CN1165146A - 绿化tamm-horsfall糖蛋白或尿调节素的方法，其纯化产物及区别二者的方法 - Google Patents
绿化tamm-horsfall糖蛋白或尿调节素的方法，其纯化产物及区别二者的方法 Download PDFInfo
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Abstract
本发明提供了用于纯化Tamm-Horsfall糖蛋白(THG)和尿调节素的方法以及区别这两种物质的方法，所述方法采用简单步骤高效率纯化出THG和尿调节素。
Description
本发明涉及用于纯化Tamm-Horsfall糖蛋白(下文中简称为“THG”)或来源于人尿的尿调节素(uromodulin)的方法，该方法的纯化产物以及利用免疫球蛋白区别这两种蛋白质的方法。
首先发现的THG是尿粘蛋白(Morner Kah，1895，Skand.Arch.Physiol.6：332)，后来有人报道THG是一种具有抑制尿中病毒的血细胞凝集作用的因子(Tamm，I.and Horsfall，E.L.，1950，Proc.Soc.Exp.Biol.Med.，74：108)。对于THG的纯化方法，将收集的尿样品贮存于4℃过夜，待上清液分离开，滗去一部分之后与氯化钠混合至浓度为0.58M，随后搅拌和离心；收集得到的沉淀并溶于水中，随后离心，并收集上清液并冻干，从而得到被认作为THG的冻干粉。随后，已采用各种各样由许多研究者设计的改进方法纯化THG，由此，沿着上文描述的方案基本上可达到纯化目的(Moonen，P.等，FEBS Lett.，226：314.Toma，G.等，1994，Biochem.Biophys.Res.Commun.，200：275)。
另一方面，从孕妇的尿中纯化了尿调节素，它是一种显示具有相同于THG氨基酸序列的糖蛋白(Muchmore，A.V.和Decker，J.M，1985，Science，229：479)。将孕妇尿加样到植物凝血素柱(Con A-Sepharose柱)上，在用α-甲基甘露糖洗脱之后，将洗脱液在水中渗析，随后将其冻干；然后将冻干液溶于磷酸缓冲盐水中，通过等电聚焦使该溶液分离，随后透过超滤膜浓缩，从而使尿调节素纯化。现在已认为其碳水化合物链或基团能激发许多功能的尿调节素是细胞因子(例如IL-1，IL-2和肿瘤坏死因子(TNF))的特异性配体(Hession，D.等，1987，Science，237：1497.Sherblom，A.P.等，1989，J.Immunol.，143：939，Brown，K.M.，等，1986，Proc.Natl.Acad.Sci.U.S.A.，83：9119，和Winkelstein，A.，等，1990，Immunopharmacology，20：201)。但是，近几年来，将仅采用盐析方法从尿中纯化出的称为“THG”，采用盐析方法从孕妇尿中纯化出的称为“尿调节素”。
THG含有其量相当于约30％的分子量的碳水化合物并且具有富含甘露糖的碳水化合物链结构，该结构可由GluNa2 Man(5-7)表示，其中GluNa和Man分别代表N-乙酰葡萄糖胺和甘露糖。已经证明Man5和Man6的碳水化合物残基特异性地与细胞因子(包括IL-1，IL-2和肿瘤坏死因子(TNF))相结合。这些发现暗示THG可能参与免疫调节机理，同时，有人报道具有相同于THG的氨基酸序列的尿调节素显示出具有比THG的效力高10倍的免疫抑制活性。近几年来还公开了一篇报道，认为来源于羊的THG与羊的IgG结合，而来源于人的THG与人IgG结合(Rhodes，D.C.J.等，1993，Kidney Int.，44：1014)。因此，认为THG除了通过细胞因子介导的免疫调节机理外，在肾小管的新的免疫调节或免疫保护中起着关键的作用。
对于THG和尿调节素的纯化，许多研究者使用上面介绍的盐析方法。然而，该方法具有不仅使目的蛋白沉淀而且使许多其他杂质与该蛋白一起沉淀的危险性，这就需求在随后的纯化步骤中增加去除所述杂质的其他步骤。为了克服这些缺点，必须使目标蛋白在较温和的操作条件下沉淀。此外，当共存在所述金属离子如Na+和Ca++时，THG和尿调节素倾向于易于在溶液中成胶，因此，冻干的THG非常不易溶于等渗盐水中。为此，在生理条件下，在该纯化方法中一开始就不掺入冻干步骤而完成THG的纯化是令人满意的。而且，如果能精确地分别分析THG和尿调节素，并因此建立分别定量测定这两种物质的方法，则可更有效地阐明其生理和临床意义而有效地运用。
本发明人发现，不论是普通的尿或孕妇的尿经过冷冻和解冻，会产生体积较大的沉淀，其中含有大量的THG，并且可以在不使用盐析方法的情况下在一个冷冻和解冻步骤中成功地使THG大量地沉淀出来。而且与盐析方法相反，该新的方法不会引起快速沉淀并且发现该方法具有使杂质不易于掺入沉淀中的优点。此外，由于通过抛弃常规方法所需的许多步骤而使纯化方法简单，本发明人发现，使用高效液相层析(下文中简称为“HPLC”)的凝胶渗透可以有效地去除杂质(主要由生色物质组成)，而透过例如孔径约0.2μm的滤膜的含有目的蛋白的收集到的组分可贮存于4℃，不需冷冻干燥。对于尿调节素，发明人已经建立了采用苯甲酸盐沉淀方法，以及上面描述的冷冻-解冻沉淀方法从孕妇尿中纯化出该物质的方案。还发现纯化的THG与人的免疫球蛋白类IgG，IgA和IgM以及IgG的所有亚类IgG1，IgG2，IgG3和IgG4以及IgG的F(ab’)2片段结合，而纯化的尿调节素与人的免疫球蛋白类IgG和IgA以及IgG亚类IgG1，IgG2和IgG4结合，但是它不易与IgM，IgG的亚类IgG3和IgG的F(ab’)2片段结合。
基于这些发现完成了本发明，并且本发明涉及用于纯化THG或尿调节素的方法，其特征在于所说方法包括将人尿冷冻，然后解冻，从而收集到所得的沉淀组分，或向人尿中加入苯甲酸钠或苯甲酸铵，然后使溶液变酸，足于使苯甲酸结晶出来，从而收集得到的沉淀组分，本发明涉及各自显示其特征的纯化的THG或尿调节素，并且涉及区别THG和尿调节素的方法，其特征在于所述方法包括以不易与尿调节素结合的免疫球蛋白例如IgG的F(ab’)2片段与样品反应，并且分析免疫球蛋白的起反应的量。
采用常规的已知技术完成对人尿的冷冻和解冻或不冷冻，从而收集在解冻溶液中形成的沉淀组分。以约2％(W/V)的比例向人尿中加入苯甲酸钠或苯甲酸铵就足够了，通过加入例如盐酸使得到的溶液调至pH3-4的酸性范围，从而使苯甲酸沉淀，其中沉淀的苯甲酸吸附所需的蛋白和沉淀物。将该沉淀的组分分离出来并且与乙醇混合，随后进行搅拌，通过溶于乙醇中而得到没有苯甲酸的沉淀。
例如，通过将得到的沉淀溶于磷酸缓冲盐水，随后凝胶渗透而去除包括生色物质等在内的杂质，进一步对上述步骤获得的沉淀组分进行纯化。
如果以该方式获得的纯化产物是纯化的THG时，它显示的特征性质是它与免疫球蛋白类的IgG的F(ab’)2片段，IgA(单体)和IgM以及所有的IgG亚类IgG1，IgG2，IgG3和IgG4结合，而如果纯化产物是纯化的尿调节素，它显示另一方面的特征特性，即与人免疫球蛋白类的IgG和IgA(单体)以及IgG亚类的IgG1，IgG2和IgG4结合，而不易于与人免疫球蛋白类的IgG的F(ab’)2片段和IgM以及IgG的亚类Ig3结合。
事实既然如此，通过采用已知方法将纯化产物固定，随后采用人免疫球蛋白类的IgM或IgG亚类的IgG3进行作用，然后用酶标记的蛋白质G分析任一种免疫球蛋白的起反应的量，或直接以酶标记的IgG的F(ab’)2片段作用于纯化产物，然后分析任一种免疫球蛋白的起反应的量。
据认为内毒素休克和脓毒病是由参与炎症的细胞因子IL-1和TNF引起，而来源于人尿的THG或尿调节素显示抗细胞因子IL-1的抑制活性，因此THG和尿调节素可用作为治疗剂有效地抵抗所述疾病。就其对免疫球蛋白的反应性而言，认为THG和尿调节素也具有有效的抗感染活性。由此已经建立了一种基于免疫球蛋白的用于区分THG和尿调节素的方法，另外，本发明可用于阐明这些蛋白的生理和临床意义。
根据本发明，采用简化的方法可高效率地纯化THG和尿调节素，从而得到显示特征性质的纯化产物，并且借助于这种特征性质显示的全部优点，可以区分THG和尿调节素。
下文中描述的实施例用于更详细地描述本发明，但是这些实施例并不是用于限制本发明，其中附图说明如下。
图1是将实施例1中采用冷冻-解冻方法纯化的人尿制备的样品进行凝胶过滤后获得的洗脱图样，箭头符号表示THG组分。
图2是在还原(道1和2)和非还原(道3和4)条件下将实施例1中纯化的THG单独地进行SDS-PAGE获得的电泳图谱的照片，其中，道1和3：分子量标记；道2和4：THG样品。
图3是采用苯甲酸钠吸附沉淀方法纯化孕妇尿制备的样品进行凝胶渗透后获得的洗脱图样，箭头符号表示尿调节素组分。
图4是在还原(道1和2)和非还原(道3和4)条件下将实施例2中纯化的尿调节素单独地进行SDS-PAGE获得的电泳图谱的照片，其中，道1和3：分子量标记；道2和4：THG样品。
图5显示如在实施例3和4中进行的固定于微平板上的THG和尿调节素与人免疫球蛋白或IgG的F(ab’)2片段反应的过程，表明基于其与人IgG的F(ab’)2片段的亲和性的不同可将THG和尿调节素分别辨认出并区分开；图5(a)表示THG和尿调节素对人IgG的亲和性；图5(b)表示THG和尿调节素对人IgG F(ab’)2的亲和性；在图5(a)和(b)中，符号“○”和“●”分别是指THG和尿调节素。
实施例1
收集正常人尿，贮存于-20℃，然后于4℃解冻，从而得到沉淀。对分离出的沉淀以3,000xg离心30分钟，滗去上清液，将沉淀溶于磷酸缓冲盐水(PBS)，随后在PBS中透析并离心。采用Amicon分离并浓缩得到的上清液，将浓缩液进行HPLC，随后在PBS状态下(TSK-300SW；由Tosoh Inc.日本制造)对流动相进行凝胶渗透(参见图1)。对空体积中洗脱出的组分进行SDS-PAGE，以鉴别分子量和纯度(参见附图2)。采用上面介绍的程序也能纯化孕妇的尿(在怀孕的第二个半月至第六个月)。
实施例2
在孕妇尿(在怀孕的第二个半月至六个月)中混入占尿体积2％的比例的苯甲酸钠，随后搅拌溶解。用16％盐酸将该溶液pH调至3.9，并搅拌60分钟，从而使得到的蛋白质沉淀有效地吸附到苯甲酸的磨成细粉的沉淀上。收集得到的凝集块沉淀，用20倍体积的冷饱和苯甲酸溶液(0.3％(W/V))洗涤，并压滤至分离出沉淀。在将沉淀与3倍体积的冷乙醇混合之后，用22％氨水将得到的溶液pH调至5.5，然后搅拌30分钟，置于4℃保持3小时，去2/3的上清液。将剩余的沉淀溶液加入到Celite Precoat(由Shiroyama Kogyo K.K.of Ja-pan生产，商标为Gemlite Super M.)，随后过滤，回收沉淀并贮存于-30℃，将过滤后得到的湿滤饼溶于PBS中并在PBS中透析，将透析液以15,000rpm离心10分钟，并利用PBS平衡的HPLC(TSK 3000-SW：由Tosoh Inc.of Japan生产)进行凝胶渗透(参见附图3)。将空体积中洗脱出的组分进行SDS-PAGE，以确定分子量和纯化纯度(参见图4)。收集主要含有尿调节素的组分，然后透过一个0.22μm滤膜，随后贮存于4℃。采用上面介绍的方法也可纯化正常人尿。
实施例3
分别用50mM碳酸钠缓冲液(下文中简称为“SCB”，由U.S.A的Sigma公司生产)(pH9.6)稀释实施例1和2中获得的纯化THG和尿调节素。将每种溶液分布到96孔平板(由Nunc Co.生产)的各个孔中，每孔加入50μl，随后置于4℃过夜。用SCB洗涤平板，将1％BSA-SCB溶液倒入每个孔中，每孔300μl，随后贮存于4℃过夜。在用20mM Tris缓冲盐水+0.0 5％吐温20(下文中简称为“TTBS”)(pH7.4)洗涤平板之后，将用1％BSA-TTBS对起始浓度3mg/ml的IgG连续2倍稀释(由Kappel Co.生产)之后加入到每个孔中，每孔50μl，随后置于4℃放置过夜，使反应完成。在用TTBS洗涤之后，将稀释3000倍的过氧化物酶标记的EIA级蛋白质G(由Biorad Co.ofU.S.A.生产)加入每孔中，每孔50μl，随后在37℃反应2小时。在用TTBS洗涤并用SCB漂洗之后，利用TMB过氧化物酶EIA底物盒(由Biorad Co.of U.S.A.生产)完成显色反应，随后用平板计数器在450nm波长处测定吸收值。如在图5(a)中说明的，发现THG和尿调节素以剂量依赖性方式与人免疫球蛋白IgG反应。
实施例4
用50mM SCB分别稀释实施例1和2中获得的纯化THG和尿调节素并分配到96孔平板的各个孔中，每孔50μl，随后贮存于4℃过夜。在用SCB洗涤平板之后，以每份300μl向各个孔中加入1％BSA-SCB溶液，随后置于4℃过夜。在用TTBS洗涤之后，用1％BSA-TTBS将起始浓度为3mg/ml的过氧化物酶标记的人IgG的F(ab’)2片段(由Rock land Co.ofU.S.A.生产)连续稀释2倍后以每份50μl加入到各个孔中，随后置于4℃过夜。在用TTBS洗涤并用SCB漂洗之后，用TMB过氧化物酶EIA底物试剂盒反应，在450nm处测定吸收值。如在图5(b)中描述的，发现THG以剂量依赖性方式与人IgG的F(ab’)2片段发生反应，而尿调节素不易于与之反应。
实施例5
以人尿和孕妇尿用作为样本，用50mM SCB稀释每个样本，并以每份50μl的量分布于96孔平板的每个孔中，随后置于4℃过夜，在用SCB洗涤平板之后，以每份300μl的量向每孔中加入1％BSA-SCB溶液，接着在4℃静置过夜。在用20mM TTBS洗涤之后，用1％BSA-TTBS将起始浓度为3mg/ml的人IgG连续稀释2倍，稀释之后，以50μl每份的量加入到每个孔中，随后置于4℃过夜，以使反应完成。在用TTBS洗涤之后，向每孔中加入50μl的稀释3000倍的过氧化物酶标记的EIA级蛋白质G，随后于37℃反应2小时。在用TTBS洗涤并用SCB漂洗之后，用TMB过氧化物酶EIA底物试剂盒完成显色反应，并在平板读数器上于450nm波长处测定吸收值，获得的结果与实施例3的结果基本相当。
实施例6
以人尿和孕妇尿用作为样本，用50mM SCB稀释每个样本，并以每份50μl的量分布于96孔平板的每个孔中，随后置于4℃过夜。在用SCB洗涤平板之后，以每份300μl的量向每孔中加入1％BSA-SCB溶液，随后于4℃静置过夜。在用20mM TTBS洗涤之后，将起始浓度为3mg/ml的稀释2倍的过氧化物酶标记的人IgG的F(ab’)2片断以50μl每份的量加入到每个孔中，随后置于4℃过夜，以使反应完成。在用TTBS洗涤并用SCB漂洗之后，用TMB过氧化物酶EIA底物试剂盒完成显色反应，并于450nm波长处测定吸收值，获得的结果与实施例4的结果基本相当。
Claims (7)
1.一种用于纯化Tamm-Horsfall糖蛋白或尿调节素的方法，其特征在于该方法包括将人尿置于较低温度下或冷冻，随后解冻，从而收集得到的沉淀组分，或者将苯甲酸钠或苯甲酸铵溶于人尿中并使溶液变酸，以便足于使苯甲酸结晶出来，从而收集得到的沉淀组分。
2.根据权利要求1所述纯化方法，其中进一步用凝胶过滤纯化得到的沉淀组分。
3.根据权利要求1或2所述纯化方法，其中以正常尿作为人尿，纯化Tamm-Horsfall糖蛋白。
4.根据权利要求1或2所述的纯化方法，其中以孕妇的尿作为人尿，纯化尿调节素。
5.一种纯化的Tamm-Horsfall糖蛋白，该糖蛋白能够与人免疫球蛋白类的IgG的F(ab’)2片段，IgA(单体)和IgM，以及与IgG所有亚类IgG1，IgG2，IgG3和IgG4相结合。
6.一种纯化的尿调节素，它能与人免疫球蛋白类的IgG和IgA(单体)，以及与IgG亚类IgG1，IgG2和IgG4相结合，但不易与人免疫球蛋白类的IgG的F(ab’)2片段和IgM以及IgG亚类IgG3相结合。
7.一种用于区分Tamm-Horsfall糖蛋白和尿调节素的方法，其特征在于所述方法包括使人免疫球蛋白类的IgM或IgG亚类IgG3作用于尚未鉴定出是Tamm-Horsfall糖蛋白还是尿调节素的样本，然后用酶标记的蛋白质G分析这种免疫球蛋白起反应的量，或者使酶标记的IgG的F(ab’)2片段直接作用于所述样本，然后分析所述IgG的F(ab’)2片段的起反应的量。
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| US20150005240A1 (en) | 2012-02-09 | 2015-01-01 | Instituto Massone S.A. | Therapeutic method of treatment for inhibiting tumor development using tamm-horsfall glycoprotein (thp) as immunostimulating drug and compositions |
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| EP0780398A2 (en) | 1997-06-25 |
| ATE241644T1 (de) | 2003-06-15 |
| DE69628397D1 (de) | 2003-07-03 |
| JPH09165398A (ja) | 1997-06-24 |
| DE69628397T2 (de) | 2003-11-27 |
| JP3839509B2 (ja) | 2006-11-01 |
| EP0780398B1 (en) | 2003-05-28 |
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| CI01 | Publication of corrected invention patent application |
Correction item: Denomination of Invention Correct: Method for purifying TAMM-HORSFALL protein or urinary regulator, purification product thereof and method for distinguishing two False: Method for greening TAMM-HORS-FALL glycoprotein or urinary regulating agent, purification product thereof and method for distinguishing two Number: 47 Page: 33 Volume: 13 |
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| ERR | Gazette correction |
Free format text: CORRECT: INVENTION NAME; FROM: METHOD FOR GREENING TAMM-HORS-FALL GLYCOPROTEIN OR UMOD, PURIFIED PRODUCT AND METHOD DISTINGUISHING THE TWO TO: PURIFIED TAMM-HORSFALL SUGAR PROTEIN OR URINE REGULATOR METHOD, PURIFIED PRODUCT AND METHOD DISTINGUISHING THE TWO |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |