CN1292001C - 核因子－κB p50亚基拮抗肽及其制备与应用 - Google Patents
核因子－κB p50亚基拮抗肽及其制备与应用 Download PDFInfo
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- CN1292001C CN1292001C CN 200510069461 CN200510069461A CN1292001C CN 1292001 C CN1292001 C CN 1292001C CN 200510069461 CN200510069461 CN 200510069461 CN 200510069461 A CN200510069461 A CN 200510069461A CN 1292001 C CN1292001 C CN 1292001C
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Abstract
本发明涉及核因子-κB p50亚基拮抗肽及其制备与应用，特别是涉及一种治疗用核因子-κB(NF-κB)p50亚基拮抗肽，更具体而言，涉及一种能够与核因子-κBp50亚基特异结合并抑制其活性的小分子肽，其氨基酸序列见序列表。
Description
技术领域
本发明涉及医药领域，特别是涉及一种治疗用核因子-κB(NF-κB)p50亚基拮抗肽，更具体而言，涉及一种能够与核因子-κB p50亚基特异结合并抑制其活性的小分子肽。本发明还涉及这种核因子-κB p50亚基拮抗肽的制备和应用。
背景技术
NF-κB是普遍存在于几乎所有细胞胞浆中的一种转录因子，是多种信号转导途径的汇聚点，具有调控多种参与免疫应答、炎症反应、病毒复制、细胞凋亡和增殖以及生长发育相关基因表达的功能。NF-κB系由两种Rel家族蛋白构成的二聚体蛋白质。根据结构、功能和合成方式等方面的差异，可将Rel蛋白家族分成两类：一类是前体蛋白p105和p100，其C末端含锚蛋白重复序列(ankin repeatmotif)，可通过ATP依赖的蛋白水解过程裂解，变为成熟的p50和p52。该类蛋白缺乏反式激活结构域，无独立激活基因转录的功能；另一类是RelA(p65)、Rel(c-Rel)、v-Rel和RelB，其C末端含有1个或多个反式激活域(transactivationdomain)，具有独立激活基因转录的功能[1]。
当细胞处于静息状态时，NF-κB主要与κB抑制蛋白(IκBs)结合形成三聚体，以无活性方式存在于细胞质中。当细胞受到前炎症细胞因子、病毒、内毒素、紫外线、氧化剂和放射线等多种外界信号刺激而启动细胞第二信使系统时，IκBs在IκB激酶(IKK)的作用下发生磷酸化并降解，NF-κB失去IκBs的严格控制而被激活，并移位进入细胞核内，其亚基形成环状结构与靶基因启动子内的特定部位结合，高效诱导促炎细胞因子(如TNF、GM-CSF、IL-1β、IL-6、IL-11、IL-17等)、趋化因子(如IL-8、RANTES、MIP-1α、MCP-2等)、粘附分子(如ICAM-1、VCAM-1、ELAM-1等)、免疫受体(如IL-2R)以及参与炎症反应级联放大的多种酶(如NO合成酶、环氧化酶等)的表达[2]。NF-κB所诱导的一些蛋白可进一步反转活化NF-κB，导致炎症反应的扩大和延续。基于上述认识，许多学者认为抑制NF-κB的转录活性将为炎性相关疾病的治疗开辟新的途径。另外，NF-κB在细胞凋亡和增殖中也发挥重要作用，其可通过直接上调凋亡抑制因子(c-IAP1、c-IAP2、和IXAP)、TNF受体相关因子(TRAF1和TRAF2)、锌指蛋白A20、超氧化锰歧化酶、Bcl-2同系物Al/Bfl-1和IEX-IL等抗凋亡基因的表达，抑制细胞的凋亡[3]。在肿瘤治疗中，化疗和放疗可使NF-κB活化，致使肿瘤细胞对化疗耐药和对放射线不敏感。因此，抑制NF-κB的转录活性，降低肿瘤细胞的抗凋亡能力不失为提高抗癌药物治疗功效的良策[4]。
由于NF-κB强大的转录调控功能，NF-κB的过渡活化在许多疾病的发生和发展过程中扮演着重要的作用：如风湿性关节炎、哮喘、慢性肾小球性肾炎、幽门螺杆菌相关性胃炎、炎症性肠道疾病等慢性炎性疾病[5，6，7，8]，脓毒症、脓毒性休克、急性肾小球性肾炎等急性炎性疾病[9]，Alzheimer′s病[10]，动脉粥样硬化[11]，系统性红斑自狼疮等身免疫性疾病[12]和癌症的发生和发展均于NF-κB过度或持续活化密切相关。另外，NF-κB也参与了鼻病毒、流感病毒、EB病毒、巨细胞病毒、腺病毒、HTL病毒和HIV病毒等在感染细胞中的转录和复制，在这些病毒的感染和致病过程中起着重要的作用[13]。
鉴于NF-κB在上述疾病的发生和发展中的潜在作用，以NF-κB及其信号传导通路中的相关蛋白为靶点，研发NF-κB相关疾病的治疗药物已成为本领域中的研究热点。目前国外有关以NF-κB为靶点的拮抗治疗策略主要包括以下几个方面[14，15，16，17]：(1)应用传统抗炎药物——糖皮质激素等激素药物直接地或间接地部分拮抗NF-κB的活性；(2)抗氧化治疗：如抗氧化剂——乙酰半胱氨酸、阿司匹林水杨酸钠和吡咯烷二硫氨基甲酸(Pyrrolidinedithiocarbamate，PDTC)等作为NF-κB的抑制剂，其中PDTC作为NF-κB的抑制剂已受到众多研究者的青睐；(3)抑制IκK的形成及活性；如靶向IκKα的反义核酸，可阻止IκKα的形成，从而缓解或减轻IκB的磷酸化及降解过程，最终达到抑制NF-κB活性的目的；NF-κB必需调节蛋白(NF-κB essential modifier，NEMO)是IκK蛋白复合物的调节蛋白，对维持IκK的功能有重要作用。May等设计了针对NEMO的抗炎多肽，该多肽包含NEMO结合域(NEMO binding domain，NBD)，它可以与NEMO结合，阻止NEMO与IκKβ的连接，从而抑制IκK的活性及其后续效应；(4)减少IκB的降解：某些蛋白酶小体抑制剂如MG101、MG115、MG132、PS-341、lactacystin以及近来发现的epoxomicin均可通过抑制IκB的降解从而拮抗NF-κB的活性，某些丝氨酸蛋白酶抑制剂如TLCK(Nalpha-p-tosyl1-L-lysinechloromethyl ketone)、TPCK(N-tosyl-L-phenylalanine chloromethyl ketone)以及分泌性白细胞蛋白酶抑制剂也具有类似的功能；(5)促进IκB的生成：应用IκBα的腺病毒表达载体，使IκBα显性失活突变体过量表达，可明显抑制NF-κB的核移位及其后续效应。(6)抑制NF-κB的合成：如针对p50和p65的反义寡脱氧核苷酸可与编码NF-κB的基因及mRNA结合，进而阻断基因的转录、翻译过程，最终抑制NF-κB的形成。设计针对NF-κB家族高保守区的核酶，可对编码NF-κB的mRNA进行切割破坏，在mRNA水平阻断NF-κB的形成。
上述以NF-κB为靶点的治疗策略在动物实验及体外细胞培养系统中已表现出不同程度的疗效。但上述措施均存在特异性欠佳的问题。事实上，要获得最佳的治疗效果并尽可能地减少系统毒性，则应注意NF-κB靶性治疗的特异性。如糖皮质激素是NF-κB的强效抑制剂，但全身应用时具有扰乱内分泌和物质代谢的副作用[18]。而抗氧化剂对NF-κB活性的抑制效应更无特异性可言[19]，动物实验表明，PDTC具有很强的毒副作用。蛋白酶小体，它除了可降解IκB外，还具有其他许多重要的功能，因此抑制蛋白酶小体活性将导致潜在的严重副作用[5]。尽管有学者认为IκK、IκB对NF-κB的调节是特异性的，但迄今的研究尚无足够的证据表明IκK、IκB对其他的蛋白分子不具备调节作用，因此就不能确切的肯定靶向IκK、IκB的措施对NF-κB活性的干预就是特异性的。抑制NF-κB的合成策略对于NF-κB是特异性的，但在一些急性炎症反应性疾病中，NF-κB的活化(而不是合成)过程(细胞质中无活性的NF-κB→活化入核→与DNA结合→启动基因表达)更为重要，因此抑制NF-κB的合成策略并非最优选择。从NF-κB的整个活化途径不难看出，NF-κB与DNA顺式元件的结合是特异性的，也是NF-κB启动靶基因转录的必经之路。只有对NF-κB与DNA顺式元件的结合实施直接的干预措施，才能达到真正意义上的特异性。而对NF-κB活化的上游途径进行干预则难以达到这一目的。
由p50和p65两亚基形成的异二聚体是NF-κB的典型代表，是NF-κB所有形式中最重要的一种，几乎存在于体内所有细胞，其含量远高于其它二聚体。此异二聚体的氨基末端可构成1个环状结构域，由其介导与DNA碱基特异性结合。晶体衍射分析显示，p50/p65异源二聚体与免疫球蛋白κ链基因增强子κB序列特异性结合的方式为：p50亚单位的Arg-54、Arg-56、Tyr-57、Glu-60、His-64和Lys-241氨基酸残基与κB序列5′端的5′-G-5G-4G-3A-2C-1-3′碱基系列特异结合；p65亚单位的Arg-33、Arg-35、Arg-36、Glu-39、和Arg-187，与κB序列3′端的5′-T+1T+2C+3C+4-3′4个碱基对特异结合。另外，由NF-kB p50/p65异源二聚体的氨基端和二聚化结构域共同构成的环状结构域，还可非特异地识别DNA核糖磷酸骨架[20]。即由p50和p65N末端共同构成的这一环状结构域形成了NF-κB的DNA结合结构域，介导其与顺式作用元件的结合，值得注意的是，这种结合为特异性结合。NF-κB激活靶基因首先需要DNA结合结构域识别靶基因启动子区域内的顺式作用元件，并与之结合，后在p65反式激活域的辅助下，激活靶基因的表达。在NF-κB(p50/p65异源二聚体)与其顺式作用元件κB基序的结合过程中，p50亚基在其中起主要作用。因此，若我们获得能够拮抗p50亚基与其顺式作用元件结合的多肽，则势必能够达到拮抗NF-κB转录活性的效果，最终达到抑制NF-κB调控靶基因表达及治疗急慢性炎症相关疾病的目的[20]。
随着20世纪80年代后期崛起的一种研究蛋白质相互作用的有力工具——酵母双杂交技术的建立、成熟和发展，对NF-κB拮抗多肽的筛选工作最终成为可能。酵母双杂交系统具有很高的灵敏度，能检测微弱而短暂的蛋白质间相互作用，且该系统中蛋白质的相互作用发生于酵母细胞胞内和/或核内。与原核细胞相比，真核酵母细胞对蛋白质的翻译和加工更加接近哺乳细胞，以其为工程菌研究蛋白质的相互作用将更佳接近哺乳细胞的生理状态，即保持蛋白质天然的折叠状态，使蛋白质间相互作用更接近哺乳细胞内的真实水平。因此，应用该系统研究蛋白质间的相互作用已得到愈来愈广泛的应用，特别对胞内靶蛋白相互作用蛋白或多肽的筛选，此系统更为首选[21]。NF-κB是核转录因子，存在于细胞浆中，但于细胞核内发挥转录调控功能。因此，我们选用酵母双杂交系统，以NF-κB p50保守结构域为诱饵蛋白，从一个16肽的随机肽库中筛选NF-κB相互作用多肽，并鉴定这些多肽的功能。目前我们已筛选获得9种NF-κB p50亚基结合肽，这里所列的是其中一种具有拮抗NF-κB转录活性的p50结合肽。
发明内容
本发明的目的是提供能够与核因子-κB p50亚基专一结合，并能够抑制核因子-κB活性及其所调控基因表达的核因子-κB拮抗肽。
本发明的另一目的是提供此核因子-κB拮抗肽的用途。
本发明的核因子-κB p50亚基拮抗肽氨基酸序列如下：
Asn Thr Arg Val Lys Lys Ser Leu Gln Arg Val Arg Gly Gly Gly Val Gly Ile Pro Thr Pro His Lys ProThr Ser Ser Gln His Lys Gln Pro Ser Pro
本发明还包括，本发明的核因子-κB p50亚基拮抗肽在制备抗核因子-κB的药物中的应用。所述抗核因子-κB的药物是治疗免疫相关疾病的药物或抗肿瘤的药物。其中所述炎性相关疾病是脓毒症、脓毒性休克、风湿性关节炎、哮喘、急慢性肾小球性肾炎、系统性红斑自狼疮等，其中所述肿瘤是肝癌、胃癌、食道癌、乳腺肿瘤、膀胱癌、前列腺肿瘤等。
本发明还包括，含有本发明的核因子-κB p50亚基拮抗肽的药物组合物。组合物中可以含有药物可接受的载体。组合物可以以任何形式的药物制剂形式存在，优选的是注射剂，最优选的是冷冻干燥注射剂，该药物制剂形式药物组合物，可以按照制剂学常规技术制备，包括将药物活性成分，本发明的多肽与药物载体混合，按照制剂学常规技术制成所需要的剂型。
具有上述氨基酸序列结构的多肽可与核因子-κB p50亚基专一结合，并阻断核因子-κB与其顺式作用元件的结合，从而抑制核因子-κB的生物学活性，即核因子-κB调控各种靶基因表达的活性。此种结构多肽既可以以多肽形式单独存在，也可与其它蛋白和多肽融和，或被甲基化、乙酰化和氨基化等修饰，或进行个别氨基酸的突变或替代，或插入蛋白质表面的特定部位，如凸环区，或与其它物质连接。无论以什么形式存在，含此结构多肽的物质可能表现出与核因子-κB p50亚基结合，并抑制核因子-κB活性的作用。
本发明通过酵母双杂交技术筛选获得与核因子-κB p50亚基新型结合多肽，并证实了此条多肽具有抑制核因子-κB生物活性的功能，这对于设计和研制新型抗核因子-κB的药物具有重要意义。
研究结果表明，本发明筛选到的多肽不仅能够竞争性阻断核因子-κB与其顺式作用元件的结合，也能够抑制核因子-κB所调控的靶基因的表达，因而可以成为新的抗核因子-κB药物或药物前体，本发明的核因子-κB拮抗肽在制造抗核因子-κB的药物中具有广泛的应用价值及广阔的市场前景。
另外，本发明的核因子-κB p50亚基结合肽作为生物制剂也有着潜在的应用价值，如作为配体用于核因子-κB的亲和纯化、作为探针用于核因子-κB的检测等等。
根据核因子-κB p50亚基结合肽氨基酸序列，可以有多种获取本发明氨基酸序列的物质的方法，如固相合成方法，溶液中合成方法，基因重组方法，以及其它化学方法合成。以上制备方法可以采用多肽合成和制备的常规方法，如教科书中的方法，优选的制备方法列在本发明的具体实施例中。
含此氨基酸序列的物质可有各种应用方式，主要包括在体外和体内抑制核因子-κB的活性，以各种给药方式实现以抑制核因子-κB活性为目的的疾病治疗，以及核因子-κB的检测或纯化等。
含本发明氨基酸序列的物质在生物医学方面的应用还包括：
1、通过合成或基因重组的方式，将含此序列的肽段与其它蛋白或肽融合或进行化学修饰，用于以抑制核因子-κB活性为目的的各种炎性相关疾病、自身免疫疾病、肿瘤以及其它相关疾病的治疗。
2、将其与琼脂糖偶联后，用于核因子-κB的亲和分离。
3、将其与指示标签偶联后，用于核因子-κB的鉴定。
附图说明
图1A为GST融合肽大肠杆菌表达结果。M为蛋白Marker；GST为pET-42a(+)空载体表达产物；1为GST-结合肽融合蛋白表达产物。
图1B为GST融合肽GST亲和层析纯化的结果。M为蛋白Marker； 1、2、3、4、5、6分别为GST(pET-42a(+)表达产物)纯化前、GST过柱后产物、GST纯化产物、GST-结合肽纯化前、GST-结合肽过柱后产物、GST-结合肽纯化产物。
图2为GST-结合肽融合蛋白与核因子-κB p50亚基结合的生物传感器的检测结果
图3为ELISA检测GST-结合肽融合蛋白与核因子-κB p50亚基的特异性结合的结果。GST为pET-42a(+)空载体表达产物；GST-结合肽为GST-结合肽融合蛋白表达产物。
图4为ELISA方法检测GST-结合肽融合蛋白对核因子-κB p50亚基与核因子-κB顺式作用元件结合的竞争抑制实验的结果曲线
图5为Pull-down实验检测GST-结合肽融合蛋白与核因子-κBp50亚基相互作用结果图。M、1、2、3、4、5、6分别为低分子量蛋白Marker、GST(pET-42a(+)空载体)表达产物、GST表达产物过柱液、GST与p50结合后的洗脱液、GST-结合肽表达产物、GST-结合肽表达产物过柱液、GST-结合肽与p50结合后的洗脱液。
图6为荧光素酶报告基因实验检测GST-结合肽融合蛋白对核因子-κB反应性荧光素酶报告基因表达的抑制效应。A、B、C、D、E、F、G、H分别为U937、U937+LPS、U937+LPS+p4-kB-Luc、U937+LPS+p4-kB-Luc+0.5μg/mL GST-结合肽、U937+LPS+p4-kB-Luc+1μg/mL GST-结合肽、U937+LPS+p4-kB-Luc+2μg/mL GST-结合肽、U937+LPS+p4-kB-Luc+4μg/mL GST-结合肽、U937+LPS+p4-kB-Luc+8μg/mL GST-结合肽。
具体实施方式
实施例1、核因子-κB p50亚基结合肽与GST的融合表达与纯化
根据结合多肽的基因序列，在保持多肽蛋白基因编码序列不变的情况下，按照大肠杆菌偏爱密码子，分别设计并合成上游基因序列含BamHI，下游基因序列含SalI粘性末端的互补DNA片段，DNA序列如下，DNA序列的合成由上海生物有限工程公司合成。FP：
5’-GATCAATACGCGGGTGAAGAAGTCGTTACAGCGGGTGAGGGGGGGGGGGGGTAGGA
ATTCCTACCCCGCACAAACCTACGTCGTCACAACACAAGCAACCTTCTCCA-3’RP：
5’-TCGATGGAGAAGGTTGCTTGTGTTGTGACGACGTAGGTTTGTGCGGGGTAGGAATTC
CTACCCCCCCCCCCCTCACCCGCTGTAACGACTTCTTCACCCGCGTATT-3’
用无菌双蒸水将上述DNA序列溶解，终浓度为20μM，后取20μl FP和20μl RP于一无菌EP管中，充分混合，后将混合物放入94℃水浴变性，室温放置让水浴自然冷却，使多肽基因序列能够缓慢退火形成双链。再用T4连接酶将多肽基因序列插入经BamHI/SalI双酶切的pET 42a载体内，并转化CaCl2法制备的E.coli DH-5α感受态细胞。挑取数个单菌落进行酶切鉴定，并测序。将鉴定正确的重组质粒pET42a/结合肽转化CaCl2法制备的E.col.BL-21感受态细胞，挑取新鲜转化的单菌落，接种于10ml含50μg/ml硫酸卡那霉素的LB培养基中，37℃振荡培养过夜。按1∶100比例将过夜菌菌液转接于200ml含50μg/ml硫酸卡那霉素的LB培养基中，37℃振荡培养至OD600≈0.5～0.6，加IPTG至终浓度为0.1-0.5mmol/L，移至30℃继续培养4h。离心收集菌体，菌体可进行纯化或冻存于-70℃。将诱导表达的菌体按原菌液1/10的体积重悬于PBS中，后加入50mmol/L PMSF 15μl后超声破碎细胞(6×10sec×40hz)，再加入终浓度为1％的Triton X-100，轻轻振荡20min，于4℃，12000r/m离心15min取上清，加DTT至终浓度为1mmol/L。同时用10倍柱体积的PBS液平衡谷胱甘肽琼脂糖4B亲和树脂，后取适量的上述上清液上柱，使GST-结合肽融合蛋白与树脂结合，再用10倍柱体积的PBS液洗去非特异结合的杂蛋白。最后用10mmol/L还原型谷胱甘肽洗脱目的蛋白，并通SDS-PAGE鉴定其纯度，纯化蛋白经蒸馏水充分透析去除还原型谷胱甘肽后冻干于-20℃保存。SDS-PAGE电泳结果表明，GST-结合肽融和蛋白为可溶性表达，表达量约为总蛋白量的24％(图1A)。经GST亲和纯化后融和蛋白纯度达到82％以上(图1B)
实施例2、生物传感器(Biosensor)检测GST-结合肽融合蛋白与核因子-κB p50亚基的结合
将羧甲基葡聚糖生物传感片装入Iasys Plus系统微射流卡盘，用60μl PBS/T(pH 7.4PBS，0.05％吐温20)冲洗3次，平衡10min，待基线走平后采集基线数据5min；将EDC和NHS按1∶1比例混合，取40μl混合液冲洗样品池2次，后再在样品池中加入40μl混合液，保留7min以激活传感片表面；用50μl PBS/T冲洗样品池3次采集基线值1min；用40μl 10mM pH5.5的乙酸缓冲液冲洗样品池3次，后再加入40μl乙酸缓冲液，采集基线值1min；然后在样品池中加入40μl用乙酸缓冲液稀释的总量约为0.5μg的p50标准蛋白；反应20min后用PBS/T冲洗3次，并采集基线值1min；用40μl 1M pH 8.0的Tris-Hcl缓冲液冲洗样品池3次，并保留3min以封闭未反应的NHS；用40μl 10mM HCl冲洗3次，除去传感片上残留的没用发生共价结合的p50标准蛋白；用50μl PBS/T冲洗3次，采集基线值1min，4℃放置待后进行结合反应。
用蛋白结合缓冲液(50mM Tris-HCl，pH7.2，100mM NaCl，10mM MgCl2，10uM ZncL2，1mM DTT，0.1％(v/v)NP40)将GST-结合肽融合蛋白稀释成1μg/mL，取50μl融合蛋白稀释液加入样品池，温度25℃，结合反应5～10min；待反应完全，用50μl蛋白结合缓冲液冲洗样品池3次，以洗脱非特异性结合，待反应曲线走平后采集反应曲线值1min；用40μL10mM HCl冲洗样品池3次，再生传感片；再用50μl蛋白结合缓冲液冲洗样品池3次，采集基线数据1min，开始新一轮的结合反应。
Biosensor结果表明，本发明的p50拮抗肽能高亲和与p50结合，见图2。
实施例3、ELISA方法检测GST-结合肽融合蛋白与核因子-κB p50亚基的特异性结合
用去离子水洗ELISA平板包被孔数次，倒置平版，并在一清洁滤纸上拍打平板，去除剩余的水份；用PBS缓冲液稀释p50蛋白，浓度为10μg/ml；加100μl稀释的p50蛋白至96孔酶联板的包被孔中，室温孵育3～4h或4℃过夜孵育，注意水份蒸发；用300μl PBS洗孔3次，去除未结合的p50蛋白；制备5％的脱脂奶粉水溶液，每孔加入200μl，室温孵育1h或4℃过夜孵育封闭包被孔；用蛋白结合缓冲液(50mM Tris-HCl，pH7.2，100mM NaCl，10mM MgCl2，10uM ZncL2，1mM DTT，0.1％(v/v)NP40)洗板5次；样品检测孔加入0.5μmol/L的GST-结合肽(蛋白结合缓冲液稀释)100μl/孔，共20孔。阴性对照孔加入0.5μmol/L的GST纯化蛋白(蛋白结合缓冲液稀释)100μl/孔，共20孔。于样品检测孔和阴性对照孔中分别加入终浓度分别为0μg/ml、20μg/ml、100μg/ml、400μg/ml的p50蛋白，各浓度行5个复孔，以此通过未包被的p50蛋白与包被的p50蛋白竞争性地p50结合肽结合来验证GST-结合肽与p50蛋白的特异性结合，室温结合1h；PBST(PBS+0.1％TWEEN-20)洗板3次后加入抗GST的单克隆抗体(1∶1000稀释于PBS中)100μl/孔，室温孵育1h；PBST洗板3次后加入HRP标记的羊抗鼠IgG(按产品说明要求稀释于PBS中)100μl/孔，室温孵育1h；PBST洗板3次后加入显色液100μl/孔避光保存显色5～10min，加入1mol/LH2SO4 50μl/终止反应，立即测定OD450。
结果表明，本发明的GST-结合肽融合蛋白可特异性地与p50结合，随着p50加入量的增加，多肽与包被蛋白的结合减少，见图3。
实施例4、ELISA方法检测GST-结合肽融合蛋白对核因子-κB p50亚基与核因子-κB顺式作用元件结合的竞争抑制实验
于包被了核因子-κB顺式作用元件的96孔酶联板(购自美国Clontech公司的MercuryTMTransfactor p50 kits)中加入150μl/孔转录因子封闭液，室温孵育15min；弃转录因子封闭液后加入50μl/孔用转录因子封闭液稀释的检测样品，样品分别为含10ng/μl的p50标准蛋白作为阳性对照、含10ng/μl的p50标准蛋白同时加入系列浓度GST融合肽以及含10ng/μl的p50标准蛋白同时加入相同系列浓度GST纯化蛋白的样品，室温孵育1h，空白对照为加入50μl转录因子封闭液；加入转录因子封闭液150μl/孔洗涤3次，每次4min，去除洗涤液；加入用转录因子封闭液以1∶500稀释的p50抗体100μl/孔，室温孵育1h；加入转录因子封闭液150μl/孔洗涤3次，每次4min，去除洗涤液；加入用转录因子封闭液以1∶1000稀释的羊抗兔IgG-HRP二抗100μl/孔，室温孵育30min；加入转录因子缓冲液250μl/孔洗涤4次，每次4min，去除洗涤液；加入TMB底物，室温孵育10min，待液体变蓝后，用100μl/孔的终止液终止反应；于450nm和630nm双波长检测OD值。p50结合肽对p50与其顺式作用元件结合的抑制百分数计算公式为：(同一浓度GST纯化蛋白OD-同一浓度GST融合肽OD)/p50标准蛋白阳性对照OD。
结果表明，本发明的GST-结合肽融合蛋白可特异性抑制p50与其顺式作用元件的结合，而这种抑制效应具有量效关系，既抑制作用随着肽浓度的升高而增加，而GST则不影响p50与其顺式作用元件的结合，如图4所示。
实施例5、Pull-down实验检测GST-结合肽融合蛋白与核因子-κB p50亚基相互作用
按PIERCE公司的GST pull-down assay试剂盒操作说明书进行。先将1ml谷胱甘肽琼脂糖4B亲和层析树脂装柱在一次性注射器的针管中，用预冷的PBS自然过滤洗涤4～6次(5mL/次)并平衡树脂；然后加入经IPTG诱导表达GST-结合肽融合蛋白的大肠杆菌超声破碎的上清液5ml，将GST-结合肽融合蛋白固化在谷胱甘肽琼脂糖4B亲和层析树脂上，用预冷的PBS自然过滤洗涤10次(5mL/次)。洗去未结合的杂蛋白；加入2mL蛋白结合缓冲液(142.5mmol/LKCl，5mmol/L MgCl2，0.2％(v/v)诺乃洗涤剂-40，10mmol/L N-2-羟乙基哌嗪-N’-2-乙磺酸，pH：7.2，1mmol/L EDTA)冰上预孵30min后，最后加入经原核重组表达获得的粗制p50蛋白5ml，自然过滤并捕获目的蛋白，再用预冷的PBS过滤洗涤10次，洗去未结合的蛋白，最后加入1mL洗脱液(10mmol/L还原型谷胱甘肽，50mmol/L Tris-HCl，pH：8.0)，室温孵育30min后，过滤。收集最后的过滤产物行SDS-PAGE分析。同时用pET-42a(+)空载体表达获得的GST蛋白做阴性对照。
实施例6、核因子-κB p50亚基拮抗肽对核因子-κB反应性报告基因表达的抑制实验。
1640培养基培养U937细胞，转染前一天将细胞密度调整为0.5～2.5×105个/mL，过夜培养；取1mL过夜培养细胞或以0.5×105个细胞/孔细胞量加入24孔培养板；取3μL GeneJuice转染试剂加入100μL无血清1640培养基中，旋涡混匀，室温放置5min；取1μg p4kB-Luc质粒加入GeneJuice/1640培养基混合物中，轻轻吹打混匀，室温放置5-15min；将上述混合物逐滴加入完全培养基中，轻轻震荡，使混合物和细胞充分混匀；37℃，5％CO2培养箱培养；转染5h后，用完全培养基进行换液培养1h后用PBS洗细胞3次，最后将细胞重悬于100μL的无血清1640培养基中；用PBS将GST-结合肽稀释成6个不同浓度(加入培养基后的终浓度分别为0、0.5、1、2、4、8μg/mL)，体积为50μL的融合蛋白液，同时用PBS将2μLChariot转染试剂稀释成50μL，将不同浓度的GST-结合肽与Chariot转染试剂混合，轻轻吹打混匀，室温放置30min。将100μL GST-结合肽/Chariot转染试剂混合液逐滴加入上述100μL无血清1640培养基重悬的U937细胞培养液中，轻轻震荡，使混合物和细胞充分混匀；37℃，5％CO2培养箱培养1h；除去转染液，将培养基换成完全培养基，同时加入终浓度为1μg/mL的LPS进行刺激，培养2h后吸取细胞500g离心5min，PBS洗涤细胞一次，除尽PBS上清；以100μl/孔加入1×CCLR细胞裂解缓冲液；涡旋EP管10-15s，4℃，12000rpm离心2min，取上清，-70℃保存或直接检测荧光素酶的活性；于96孔检测板中加入100μL/孔荧光素酶检测试剂后，再加入100μL/孔细胞裂解液后立刻读数，并打印或记录数据。
结果表明，本发明的GST-结合肽融合蛋白对核因子-κB反应性荧光素酶报告基因的表达具有抑制作用，且这种抑制作用具有量效关系，既抑制作用随着蛋白浓度的升高而增加，而对照GST蛋白则不具有这种抑制作用，如图5所示。由此说明，我们筛选获得的p50结合肽具有抑制核因子-κB转录活性的功能，既具有抑制核因子-κB调控炎症介质等基因表达的功能。
参考文献
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序列表
<110>中国人民解放军第三军医大学第三附属医院
<120>核因子-κB p50亚基拮抗肽及其制备与应用
<160>1
<210>1
<211>34
<212>PRT
<213>人工序列
<220>
<400>1
Asn Thr Arg Val Lys Lys Ser Leu Gln Arg Val Arg Gly Gly Gly
1 5 10 15
Val Gly Ile Pro Thr Pro His Lys Pro Thr Ser Ser Gln His Lys
20 25 30
Gln Pro Ser Pro
34
Claims (10)
1.一种特异性地与核因子-κB p50亚基结合，并特异性地拮抗核因子-κB活性的多肽，氨基酸序列如下：
Asn Thr Arg Val Lys Lys Ser Leu Gln Arg Val Arg Gly Gly Gly Val Gly
Ile Pro Thr Pro His Lys Pro Thr Ser Ser Gln His Lys Gln Pro Ser Pro。
2.权利要求1的多肽在制备抗核因子-κB的药物中的应用。
3.权利要求2的应用，所述抗核因子-κB的药物是治疗炎性免疫相关疾病的药物或抗肿瘤的药物。
4.权利要求3的应用，其中所述炎性相关疾病是脓毒症、脓毒性休克、风湿性关节炎、哮喘、急慢性肾小球性肾炎、系统性红斑自狼疮，其中所述肿瘤是肝癌、胃癌、食道癌、乳腺肿瘤、膀胱癌、前列腺肿瘤。
5.含有权利要求1的多肽的药物组合物。
6.权利要求5的药物组合物，其中含有药物可接受的载体。
7.权利要求5的药物组合物，以任何形式的药物制剂形式存在。
8.权利要求5的药物组合物，是注射剂。
9.权利要求5的药物组合物，是冷冻干燥注射剂。
10.权利要求1的多肽的制备方法，其特征在于，所述方法选自常规的固相合成方法、溶液中合成方法，基因重组方法或化学合成方法。
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