRKRRL 394
垂体同源框3                                               58 LKKKQRRQ 65
                                                          59 KKKQRR 64
                                                          112 NRRAKW 117
                                                          118 RKRERS 123
                                                          59 KKKQRR 64
                                                          112 NRRAKW 117
                                                          118 RKRERS 123
HPRL-3                                                    136 KRRGRI 141
                                                          136 KRRGRI 141
Advillin                                                  812 MKKEKG 817
                                                          812 MKKEKG 817
Nuclear LIM interactor-interacting factor 1               32 GRRARP 37
                                                          109 LKKQRS 114
                                                          32 GRRARP 37
                                                          109 LKKQRS 114
Core histone macro-H2A.1                                  5 GKKKSTKT 12
                                                          114 AKKRGSKG 121
                                                          70 NKKGRV 75
                                                          132 AKKAKS 137
                                                          154 ARKSKK 159
                                                          302 DKKLKS 307
                                                          70 NKKGRV 75
                                                          132 AKKAKS 137
                                                          154 ARKSKK 159
                                                          302 DKKLKS 307
绒毛蛋白-样蛋白                                           180 KRRRNQKL 187
                                                          179 EKRRRN 184
                                                          179 EKRRRN 184
BETA-细丝蛋白                                             254 PKKARA 259
                                                          2002 ARRAKV 2007
                                                          254 PKKARA 259
                                                          2002 ARRAKV 2007
三重基序蛋白TRIM31 alpha                                  290 LKKFKD 295
                                                          290 LKKFKD 295
核受体辅阻抑物1                                           106 SKRPRL 111
                                                          299 ARKQRE 304
                                                          330 RRKAKE 335
                                                          349 IRKQRE 354
                                                          412 QRRVKF 417
                                                          497 KRRGRN 502
                                                          580 RRKGRI 585
                                                          687 SRKPRE 692
                                                          2332 SRKSKS 2337
                                                          106 SKRPRL 111
                                                          299 ARKQRE 304
                                                          330 RRKAKE 335
                                                          349 IRKQRE 354
                                                          412 QRRVKF 417
                                                          497 KRRGRN 502
                                                          580 RRKGRI 585
                                                          687 SRKPRE 692
                                                          2332 SRKSKS 2337
脑表达的环指蛋白                                          432 KRRVKS 437
                                                          432 KRRVKS 437
PB39                                                      231 TKKIKL 236
                                                          231 TKKIKL 236
精子顶体蛋白(Sperm acrosomal protein)                     48 FRKRMEKE 55
                                                          24 RRKARE 29
                                                          135 KRKLKE 140
                                                          213 KKRLRQ 218
                                                          24 RRKARE 29
                                                          135 KRKLKE 140
                                                          213 KKRLRQ 218
小泡运输(VESICLE TRAFFICKING)蛋白SEC22B                   177 SKKYRQ 182
                                                          177 SKKYRQ 182
核仁转录因子1                                             79 VRKFRT 84
                                                          102 GKKLKK 107
                                                          125 EKRAKY 130
                                                          147 SKKYKE 152
                                                          156 KKKMKY 161
                                                          240 KKRLKW 245
                                                          451 KKKAKY 456
                                                          523 EKKEKL 528
                                                          558 SKKMKF 563
                                                          79 VRKFRT 84
                                                          102 GKKLKK 107
                                                          125 EKRAKY 130
                                                          147 SKKYKE 152
                                                          156 KKKMKY 161
                                                          240 KKRLKW 245
                                                          451 KKKAKY 456
                                                          523 EKKEKL 528
                                                          558 SKKMKF 563
丛蛋白(Plexin)-B3                                         248 FRRRGARA 255
3型连接蛋白(Junctophilin)                                 626 QKRRYSKG 633
Plaucible mixed-lineage kinase protein                    773 YRKKPHRP 780
                                                          312 ERRLKM 317
                                                          312 ERRLKM 317
脂肪酸结合蛋白4，脂肪细胞                                 78 DRKVKS 83
                                                          105 IKRKRE 110
                                                          78 DRKVKS 83
                                                          105 IKRKRE 110
exostoses(复数)1                                          78 SKKGRK 83
                                                          78 SKKGRK 83
含DHHC-结构域且富含半胱氨酸的蛋白                         64 HRRPRG 69
                                                          64 HRRPRG 69
Myb原癌基因-蛋白                                          2 ARRPRH 7
                                                          292 EKRIKE 297
                                                          523 LKKIKQ 528
                                                          2 ARRPRH 7
                                                          292 EKRIKE 297
                                                          23 LKKIKQ 528
长链脂肪酸-CoA连接酶2                                     259 RRKPKP 264
                                                          259 RRKPKP 264
突触融合蛋白(syntaxin)1B2                                 260 ARRKKI 265
                                                          260 ARRKKI 265
Dachshund 2                                               162 ARRKRQ 167
                                                          516 QKRLKK 521
                                                          522 EKKTKR 527
                                                          162 ARRKRQ 167
                                                          516 QKRLKK 521
                                                          522 EKKTKR 527
DEAD/DEXH螺旋酶DDX31                                      344 EKRKSEKA 351
                                                          760 TRKKRK 765
                                                          760 TRKKRK 765
雄激素受体                                                628 ARKLKK 633
                                                          628 ARKLKK 633
视黄酸受体alpha                                           364 RKRRPSRP 371
                                                          163 NKKKKE 168
                                                          363 VRKRRP 368
                                                          163 NKKKKE 168
                                                          363 VRKRRP 368
驱动蛋白重链(Kinesin heavy chain)                         340 WKKKYEKE 347
                                                          605 VKRCKQ 610
                                                          864 EKRLRA 869
                                                          605 VKRCKQ 610
                                                          864 EKRLRA 869
二泛素(DIUBIQUITIN)                                       30 VKKIKE 35
                                                          30 VKKIKE 35
BING1蛋白                                                 519 NKKFKM 524
                                                          564 ERRHRL 569
                                                          519 NKKFKM 524
                                                          564 ERRHRL 569
粘着斑激酶1                                               664 SRRPRF 669
                                                          664 SRRPRF 669
EBN2蛋白                                                  20 TKRKKPRR 27
                                                          13 PKKDKL 18
                                                          20 TKRKKP 25
                                                          47 NKKNRE 52
                                                          64 LKKSRI 69
                                                          76 PKKPRE 81
                                                          493 SRKQRQ 498
                                                          566 VKRKRK 571
                                                          13 PKKDKL 18
                                                          20 TKRKKP 25
                                                          47 NKKNRE 52
                                                          64 LKKSRI 69
                                                          76 PKKPRE 81
                                                          493 SRKQRQ 498
                                                          566 VKRKRK 571
CO16蛋白                                                  33 ARRLRR 38
                                                          115 PRRCKW 120
                                                          33 ARRLRR 38
                                                          115 PRRCKW 120
犬尿氨酸3-单加氧酶                                        178 MKKPRF 183
                                                          178 MKKPRF 183
MLN 51蛋白                                                4 RRRQRA 9
                                                          255 PRRIRK 260
                                                          407 ARRTRT 412
                                                          4 RRRQRA 9
                                                          255 PRRIRK 260
                                                          407 ARRTRT 412
MHC II类抗原                                              99 QKRGRV 104
MHC II类抗原                                              99 QKRGRV 104
转化型酸性含卷曲螺旋的蛋白1
(Transforming acidic coiled-coil-containing protein 1)    225 SRRSKL 230
                                                          455 PKKAKS 460
                                                          225 SRRSKL 230
                                                          455 PKKAKS 460
神经内分泌特异性蛋白VGF                                   479 EKRNRK 484
                                                          479 EKRNRK 484
有机阳离子转运蛋白                                        230 GRRYRR 235
                                                          535 PRKNKE 540
                                                          230 GRRYRR 235
                                                          535 PRKNKE 540
DNA聚合酶theta                                            215 KRRKHLKR 222
                                                          214 WKRRKH 219
                                                          220 LKRSRD 225
                                                          1340 GRKLRL 1345
                                                          1689 SRKRKL 1694
                                                          214 WKRRKH 219
                                                          220 LKRSRD 225
                                                          1340 GRKLRL 1345
                                                          1689 SRKRKL 1694
CDC45-相关蛋白                                            169 MRRRQRRE 176
                                                          155 EKRTRL 160
                                                          170 RRRQRR 175
                                                          483 NRRCKL 488
                                                          155 EKRTRL 160
                                                          170 RRRQRR 175
                                                          483 NRRCKL 488
氯化物胞内通道蛋白2                                       197 AKKYRD 202
                                                          197 AKKYRD 202
甲基-CpG结合蛋白                                          85 KRKKPSRP 92
                                                          83 SKKRKK 88
                                                          318 QKRQKC 323
                                                          354 YRRRKR 359
                                                          83 SKKRKK 88
                                                          318 QKRQKC 323
                                                          354 YRRRKR 359
蛋白激酶C，eta型                                          155 RKRQRA 160
                                                          155 RKRQRA 160
核内不均一核糖核蛋白H                                     71 LKKDRE 76
                                                          169 LKKHKE 174
                                                          71 LKKDRE 76
                                                          169 LKKHKE 174
ORF2                                                      11 SRRTRW 16
                                                          155 ERRRKF 160
                                                          185 LRRCRA 190
                                                          530 SRRSRS 535
                                                          537 GRRRKS 542
                                                          742 ERRAKQ 747
                                                          11 SRRTRW 16
                                                          155 ERRRKF 160
                                                          185 LRRCRA 190
                                                          530 SRRSRS 535
                                                          537 GRRRKS 542
                                                          742 ERRAKQ 747
F-box only protein 24                                     9 LRRRRVKR 16
                                                          9 LRRRRV 14
                                                          29 EKRGKG 34
                                                          9 LRRRRV 14
                                                          29 EKRGKG 34
富含亮氨酸的神经元蛋白                                    51 NRRLKH 56
                                                          51 NRRLKH 56
RER1蛋白                                                  181 KRRYRG 186
                                                          181 KRRYRG 186
Nephrocystin                                              3 ARRQRD 8
                                                          430 PKKPKT 435
                                                          557 NRRSRN 562
                                                          641 EKRDKE 646
                                                          3 ARRQRD 8
                                                          430 PKKPKT 435
                                                          557 NRRSRN 562
                                                          641 EKRDKE 646
腺苷酸激酶同功酶2，线粒体                                 60 GKKLKA 65
                                                          116 KRKEKL 121
                                                          60 GKKLKA 65
                                                          116 KRKEKL 121
十氯酮还原酶(Chlordecone reductase)                       245 AKKHKR 250
                                                          245 AKKHKR 250
Metaxin 2                                                 166 KRKMKA 171
                                                          166 KRKMKA 171
成对的中胚层同源框蛋白1                                   89 KKKRKQRR 96
                                                          88 EKKKRK 93
                                                          94 QRRNRT 99
                                                          144 NRRAKF 149
                                                          88 EKKKRK 93
                                                          94 QRRNRT 99
                                                          144 NRRAKF 149
环指蛋白                                                  174 LKRKWIRC 181
                                                          8 TRKIKL 13
                                                          95 MRKQRE 100
                                                          8 TRKIKL 13
                                                          95 MRKQRE 100
Ataxin 7                                                  55 PRRTRP 60
                                                          377 GRRKRF 382
                                                          704 GKKRKN 709
                                                          834 GKKRKC 839
                                                          55 PRRTRP 60
                                                          377 GRRKRF 382
                                                          704 GKKRKN 709
                                                          834 GKKRKC 839
生长捕获特异性蛋白1(Growth-arrest-specific protein 1)     169 ARRRCDRD 176
SKAP55蛋白                                                115 EKKSKD 120
                                                          115 EKKSKD 120
丝氨酸棕榈酰转移酶1                                       232 PRKARV 237
                                                          232 PRKARV 237
丝氨酸棕榈酰转移酶2                                       334 KKKYKA 339
                                                          450 RRRLKE 455
                                                          334 KKKYKA 339
                                                          450 RRRLKE 455
Synaptopod in                                             405 KRRQRD 410
                                                          405 KRRQRD 410
Alpha-tectorin                                            1446 TRRCRC 1451
                                                          2080 IRRKRL 2085
                                                          1446 TRRCRC 1451
                                                          2080 IRRKRL 2085
长形转录因子C-MAF                                         291 QKRRTLKN 298
Usher综合症Iia型蛋白                                      1285 MRRLRS 1290
                                                          1285 MRRLRS 1290
MSin3A相关多肽p30                                         95 QKKVKI 100
                                                          124 NRRKRK 129
                                                          158 LRRYKR 163
                                                          95 QKKVKI 100
                                                          124 NRRKRK 129
                                                          158 LRRYKR 163
Igdelta链C区                                              142 KKKEKE 147
                                                          142 KKKEKE 147
甲状腺激素受体相关蛋白复合体组分TRAP100                   383 AKRKADRE 390
                                                          833 KKRHRE 838
                                                          833 KKRHRE 838
P60katanin                                                369 LRRRLEKR 376
                                                          326 SRRVKA 331
                                                          326 SRRVKA 331
转录因子jun-D                                             286 RKRKLERI 293
                                                          273 RKRLRN 278
                                                          285 CRKRKL 290
                                                          273 RKRLRN 278
                                                          285 CRKRKL 290
固醇/视黄醇脱氢酶                                         152 VRKARG 157
                                                          152 VRKARG 157
糖原[淀粉]合酶，肝                                        554 DRRFRS 559
                                                          578 SRRQRI 583
                                                          554 DRRFRS 559
                                                          578 SRRQRI 583
雌激素-相关受体gamma                                      173 TKRRRK 178
                                                          353 VKKYKS 358
                                                          173 TKRRRK 178
                                                          353 VKKYKS 358
神经视网膜-特异性亮氨酸拉链蛋白                           162 QRRRTLKN 169
胞浆型磷脂酶A2-gamma                                      514 NKKKILRE 521
                                                          31 LKKLRI 36
                                                          218 FKKGRL 223
                                                          428 CRRHKI 433
                                                          31 LKKLRI 36
胞浆型磷脂酶A2-gamma                                      218 FKKGRL 223
                                                          428 CRRHKI 433
GLE1                                                      415 AKKIKM 420
                                                          415 AKKIKM 420
Multiple exostoses type II protein EXT2.I                 296 VRKRCHKH 303
                                                          659 RKKFKC 664
                                                          659 RKKFKC 664
循环的-AMP-依赖性转录因子ATF-7                            86 EKKARS 91
                                                          332 GRRRRT 337
                                                          344 ERRQRF 349
                                                          86 EKKARS 91
                                                          332 GRRRRT 337
                                                          344 ERRQRF 349
蛋白激酶/endoribonulcease                                 886 LRKFRT 891
                                                          886 LRKFRT 891
转录因子E2F6                                              23 RRRCRD 28
                                                          59 VKRPRF 64
                                                          98 VRKRRV 103
                                                          117 EKKSKN 122
                                                          23 RRRCRD 28
                                                          59 VKRPRF 64
                                                          98 VRKRRV 103
                                                          117 EKKSKN 122
MAP激酶活化的死亡结构域蛋白                               1333 IRKKVRRL 1340
                                                          160 KRRAKA 165
                                                          943 MKKVRR 948
                                                          1034 DKRKRS 1039
                                                          1334 RKKVRR 1339
                                                          1453 TKKCRE 1458
                                                          160 KRRAKA 165
                                                          943 MKKVRR 948
                                                          1034 DKRKRS 1039
                                                          1334 RKKVRR 1339
                                                          1453 TKKCRE 1458
孤儿核受体PXR(Orphan nuclear receptor PXR)                126 KRKKSERT 133
                                                          87 TRKTRR 92
                                                          125 IKRKKS 130
                                                          87 TRKTRR 92
                                                          125 IKRKKS 130
LENS上皮细胞衍生的生长因子                                149 RKRKAEKQ 156
                                                          286 KKRKGGRN 293
                                                          145 ARRGRK 150
                                                          178 PKRGRP 183
                                                          285 EKKRKG 290
                                                          313 DRKRKQ 318
                                                          400 LKKIRR 405
                                                          337 VKKVEKKRE 345
                                                          145 ARRGRK 150
                                                          178 PKRGRP 183
                                                          285 EKKRKG 290
                                                          313 DRKRKQ 318
                                                          400 LKKIRR 405
LIM同源框蛋白辅因子                                       255 TKRRKRKN 262
                                                          255 TKRRKR 260
                                                          255 TKRRKR 260
MULTIPLE MEMBRANE SPANNING RECEPTOR TRC8                  229 WKRIRF 234
                                                          229 WKRIRF 234
转录因子SUPT3H                                            172 DKKKLRRL 179
                                                          169 MRKDKK 174
                                                          213 NKRQKI 218
                                                          169 MRKDKK 174
                                                          213 NKRQKI 218
GEMININ                                                   50 KRKHRN 55
                                                          104 EKRRKA 109
                                                          50 KRKHRN 55
                                                          104 EKRRKA 109
细胞循环调节因子p78                                       165 EKKKVSKA 172
                                                          124 IKRKKF 129
                                                          188 TKRVKK 193
                                                          381 DRRQKR 386
                                                          124 IKRKKF 129
                                                          188 TKRVKK 193
                                                          381 DRRQKR 386
淋巴细胞抗原6复合体，locus D                              61 QRKGRK 66
                                                          85 ARRLRA 90
                                                          61 QRKGRK 66
                                                          85 ARRLRA 90
Delta 1-吡咯啉-5-羧酸酯合成酶                             455 LRRTRI 460
                                                          455 LRRTRI 460
B细胞连接蛋白BLNK                                         36 IKKLKV 41
                                                          36 IKKLKV 41
B细胞连接蛋白BLNK-S                                       36 IKKLKV 41
                                                          36 IKKLKV 41
致命性Alzheimer抗原                                       5 ARRRRKRR 12
                                                          16 PRRRRRRT 23
                                                          93 WKKKTSRP 100
                                                          5 ARRRRK 10
                                                          16 PRRRRR 21
                                                          26 PRRPRI 31
                                                          35 TRRMRW 40
                                                          5 ARRRRK 10
                                                          16 PRRRRR 21
                                                          26 PRRPRI 31
                                                          35 TRRMRW 40
瞬时受体潜在通道4zeta剪接变体                             505 CKKKMRRK 512
                                                          506 KKKMRR 511
                                                          676 HRRSKQ 681
                                                          506 KKKMRR 511
                                                          676 HRRSKQ 681
Myofibrillogenesis调节因子MR-2                            65 RKRGKN 70
                                                          65 RKRGKN 70
含磷酸酶锚定蛋白2c的SH2结构域                             269 IKKRSLRS 276
免疫球蛋白超家族，成员3                                   394 SKRPKN 399
                                                          394 SKRPKN 399
Meis(小鼠)同系物3                                         112 PRRSRR 117
                                                          120 WRRTRG 125
                                                          112 PRRSRR 117
                                                          120 WRRTRG 125
Deleted in azoospermia 2                                  105 GKKLKL 110
                                                          114 IRKQKL 119
                                                          105 GKKLKL 110
                                                          114 IRKQKL 119
Centaurin gamma3                                          543 NRKKHRRK 550
                                                          544 RKKHRR 549
                                                          544 RKKHRR 549
Pre-B-细胞白血病转录因子-1                                233 ARRKRR 238
                                                          286 NKRIRY 291
                                                          233 ARRKRR 238
                                                          286 NKRIRY 291
60S核糖体蛋白L13a                                         112 DKKKRM 117
                                                          158 KRKEKA 163
                                                          167 YRKKKQ 172
                                                          112 DKKKRM 117
                                                          158 KRKEKA 163
                                                          167 YRKKKQ 172
WD40-和蛋白3FYVE-结构域                                   388 IKRLKI 393
                                                          388 IKRLKI 393
LENG1蛋白                                                 34 RKRRGLRS 41
                                                          84 SRKKTRRM 91
                                                          1 MRRSRA 6
                                                          33 ERKRRG 38
                                                          85 RKKTRR 90
                                                          1 MRRSRA 6
                                                          33 ERKRRG 38
                                                          85 RKKTRR 90
MRIP2                                                     375 NKKKHLKK 382
G蛋白偶联受体                                             430 EKKKLKRH 437
                                                          290 WKKKRA 295
                                                          395 RKKAKF 400
                                                          431 KKKLKR 436
                                                          290 WKKKRA 295
                                                          395 RKKAKF 400
                                                          431 KKKLKR 436
                                                          143 LKKFRQ 148
                                                          228 LRKIRT 233
                                                          143 LKKFRQ 148
                                                          228 LRKIRT 233
                                                          232 QKRRRHRA 239
                                                          232 QKRRRH 237
                                                          232 QKRRRH 237
精子离子通道                                              402 QKRKTGRL 409
A-激酶锚定蛋白                                            2232 KRKKLVRD 2239
                                                          2601 EKRRRERE 2608
                                                          2788 EKKKKNKT 2795
                                                          370 RKKNKG 375
                                                          1763 SKKSKE 1768
                                                          2200 EKKVRL 2205
                                                          2231 LKRKKL 2236
                                                          2601 EKRRRE 2606
                                                          2785 EKKEKK 2790
                                                          1992 QKKDVVKRQ 2000
                                                          370 RKKNKG 375
                                                          1763 SKKSKE 1768
                                                          2200 EKKVRL 2205
                                                          2231 LKRKKL 2236
                                                          2601 EKRRRE 2606
                                                          2785 EKKEKK 2790
淋巴细胞特异性蛋白LSP1                                    315 GKRYKF 320
                                                          315 GKRYKF 320
类似于信号传递淋巴细胞活化分子(智人)                      261 RRRGKT 266
                                                          261 RRRGKT 266
皮质素-4-磺基转移酶-1                                     242 VRRYRA 247
                                                          242 VRRYRA 247
Moesin                                                    291 MRRRKP 296
                                                          325 EKKKRE 330
                                                          291 MRRRKP 296
                                                          325 EKKKRE 330
A-Raf原致肿瘤基因丝氨酸/苏氨酸-蛋白激酶                   288 KKKVKN 293
                                                          358 LRKTRH 363
                                                          288 KKKVKN 293
                                                          358 LRKTRH 363
细胞色素P4502C18                                          117 GKRWKE 122
                                                          117 GKRWKE 122
                                                          117 GKRWKE 122
                                                          156 LRKTKA 161
                                                          117 GKRWKE 122
                                                                          156 LRKTKA 161
蛋白酪氨酸磷酸酶，3型非受体                                               594 IRRRAVRS 601
                                                                          263 FKRKKF 268
                                                                          388 IRKPRH 393
                                                                          874 VRKMRD 879
                                                                          263 FKRKKF 268
                                                                          388 IRKPRH 393
                                                                          874 VRKMRD 879
类似于激肽释放酶7(chymotryptic，角质层)                                   15 VKKVRL 20
                                                                          15 VKKVRL 20
激素敏感性脂肪酶                                                          703 ARRLRN 708
                                                                          703 ARRLRN 708
40S核糖体蛋白S30                                                          25 KKKKTGRA 32
                                                                          23 EKKKKK 28
                                                                          23 EKKKKK 28
锌指蛋白91                                                                617 LRRHKR 622
                                                                          617 LRRHKR 622
NNP-1蛋白                                                                 320 NRKRLYKV 327
                                                                          387 ERKRSRRR 394
                                                                          432 QRRRTPRP 439
                                                                          454 EKKKKRRE 461
                                                                          29 VRKLRK 34
                                                                          355 GRRQKK 360
                                                                          361 TKKQKR 366
                                                                          388 RKRSRR 393
                                                                          454 EKKKKR 459
                                                                          29 VRKLRK 34
                                                                          355 GRRQKK 360
                                                                          361 TKKQKR 366
                                                                          388 RKRSRR 393
                                                                          454 EKKKKR 459
甲硫氨酰-tRNA合成酶                                                       725 WKRIKG 730
                                                                          725 WKRIKG 730
ELMO2                                                                     560 NRRRQERF 567
脑脊膜瘤-表达的抗原6/11                                                   432 RKRAKD 437
                                                                          432 RKRAKD 437
I型β肌醇多磷酸4-磷酸酶(Inositol polyphosphate 4-phosphatase type I-beta) 375 LRKKLHKF 382
                                                                          829 ARKNKN 834
                                                                          829 ARKNKN 834
                                                                          815 SKKRKN 820
                                                          815 SKKRKN 820
C7ORF12                                                   40 SRRYRG 45
                                                          338 HRKNKP 343
                                                          40 SRRYRG 45
                                                          338 HRKNKP 343
Rap鸟嘌呤核苷酸交换因子                                   138 SRRRFRKI 145
                                                          1071 QRKKRWRS 1078
                                                          1099 HKKRARRS 1106
                                                          139 RRRFRK 144
                                                          661 SKKVKA 666
                                                          930 LKRMKI 935
                                                          1071 QRKKRW 1076
                                                          1100 KKRARR 1105
                                                          1121 ARKVKQ 1126
                                                          139 RRRFRK 144
                                                          661 SKKVKA 666
                                                          930 LKRMKI 935
                                                          1071 QRKKRW 1076
                                                          1100 KKRARR 1105
                                                          1121 ARKVKQ 1126
Sigma 1C衔接蛋白                                          27 ERKKITRE 34
Alsin                                                     883 GRKRKE 888
                                                          883 GRKRKE 888
NOPAR2                                                    14 LKRPRL 19
                                                          720 VKREKP 725
                                                          14 LKRPRL 19
                                                          720 VKREKP 725
AT-结合转录因子1                                          294 SKRPKT 299
                                                          961 EKKNKL 966
                                                          1231 NKRPRT 1236
                                                          1727 DKRLRT 1732
                                                          2032 QKRFRT 2037
                                                          2087 EKKSKL 2092
                                                          2317 QRKDKD 2322
                                                          2343 PKKEKG 2348
                                                          294 SKRPKT 299
                                                          961 EKKNKL 966
                                                          1231 NKRPRT 1236
                                                          1727 DKRLRT 1732
                                                          2032 QKRFRT 2037
                                                          2087 EKKSKL 2092
                                                          2317 QRKDKD 2322
                                                          2343 PKKEKG 2348
Suppressin                                                232 YKRRKK 237
                                                          232 YKRRKK 237
Midline 1蛋白                                             100 TRRERA 105
                                                          494 HRKLKV 499
                                                          100 TRRERA 105
                                                          494 HRKLKV 499
高迁移率族蛋白2a(High mobility group protein 2a)          6 PKKPKG 11
                                                          84 GKKKKD 89
                                                          6 PKKPKG 11
                                                          84 GKKKKD 89

Claims (22)

1、一种将GAG结合位点导入蛋白的方法，其特征在于：
-鉴定出蛋白中对于维持结构而言非必需的区，
-将至少一个碱性氨基酸导入所述位点和/或在所述位点中删除至少一个体积大的和/或酸性的氨基酸，
其中所述GAG结合位点具有Kd≤10μM的GAG结合亲和力，优选Kd≤1μM，更优选Kd≤0.1μM。
2、根据权利要求1所述方法获得的蛋白。
3、修饰的GAG结合蛋白，其特征在于：所述蛋白中的GAG结合区通过取代，插入，和/或删除了至少一个氨基酸而进行了修饰，从而增加了在所述GAG结合区内的碱性氨基酸的相对量，和/或降低了在所述GAG结合区内、优选在溶剂暴露位置的体积大的和/或酸性的氨基酸的量，其特征还在于：所述蛋白的GAG结合亲和力比相应野生型蛋白的GAG结合亲和力提高。
4、权利要求2或3所述的修饰蛋白，其特征在于：将选自Arg，Lys和His的至少一个碱性氨基酸插入所述GAG结合区。
5、权利要求2-4的任一项所述的修饰蛋白，其特征在于：所述蛋白是趋化因子，优选IL-8，RANTES或MCP-1。
6、权利要求5所述的修饰蛋白，其特征在于：所述GAG结合区是C末端的α-螺旋。
7、权利要求6所述的修饰蛋白，其特征在于：IL-8中的位置17，21，70和/或71被Arg，Lys，His，Asn和/或Gln取代。
8、权利要求2-7的任一项所述的修饰蛋白，其特征在于：增加的GAG结合亲和力是增加了对硫酸类肝素和/或肝素的结合亲和力。
9、权利要求2-8的任一项所述的修饰蛋白，其特征在于：对另外的生物活性区进行了修饰，从而抑制或下调了所述蛋白的另外的生物活性。
10、权利要求9所述的修饰蛋白，其特征在于：所述另外的生物活性区通过删除、插入和/或取代来修饰，优选用丙氨酸、空间相似残基和/或静电相似残基通过删除、插入和/或取代来修饰。
11、权利要求9或10所述的修饰蛋白，其特征在于：所述蛋白是趋化因子，所述另外的生物活性是白细胞活性。
12、权利要求11所述的修饰蛋白，其特征在于：所述蛋白是IL-8，所述另外的生物活性区位于前10个N-末端氨基酸内。
13、权利要求12所述的修饰蛋白，其特征在于：所述蛋白是删除了前6个N-末端氨基酸的IL-8突变体。
14、权利要求13所述的修饰蛋白，其特征在于：所述蛋白是选自del6F17RE70KN71R，del6F17RE70RN71K和del6E70KN71K的IL-8突变体。
15、分离的多核酸分子，其特征在于：它编码权利要求2-14的任一项所述的蛋白。
16、分离的多核酸分子，其特征在于：它在严谨条件下与权利要求15所述的DNA分子杂交。
17、载体，其特征在于：它包括权利要求15或16所述的分离的DNA分子。
18、重组细胞，其特征在于：它被权利要求17所述的载体稳定转染。
19、药物组合物，其特征在于：它包括权利要求2-14的任一项所述的蛋白，权利要求15或16所述的多核酸，或权利要求17所述的载体和药学上可接受的载体。
20、权利要求2-14的任一项所述的蛋白，权利要求15或16所述的多核苷酸或权利要求17所述的载体在抑制或遏制相应野生型蛋白的生物活性的方法中的用途。
21、权利要求2-14的任一项所述的蛋白，权利要求15或16所述的多核苷酸或权利要求17所述的载体在制备用于治疗炎症病症的药物的方法中的用途。
22、权利要求21所述的用途，其特征在于：所述炎症病症选自类风湿性关节炎，牛皮癣，骨关节炎，哮喘，阿尔茨海默氏症和多发性硬化。
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