CN1898261B - 新的磷酸结合蛋白、含有它的药物组合物及其用途 - Google Patents
新的磷酸结合蛋白、含有它的药物组合物及其用途 Download PDFInfo
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Abstract
本发明涉及一种蛋白质,特征在于,其包含或其组成为:(i)序列SEQ ID NO:1;(ii)或衍生自序列SEQ ID NO:1的任何序列,尤其是通过置换、缺失或添加1或多个氨基酸而衍生得到的序列,条件是所述衍生序列结合磷酸;(iii)或与序列SEQ ID NO:1同源的任何序列,优选地与序列SEQ ID NO:1有至少大约80%同源性,条件是所述同源序列可结合磷酸,或(iv)以上定义的序列之一的任何片段,条件是所述片段可结合磷酸,尤其是由序列SEQ ID NO:1中的至少大约20个连续氨基酸组成的任何片段。
Description
技术领域
本发明的主题是从人类血清中获得的新的磷酸结合蛋白(phosphate-binding protein)、含有它的药物组合物以及其用途,尤其是在高磷酸盐血症(hyperphosphataemia)和心血管疾病或关节炎的治疗框架中的用途。
背景技术
磷酸是一种参与众多生物学机制的极其重要的分子。磷酸尤其存在于磷脂、能量产生机制(ATP,ADP)、细胞信号传导过程、遗传物质的组成及骨(以磷酸钙的形式)中。
高磷酸盐血症是一种与生物体中磷酸过量相关的病症,其尤其通过促进动脉粥样硬化和动脉钙化的过程,造成心血管疾病危险增加(Dorozhkin和Epple,2002;Amann等,2003;Blazheevich等,1975)。当关节中发生钙化时,高磷酸盐血症也可以引起关节炎(假性痛风)。
高磷酸盐血症期间血清中产生的磷酸钙盐沉积在软组织中,并且在不同组织:血管(脑或心血管意外)、关节(假性痛风)、晶状体、肾间质组织(肾钙质沉着症)、皮下(瘙痒病(pruritis))、肺和胰腺中异位钙化。
因此,在患有肾功能不全的个体中有半数的死亡由与高磷酸盐血症有关的心血管疾病引起。在此方面,某些在肠腔中络合磷酸的磷酸螯合剂目前被用作药物。然而,并非所有这些螯合剂均是生理性的。这导致与其应用相关的某些并发症或限制。
含有镁的制剂由于出现消化道紊乱(腹泻)而受到限制,并且由于高镁血症的危险而被禁止。类似地,由于铝中毒危险(小红细胞性低血色素贫血、软骨病、肌病、痴呆),必需避免因其有效性而长期使用的氢氧化铝处方,或者至少限制到非常短的时期。
钙盐处方是纠正低血钙和高磷酸盐血症(hyperphos phoraemia)的最佳手段,这一方面可使小肠吸收的钙量增加而不论是否钙三醇缺乏,且另一方面可使以磷酸钙的形式在肠腔中络合磷,而磷酸钙在粪便中排除。然而,含钙螯合剂的主要缺点是诱导高血钙,在某些系列中已经在20%患者中注意到此现象。此危险已经导致了能够限制高磷酸盐血症的其它产物的开发。
本发明的目的是提供结合磷酸的新生理性蛋白质螯合剂,从而无需使用其它能导致并发症的离子并提供比目前螯合剂更宽的应用前景。
发明内容
本发明涉及蛋白质,所述蛋白质的特征在于其包含或其组成是:
-序列SEQ ID NO:1,
-或衍生自序列SEQ ID NO:1的任何序列,尤其是通过置换、缺失或添加1或多个氨基酸得到的序列,条件是所述衍生的序列可结合磷酸,
-或与序列SEQ ID NO:1同源的任何序列,优选地与序列SEQ IDNO:1有至少大约80%同源性,条件是所述同源序列可结合磷酸,
-或以上定义的序列之一的任何片段,条件是所述片段可结合磷酸,尤其是由序列SEQ ID NO:1中的至少大约20个连续氨基酸组成的任何片段。
本发明涉及以上定义的蛋白质,其特征在于该蛋白质包含或其组成是:
-序列SEQ ID NO:2或序列SEQ ID NO:3,
-或者衍生自序列SEQ ID NO:2或SEQ ID NO:3的任何序列,尤其是通过置换、缺失或添加1或多个氨基酸得到的序列,条件是所述衍生序列可结合磷酸,
-或与序列SEQ ID NO:2或SEQ ID NO:3同源的任何序列,优选地与序列SEQ ID NO:2或SEQ ID NO:3有至少大约80%同源性,条件是所述同源序列可结合磷酸,
-或以上定义的序列之一的任何片段,条件是所述片段可结合磷酸,尤其是由序列SEQ ID NO:2或SEQ ID NO:3中的至少大约20个连续氨基酸组成的任何片段。
序列SEQ ID NO:2对应于人磷酸结合蛋白质。该新蛋白已经从人类血浆中分离,其三维结构显示其属于“磷酸结合蛋白”(PBP)类。以下也将其称作HPBP(人磷酸结合蛋白)。
序列SEQ ID NO:3对应于与序列SEQ ID NO:2同源的蛋白质,该蛋白质与序列SEQ ID NO:2有大约90%的百分同一性并与SEQ ID NO:2具有相同的磷酸结合性质。
本发明序列的磷酸结合性质可以通过如下磷酸结合试验通过放射性标记来验证:
使蛋白质结合在硝化纤维素膜上(通过抽吸进行点印迹)。膜在放射性缓冲液(32P(10mCi/ml,Amersham-Biosciences)2M;Tris 50mM;pH 8.0)中孵育。
在Tris 50mM缓冲液,pH 8.0中快速漂洗该膜2×1分钟。通过用膜曝光胶片(大约45min),可以检测结合放射性磷酸的区(见之后的图3)。
本发明还涉及编码以上定义的蛋白质的核苷酸序列。
本发明还涉及含有上述核苷酸序列的重组载体,尤其是质粒、粘粒、噬菌体或病毒DNA。
根据一个有利的实施方案,本发明涉及以上定义的重组载体,该载体含有使插入所述载体的上述核苷酸序列所编码的多肽在宿主细胞中表达所必需的元件。
本发明还涉及宿主细胞,该宿主细胞尤其选自细菌、病毒、酵母、真菌、植物或哺乳动物细胞,且所述宿主细胞被转化,尤其是使用以上定义的重组载体转化。
本发明还涉及含有以上定义的蛋白质,尤其是SEQ ID NO:2或SEQ ID NO:3作为活性成分并组合含有可药用载体的药物组合物。
本发明还涉及以上定义的药物组合物,其中本发明的蛋白质,尤其是SEQ ID NO:2或SEQ ID NO:3,与具有对氧磷水解活性的对氧磷酶蛋白质变体组合。
在对氧磷酶变体中,可以提及人或非人来源的变体PON1、PON2、PON3,例如SEQ ID NO:4(人PON1;Hassett等,1991)、SEQ ID NO:5(人PON2;Primo-Parmo等,1996)、SEQ ID NO:6(人PON3;Reddy等,2001)、SEQ ID NO:7(兔PON1;Hassett等,1991)、SEQ ID NO:8(大鼠PON1;Rodrigo等,1997)、SEQ ID NO:9(小鼠PON1;Sorenson等,1995)、SEQ ID NO:10(小鼠PON2;Primo-Parmo等,1996)和SEQ ID NO:11(小鼠PON3;Primo-Parmo等,1996)。
本发明还涉及以上定义的蛋白质,尤其是SEQ ID NO:2或SEQ IDNO:3,在制备旨在用于预防或治疗与高磷酸盐血症相关的疾病,例如心血管疾病和关节炎(假性痛风)的药物中的用途。
术语“高磷酸盐血症”指生物体中磷酸(phosphate)过量。更精确地,高磷酸盐血症定义为血浆中磷酸浓度增加到高于1.44mmol/l(45mg/1),所述量通过总磷酸测定(在矿化步骤后进行比色法测定)而获得。
根据一个有利的实施方案,本发明蛋白质可以静脉内施用以便能够长期(大约1周)结合最大量的磷酸。通过随后清除该蛋白质,可以由此快速地消除大量磷酸。这使得可以加大透析期之间的间隔和缩短透析期。
本发明更尤其涉及以上定义的蛋白质,尤其是SEQ ID NO:2或SEQ ID NO:3,在心血管疾病预防或治疗框架中的应用。
本发明还涉及本发明蛋白质,尤其是SEQ ID NO:2或SEQ ID NO:3所表示的蛋白质,与诸如对氧磷酶蛋白质变体等蛋白质的组合在杀虫剂或神经毒剂(nerve agents)(例如,梭曼(soman)、VX、塔崩(tabun)或沙林(sarin))所引起的中毒的预防和治疗框架中或在动脉粥样硬化的治疗框架中的应用。
本发明还涉及包含至少一种以上定义的蛋白质(尤其是SEQ IDNO:2或SEQ ID NO:3)和至少一种对氧磷酶蛋白质变体的组合产品,其中所述以上定义的蛋白质和对氧磷酶蛋白质变体同时或分开使用或者分散在一段时间内使用,以旨在预防或治疗由杀虫剂或神经毒剂如梭曼、VX、塔崩或沙林引起的中毒。
本发明蛋白质(尤其是SEQ ID NO:2)与对氧磷酶蛋白质变体的组合施用使得可以增加对氧磷酶的稳定性,尤其是在预防或治疗由杀虫剂或神经毒剂所引起的中毒的框架中。
本发明还涉及以上定义的蛋白质测定方法,特征在于其包括如下阶段:
-将抗本发明蛋白质的不同表位的兔单克隆抗体(抗-HPB)固定在平板上,向所述平板施加含有所述蛋白质(HPB)的待分析人类血清,
-漂洗和洗涤平板,
-向所述平板施加过氧化物酶标记的针对兔抗体的抗体(抗-IGrabbit-per)30分钟,以在兔单克隆抗体、本发明蛋白质和上述针对兔抗体的抗体之间形成三元复合体(抗-HPB-HPB-抗-IGrabbit-per),
-漂洗和洗涤平板,
-使固定于平板上的过氧化物酶与其底物(商业试剂盒,化学发光过氧化物酶底物(Sigma))反应,在与3,3’,5,5’-四甲基联苯胺(TMB,Sigma)反应30分钟结束时终止反应,
-使用分光光度计测量前述步骤中形成的产物在450nm的光密度,将此测量结果和标准曲线比较,从而可以确定血清中本发明蛋白质(HPB)的浓度。
因此,上述测定方法使用ELISA型免疫测定方法(Engvall等,1971)。
可以使用其它方法测定血浆中本发明蛋白质的浓度,例如:
-电泳方法,或者
-该蛋白质活性的定量。
本发明还涉及上述定义的测定方法的如下用途,
-用于体外诊断与高磷酸盐血症相关的疾病,尤其是当根据以上定义的方法测定的上述蛋白质(尤其是SEQ ID NO:2或SEQ ID NO:3)的量少于健康个体血液中正常存在的该蛋白质的量时,或者
-用于体外诊断与低磷酸盐血症相关的疾病,尤其是当根据以上定义的方法测定的上述蛋白质(尤其是SEQ ID NO:2或SEQ ID NO:3)的量多于健康个体血液中正常存在的该蛋白质的量时,或者
-用于体外诊断个体对此类病状的易感性。
本发明蛋白质的水平是心血管疾病危险易感性的指标。因此,具有低水平的所述蛋白质的个体将具有更高水平的游离磷酸,该磷酸将与血浆中钙一起沉淀以形成磷酸钙块,磷酸钙块是加剧尤其是心血管疾病或关节炎风险的因子。
该蛋白质的异常水平也是已存在的病状的征兆。例如,高磷酸盐血症能够触发蛋白质产量的增加以限制磷酸水平。低水平也能够揭示功能失调。
本发明还涉及上述的用途,用于在体外诊断与高磷酸盐血症相关的疾病(例如心血管疾病,尤其是与动脉粥样化斑块(atheromaplaques)的形成相关的心血管疾病),或者用于体外诊断个体发生上述疾病之一的易感性。
本发明还涉及上述用途,用于体外诊断与低磷酸盐血症相关的疾病、或者用于体外诊断个体发生这些疾病的易感性。
在表征与低磷酸盐血症相关的疾病的临床或生理学征兆中,可以提及的是:
-骨的脱矿质化,
-低磷酸盐血症的肌肉表现,其包括影响骨骼肌的近端肌病和影响平滑肌的吞咽困难和肠梗阻,
-由于ATP缺乏而致的心肺功能缺陷(cardio pulmonarydeficiencies),和
-代谢性脑病变。
附图说明
图1表示纯化人对氧磷酶和本发明蛋白质SEQ ID NO:2中得到的最终级分的SDS-PAGE凝胶。
A道对应于分子量标记,B、C和D道对应于来源于不同袋的人类血浆的三个不同纯化产物。所有三者均包含人对氧磷酶和磷酸结合蛋白。
图2图示与磷酸分子结合的本发明蛋白质SEQ ID NO:2的示意性结构。
图3对应于本发明蛋白质SEQ ID NO:2的磷酸结合试验。
A至F道对应于来源于不同袋的人类血浆的本发明蛋白质的不同批次纯化产物;G道对应于1mg/ml的溶菌酶,H道对应于β-乳球蛋白。
图4是本发明蛋白质SEQ ID NO:2和对氧磷酶的混合物的双向电泳凝胶。
图5是本发明的SEQ ID NO:2的结晶蛋白质分子坐标。
实验部分
蛋白质的分离
蛋白质SEQ ID NO:2根据Gan等的如下方法(1991),从人血浆获得:
从Lyon-Beynost的Establissement de Transfusion Sanguine提供的冰冻血浆(大约200ml)袋中纯化蛋白质SEQ ID NO:2。向血浆中加入1M(1%v/v)CaCl2,形成血纤蛋白凝块,通过过滤从血清中将之分离。然后血清与利用缓冲液A(Tris/HCl 50mM,CaCl2 1mM,NaCl4M,pH 8)平衡的400ml亲和凝胶(Cibacron 3GA-琼脂糖,C-1535,Sigma)混和。在这些条件下,主要吸附HDL(“高密度脂蛋白”)。孵育6至8小时后,通过在2号烧结的多孔圆盘上过滤除去凝胶上未吸附的蛋白质。持续进行该洗涤,直到洗脱液中不再检测到蛋白质(280nmUV吸收)。然后用缓冲液B(Tris/HCl 50mM,CaCl2 1mM,pH 8)平衡凝胶,再将凝胶放入XK 50/30柱(Pharmacia)中。通过向缓冲液B中加入1g/l脱氧胆酸钠和0.1%Triton X-100,进行洗脱。将显示芳基酯酶活性的级分注射在50ml阴离子交换凝胶(DEAE Sepharose FastFlow,Pharmacia)上,其中所述凝胶放置在XK 26/70柱(Pharmacia)中并已经用缓冲液B和0.05%Triton X-100平衡。利用NaCl进行梯度洗脱。在87.5mM达到第一平台从而除去apo A-I(与对氧磷酶结合的蛋白质)、和大多数污染性蛋白质。人类对氧磷酶(PON1)大约在140mM NaCl浓度下洗脱。所有保留级分均显示对氧磷酶和芳基酯酶活性,这些活性根据以下试验验证。不合并这些洗脱的级分。所获级分的SDS-PAGE凝胶显示包含在38kDa至45kDa之间的条带(见图1)。每次纯化并不总是得到相同的表观质量分布。这种轻微的异质性可以由PON1上存在2个糖基化链来解释。
在这些批次中,除了PON1,还通过结晶、通过用C12-麦芽糖苷替代Triton以及使用硫酸铵作为沉淀剂分离出另一蛋白质。所获未知蛋白质的晶体通过放射晶体学进行表征,其对应于本发明序列SEQ ID NO:2。结晶是目前纯化该蛋白质的唯一现有方法。
在1M NaCl存在下,在甘氨酸50mM/NaOH,CaCl2 1mM缓冲液(pH 10.5)中测量对氧磷酶活性,并通过恒温调节在25℃的双光束分光光度计(Shimadzu UV 160A)确定该活性。根据412nm的吸光度变化,确定水解速度,其对应于作为时间的函数通过水解对氧磷释放的对硝基苯酚的形成,ε=18290M-1cm-1(Smolen,1991)。
在Tris 50mM/HCl,CaCl2 1mM缓冲液,pH 8中测量芳基酯酶活性,并通过恒温调节在25℃的双光束分光光度计(Shimadzu UV 160A)确定该活性。根据270nm的吸光度变化,确定水解速度,其对应于作为时间的函数通过水解乙酸苯基酯释放的苯酚的形成,ε=1310M-1cm-1(Smolen,1991)。
结构
在ESRF的BM30线(Grenoble)上收集X射线衍射数据
将晶体浸在含有铀盐的溶液中,获得重原子盐衍生物。
用XDS2000程序(Kabsch,1993)和CCP4软件包(COLLABORATIVECOMPUTATIONAL PROJECT,NUMBER 4,1994,“CCP4软件包:蛋白质晶体学程序”,Acta Cryst。D50,760-763)对图像进行求积分、分级和组合。
使用CNS(BRUNGER,1998)和SnB(Weeks,1999)程序,以定位铀原子。使用SHARP程序(Copyright2001-2002,The BusterDevelopment Group)以便通过SIRAS技术获得相位。
通过ARP/wARP(Perrakis,1997)程序,自动在电子密度图中构建了372个氨基酸。然后通过CNS程序修正此第一个模型。
由于电子密度图有非常良好的质量,故可以赋予该蛋白质的一级结构80%的可靠性。还可以定位磷酸分子。
所获结构根本不对应于人类对氧磷酶。通过从电子密度鉴定氨基酸所获得的序列说明,此人类蛋白质以及其基因在先前从未有过描述。因此,其是一种新蛋白质。
本发明蛋白质的结构显示出与大肠杆菌(Escherichia coii)磷酸结合蛋白质极大的同源性。此细菌中的该蛋白质的作用是跨周质转运磷酸。该细菌蛋白质存在于许多原核生物中但是未在真核生物中被发现。
电子密度还显示出磷酸分子与本发明新蛋白质结合,且结合方式与在大肠杆菌中的相同。
因此,可以得出如下结论:来源于人类血浆的经表征的本发明蛋白质与该细菌蛋白质具有极大的同源性并且其能够结合并转运磷酸。
测序
在凝胶中消化
使用SDS-PAGE(未加热)通过电泳凝胶分离对氧磷酶-HPBP混合物。切出70kDa区域中对应于HPBP的几个条带。
利用自动消化系统MassPrep Station(Waters Manchester,UK),消化这些条带中包含的蛋白质。用50μl的25mM碳酸氢铵(NH4HCO3)和50μl乙腈洗涤凝胶条带2次。用50μl 10mM二硫苏糖醇溶液在57℃还原半胱氨酸,并用50μl 55mM碘代乙酰胺酰化。用乙腈脱水后,用10μl 12.5ng/μl改良的猪胰蛋白酶(Promega,Madisson,WI,U.S.A)或者用Lysobacter enzymogenes的Lys-C(Roche AppliedScience,Penzberg,Germany)在25mM NH4HCO3中酶促消化该蛋白质。消化在室温下过夜进行。用60%乙腈溶液和5%甲酸抽提切割的肽。
质谱分析
MALDI-MS和MALDI-MS/MS
在UltraflexTM TOF/TOF(Bruker,Daltonik GmbH,Bremen,德国)上进行MALDI-TOF质量测量。该仪器以25KV的最高加速电压以反射模式(reflectron mode)使用。使用α-氰基-4-羟基肉桂酸作为基质,制备在不锈钢靶上干燥的标准滴来准备样品。
仅使用肽(缓激肽1-7(m/z=757.400)、人血管紧张素肽II(m/z=1046.542)、人血管紧张素肽I(m/z=1296.685)、P物质(m/z=1347.735)、铃蟾肽(m/z=1619.822)、肾素(m/z=1758.933)、ACTH1-17(m/z=2093.087)和ACTH 18-39(m/z=2465.199))的已知溶液的单一同位素电荷峰,外部校准MALDI-MS谱。使用Flexanalysis 2.0程序自动注释这些单一同位素肽的质量。
利用分段的离子前体的“激光诱导分解”(LID),不经在气相中的额外碰撞,获得亚稳态离子,通过分析该亚稳态离子获得了MS/MS谱。离子前体加速至8kV并利用时控离子门选择。这些碎片进一步在LIFT室中加速达到19kV,在通过离子反射器后测量其质量。
利用Full DeNovo Sequencing程序(Biotools,Bruker DaltonikGmbH,Bremen,德国),对这些MS/MS谱之每一个进行从头测序。
Nano LC-MS/MS
使用CapLC(Waters,Manchester,UK)进行Nano LC-MS/MS分析,其中所述CapLC与Q-TOF II正交四极杆(orthogonal hybridquadrupole)所加速的飞行时间质谱仪(Micromass,Manchester,UK)偶联。使用毛细管(Pepmap C18,75μm i.d.,15cm长,LC Packings)在200nL/min流速(通过分配前置柱保持恒定)下完成反相色谱分离。使用2pmol/μl GFP校准。
通过自动在MS模式和MS/MS模式间进行转换的MassLynx程序(Micromass,Manchester,UK),控制质量数据获取。
产生的MS/MS谱逐个地从头测序,以便获得部分或完整序列。使用能够完全地处理.pkl文件以对每一个MS/MS谱从头自动测序的PepSeq程序(MassLynx,Micromass)和PEAKS Studio程序(Bioinformatics Solutions,Waterloo,Canada),给出这些解释。
磷酸结合
根据如下试验证实本发明蛋白质SEQ ID NO:2对磷酸的结合:
将200μl本发明蛋白质SEQ ID NO:2(图3的道A-F)或1mg/ml溶菌酶(G道)或β-乳球蛋白加至硝化纤维素上(通过抽吸方式进行点印迹)
混和物在包含Tris 50mM;pH 8.0;32P(10mCi/ml)2mM的混合中孵育2小时30分钟。
然后用Tris 50mM pH 8.0漂洗2次1分钟,然后室温下暴露混合物45分钟。
然后可见(见图3),本发明蛋白质已经结合放射性磷酸(A至F道),而试验对照不与放射性磷酸结合(G和H道)。
蛋白质SEQ ID NO:2的角色和用途
为了分析该蛋白在血浆中的浓度,可以使用的方法是:
-电泳方法,
-蛋白质的纯化,
-该蛋白质的活性定量,
-使用抗该蛋白质的多克隆/单克隆抗体,免疫测定该蛋白质。
与对氧磷酶联合
双向电泳
前述方案中描述的纯化的蛋白质(40μg)与100μl含有9.8M尿素、4%(v/v)Triton X100、2mM三丁基膦、0.2%(v/v)ampholine 3-10(Bio-Lytes 3-10;Bio-Rad)和0.001%(m/v)溴酚兰的溶液混和。使用Ready-to-Use聚丙烯酰胺凝胶条(IPG-Strips;Bio-Rad)(T:4%;C:3%)。Ampholine以共价方式结合聚丙烯酰胺,以致具有预先确立的线性pH梯度。所用pH梯度为3.0至10.0。
1.等电聚焦(IEF)
使这些凝胶条与蛋白质样品在Protean IEF Cell装置(Bio-Rad)中接触,20℃活跃地重新水化(50V恒定)15小时。然后分3个阶段在20℃进行等电聚焦。首先,施加250V低电压15分钟;第二,经2小时自250V至4000V程序化梯度上升(安培数限制在50μA条)。第三,将电压在4000V恒定保持4小时。迁移后,凝胶条储存在-20℃。
根据前面的纯化方案,本发明HBPB与人类对氧磷酶(PON)(Fokine等,2003)共纯化。通过用上述方案制备双向凝胶,通过N端测序鉴定了2个点,分别为本发明蛋白质HPBP和人类对氧磷酶(见图4)。两个蛋白质具有几乎相同的分子量(大约40kDa)和不同的等电点(HPBP为6.9-8.5,PON1为4-5)。考虑到为了在凝胶上成功分离这2种蛋白质必需使用极端条件(9M尿素和4% triton)而且具有十分不同的等电点的这2个蛋白质在经过阴离子交换柱(DEAE sepharose)后仍保持被共纯化的状态,故得到结论:这两个蛋白质通过形成复合物而联合。
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Claims (17)
1.一种蛋白质,特征在于其组成为序列SEQ ID NO:2。
2.编码权利要求1中定义的蛋白质的核苷酸序列。
3.含有权利要求2的核苷酸序列的重组载体。
4.权利要求3的重组载体,所述重组载体是质粒,粘粒,噬菌体或病毒DNA。
5.权利要求3的重组载体,该载体含有使插入所述载体的权利要求2的核苷酸序列所编码的多肽在宿主细胞中表达所必需的元件。
6.宿主细胞,其被权利要求3-5任一项的重组载体转化。
7.权利要求6的宿主细胞,其选自细菌、真菌细胞、植物细胞、或哺乳动物细胞。
8.含有权利要求1的蛋白质作为活性成分并组合含有可药用载体的药物组合物。
9.序列SEQ ID NO:2所表示的蛋白质与SEQ ID NO:4所表示的对氧磷酶蛋白质变体联合在制备药物中的用途,所述药物旨在用于预防或治疗关节炎或与高磷酸盐血症相关的疾病,或者在预防或治疗由杀虫剂或神经毒剂引起的中毒的框架中使用,或者在治疗动脉粥样硬化的框架中使用。
10.权利要求9的用途,所述与高磷酸盐血症相关的疾病是心血管疾病。
11.权利要求9的用途,所述杀虫剂或神经毒剂是梭曼、VX、沙林或塔崩。
12.蛋白质SEQ ID NO:2的抗体用于制备试剂的用途,所述试剂用于通过免疫测定该蛋白质,分析该蛋白质的浓度。
13.蛋白质SEQ ID NO:2的抗体用于制备诊断剂的用途,其中所述诊断剂
用于体外诊断与高磷酸盐血症相关的疾病,或者
用于体外诊断与低磷酸盐血症相关的疾病,或者
用于体外诊断个体对此类病状的易感性。
14.权利要求13的用途,其中所述诊断剂用于在体外诊断与高磷酸盐血症相关的疾病或者用于体外诊断个体发生上述疾病之一的易感性。
15.权利要求14的用途,其中所述与高磷酸盐血症相关的疾病是心血管疾病。
16.权利要求15的用途,其中所述心血管疾病是与动脉粥样斑块的形成相关的心血管疾病。
17.权利要求13的用途,其中所述诊断剂用于体外诊断与低磷酸盐血症相关的疾病、或者用于体外诊断个体发生上述疾病之一的易感性。
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Non-Patent Citations (3)
Title |
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EBI ACCESSION NO.P35482.DATABASE UNIPROT.2003,序列表. * |
KAWASAKI K. ET AL.Mineralized tissue and vertebrate evolution: the secretorycalcium-bingd phosphoprotein gene cluster.P.N.A.S100 7.2003,100(7),第4060-4065. |
KAWASAKI K. ET AL.Mineralized tissue and vertebrate evolution: the secretorycalcium-bingd phosphoprotein gene cluster.P.N.A.S100 7.2003,100(7),第4060-4065. * |
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IL175319A0 (en) | 2006-09-05 |
CA2543935C (fr) | 2014-12-02 |
JP4728246B2 (ja) | 2011-07-20 |
FR2861731B1 (fr) | 2006-02-24 |
JP2008502305A (ja) | 2008-01-31 |
IL175319A (en) | 2013-07-31 |
US20070196879A1 (en) | 2007-08-23 |
WO2005042572A1 (fr) | 2005-05-12 |
ES2341174T3 (es) | 2010-06-16 |
FR2861731A1 (fr) | 2005-05-06 |
CN1898261A (zh) | 2007-01-17 |
CA2543935A1 (fr) | 2005-05-12 |
US7718773B2 (en) | 2010-05-18 |
ATE455792T1 (de) | 2010-02-15 |
DE602004025265D1 (de) | 2010-03-11 |
EP1678204B1 (fr) | 2010-01-20 |
EP1678204A1 (fr) | 2006-07-12 |
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