CN1129944A - 新型蛋白质及其制备方法 - Google Patents
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- CN1129944A CN1129944A CN95190587A CN95190587A CN1129944A CN 1129944 A CN1129944 A CN 1129944A CN 95190587 A CN95190587 A CN 95190587A CN 95190587 A CN95190587 A CN 95190587A CN 1129944 A CN1129944 A CN 1129944A
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Abstract
来自人成纤维细胞,具有序列表中序列号1的氨基酸序列为N-末端氨基酸序列、分子量为约15KD(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳,在还原和非还原条件下)、具有成骨细胞增殖活性的蛋白质。具有序列号9的氨基酸序列、有成骨细胞增殖活性的蛋白质。培养人成纤维细胞,用阳离子交换体和肝素吸附精制,或用基因工程方法制备该蛋白质的方法。该蛋白质用于骨质疏松症等疾病的治疗或免疫学诊断用的抗原蛋白。
Description
技术领域
本发明涉及显示成骨细胞增殖活性的新型蛋白质、即成骨细胞增殖因子II(basic Osteoblast Growth Factor-II,以下称作b OGF-II)及其制备方法。
背景技术
人的骨胳是不断反复地吸收与再形成的,在此过程中起核心功能的细胞是起骨形成作用的成骨细抱和起骨吸收作用的破骨细胞。因这些细胞所担当的骨代谢的异常而发生的疾病可列举骨质疏松症为代表。这种疾病是由破骨细胞产生的骨吸收超过成骨细胞产生的骨形成而引起的疾病,然而,关于这种疾病的发生机制,目前仍未完全阐明。这种疾病发生骨痛,而因骨脆,成为骨折的原因。从而,随着高龄人口的增加,这种疾病成为因骨折而卧床老人的发生原因,也已成为社会问题,其治疗药的开发正成为当务之急。这样的骨代谢异常所致骨量减少症,期望通过抑制骨吸收、促进骨形成或改善它们的平衡而加以治疗。
骨形成,期望通过提当骨形成的细胞的增殖、分化或激活而得以促进。近年来,对具有这种生理活性的蛋白质(细胞分裂物质)的关注增多,并进行了集中精力的研究。据报导,促进分化为骨细胞的成骨细胞的增殖的细胞分裂物质有成纤维细胞增殖因子系列(fibroblastgrowth factor;FGF:Rodan S.B.et al.,Endocrinology vol.121,p1917,1987)、胰岛素样增殖因子-I(insulin like growth factor-I;IGF-I;Hock J.M.et al.,Endocrinology vol.122,p254,1988)、胰岛素样增殖因子-II(IGF-II;McCarthy T.et al.,En-docrinology vol.124,p301,1989)、和异位骨形成因子系列(bonemorphogenic protein;BMP:Sampath T.K.et al.,J.Biol Chem.vol.267,p20532,1992、Knut sen R.et al.,Biochem,Biophys.Res.Commun.vol.194,p1352,1993、及山口朗他、实验医学vol.10,p2003,1992)等;促进成骨细胞分化的细胞分裂物质有转化增殖因子-β(transforming growth factor-β;TGF-β;Centre Ila M.et al.,J.Biol.Chem.vol.262,p2869,1987)、胰岛素样增殖因子、及异位骨形成因子系列(Takuwa Y.et al.,Biochem.Biophys.Res,Commun.vol.174,p96,1991、及Knutsen R.et al.,Biochem Biophys.Res.Commun.vol.194,p1352,1993)等。这些生理活性蛋白质因促进骨形成而成为骨量减少症的改善剂的可能性是人们所期望的,生理活性骨白质异位骨形成因子系列等,就上述生理活性蛋白质的一部分而言,作为骨代谢改善剂,正在进行临床试验。
目前,作为用于治疗与骨有关的疾病及缩短治疗周期的医药品,在临床上使用的有维生素D3、降钙素及其衍生物、雌二醇等激素制剂、核黄素或钙制剂等。然而,采用这医药品的治疗法,其效果和治疗结果是不能令人满意的,期望开发代替这些医药品的新治疗药。上述骨代谢是由骨形成和骨吸收的平衡来调节的,通过促进成骨细胞增殖使骨新生活跃的生理活性蛋白质被期望成为骨质疏松等骨量减少症的治疗药。
发明的揭示
鉴于这样的现状,本发明者们广泛寻求成骨细胞增殖促进因子,锐意探索的结果,发现新型的蛋白质性成骨细胞增殖因子,而且积聚了高浓度的本发明蛋白质,确立了效率好的精制方法。然后,基于所得天然型蛋白质氨基酸序列的信息,成功地进行了编码本蛋白质的cDNA的克隆化。而且,用此cDNA,通过基因工程的方法产生了成骨细胞增殖因子。从而,本发明的课题是提供新型的成骨细胞增殖因子(蛋白质)及其有效的制备方法。
本发明者们测定了各种动物细胞培养液中成骨细胞增殖促进活性,发现人成纤维细胞IMR-90(ATCC-CCL186)培养液中存在高的成骨细胞增殖促进活性。又研究了IMR-90细胞的培养法,用氧化铝陶瓷片作为细胞载体,确定了在培养液中积聚高浓度成骨细胞增殖促进因子的条件。而且,通过发现离子交换柱和/或肝素柱高效组合的条件,确立了效率好的bOGF-I的精制方法。
本发明者们进一步确定了这种天然型bOGF-II的氨基酸序列,在此氨基酸序列的基础上设计DNA引物,用此引物以PCR法得到b OGF-II的cDNA片断。用此cDNA片断作为探针,用杂交法筛选IMP-90细胞的重组DNA基因库,成功地得到编码本发明蛋白质全长的cDNA。进一步将此cDNA组成质粒,用此质粒对宿主细胞进行性状转导,确定了从培养此性状转导细胞回收采集bOGF-II的方法。
本发明涉及的蛋白质特征在于:来自人成纤维细胞;在还原和非还原条件下,在十二烷基磺酸钠-聚丙烯酰胺凝胶电泳中分子量为约15KD;对阳离子交换体和肝素具有强的亲和性;70℃加热处理10分钟,成骨细胞增殖活性降低,90℃加热处理10分钟则失活。本发明蛋白质bOGF-II的结构与已知成骨细胞增殖因子在N末端氨基酸序列上明显不同。本发明的蛋白质bOGF-II的N-末端氨基酸序列表示为序列表中序列1和2号。
本发明还涉及蛋白质bOGF-II的制备方法,其特征在于:培养人成纤维细胞,将培养液通过肝素柱层析处理,洗脱吸附部分,将洗脱液进行阴离子交换柱层析处理,得非吸附部分,将此部分上阳离子交换柱,再用肝素柱精制,收集上述蛋白质。
本发明中的柱处理与不仅只是使培养液等在肝素葡聚糖柱等中流下,而且将培养液分批与肝素葡聚糖等混合进行柱处理具有同样的效果。
本发明的蛋白质bOGF-II可从人成纤维细胞培养液中以高效、高收率地分离精制。从此原料制备bOGF-II,与从机体物质而来的蛋白性物质的分离上广泛使用的常规方法同样地,可按照利用目的物bOGF-II的物理、化学性质的各种精制方法来进行。作为浓缩方法,可列举超滤、冷冻干燥和盐析等常规生化处理方法;作为精制方法,可将离子交换层析、亲和层析、凝胶过滤、疏水性层析、反相层析、制备用电泳等常规的蛋白性物质精制上利用的各种方法组合起来加以使用。特别令人满意的是可用IMP-90作为人成纤维细胞。且,作为原料的人成纤维细胞IMR-90培养液可使用将人成纤维细胞IMR-90吸附在陶瓷上,用添加了5%小牛血清的DMEM培养基作为培养液,在转瓶中放置培养一周所得的培养液。在进行精制处理中,以在缓冲液中添加0.1%CHAPS(3-〔(3-cholamidopropyl)-dimethylammoniol-1-propane sulfonate〕进行精制为好。
本发明蛋白质的精制方法为:先将培养液上肝素层析柱(肝素葡聚糖CL6B,Pharmacia公司制),用含2M NaCl的20mMTris-HCl缓冲液(pH7.5)洗脱,得到肝素吸附性OGF部分,将此部分上Q阴离子交换柱(HiLoad-Q/FF,Pharmacia公司制),收集其非吸附部分,可得到肝素吸附性碱性的bOGF部分。所得的bOGF活性部分用S阳离子交换柱层析(HiLoad-S/HP,Pharmacia公司制),在表观上分成3个活性峰,按以较低温度的NaCl洗脱的顺序,分别为bOGF-I(0.15M)、bOGF-II(0.35M)、bOGF-III(0.55M)。
本发明的bOGF-II可按上述方法,即再重复进行肝素柱层析(肝素-5PW,ト-ソ-公司制)加以分离精制,此物质以前述特征加以鉴定。bOGF-II经70℃热处理10分钟灭活,从肝素层析柱以约1.8M的NaCl洗脱,再用抗碱性成纤维细胞增殖因子(basic Fibrob-last Growth Factor)中和抗体中和其活性,判断其为碱性成纤维细胞增殖因子的可能性很高。
本发明的bOGF-II蛋白质的基因工程制备方法为:根据天然型bOGF-II的氨基酸序列设计引物,用此引物以PCR法得到bOGF-II的cDNA片断;然后,用此cDNA片断作为探针,用杂交法筛洗IMR-90细胞的重组基因库,得到编码bOGF-II全长的cD-NA;再将此bOGF-II cDNA插入具有合适表达用启动子的质粒,在哺乳动物细胞(例如中国地鼠卵巢细胞)等真核细胞、细菌(如大肠肝菌)等原核细胞中的合适宿主中转染此表达质粒;通过培养该种细胞,从培养液或细胞中回收精制本发明蛋白质bOGF-II。
本发明涉及的蛋白质具有这样的bOGF-IIcDNA,从其推定的氨基酸序列或含有这种氨基酸序列作为部分序列,或与此氨基酸序列具有80%以上同源性,具有成骨细胞增殖活性。
用成骨细胞株或正常成骨细胞作为靶细胞,用其细胞数的增加和3H-胸腺嘧啶掺入促进试验,可测定OGF活性。作为理想的靶细胞,可用小鼠成骨细胞株MC3T3-E1(J.Oral.Biol.23,899,1981和J.Cell.Biol.96,191,1983)。该细胞株是具有维生素D3和甲状旁腺激素反应性,据报告在体外以类似于体内的过程灰质化的细胞株。在活性的测定上,较宜用无血清培养基,通过3H-胸腺嘧啶的掺入促进试验,可以高灵敏度进行正确的测定。
本发明的蛋白质bOGF-II作为以骨质疏松症等骨量减少症或其他骨代谢异常疾患的治疗或改善为目的的医药组合物,及作为抗原用于这些疾患的免疫学诊断,都是有用的。
本发明的蛋白质可制成制剂,经口或非经口投与。即,制剂是含成骨细胞增殖因子为有效成分的医药组合物,能对人安全地投药。医药组合物的形态,可列举注射用组合物、点滴用组合物、栓剂、鼻内给药剂、舌下剂、经皮吸收带等。注射用组合物为药理有效量的本发明的成骨细胞增殖促进因子和制药学上允许的载体的混合物,其中可用氨基酸、糖类、纤维素衍生物和其它有机/无机化合物等通常添加在注射用组合物中的赋形剂/激活剂。配制含本发明的成骨细胞增殖促进因子与这些赋形剂/激活剂的注射剂时,必要时添加pH调节剂、缓冲剂、稳定剂、加溶剂等,按常法制成各种注射剂。
图面的简单说明。
图1表示采用阳离子交换柱层析(HiLoad-S/Hp,2.6×10cm,Pharmacia公司制)bOGF的洗脱图型。
图中峰1、峰2和峰3分别表示bOGF-I、bOGF-II和bOGF-III。
图2表示采用亲和层析(肝素5PW,0.8×7.5cm,ト-ソ-公司制),本发明的bOGF-II的洗脱图型。
图3表示在非还原条件下采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),本发明的bOGF-II的电泳图型。
图中各行分别表示:1.标准分子量蛋白,2.级份A,3.级份B,4.级份C,5.级份D,6.级份E。
关于级份A-E,参照图2。
图4表示用反相柱层析(OD-300,C18,2.1×200mm,Ambrite公司制)内肽酶Asp-N(Boehringer公司制)消化的还原PE化bOGF-II来源的肽的洗脱图型。
图5表示bOGF-II的成骨细胞增殖促进活性。
图6表示质粒pQE30-OGF-II的结构。
图中6His为6个组氨酸区域,Phage T5 promoter为噬菌体T5启动子,bIa为耐氨苄青霉素基因,Ori为大肠杆菌上的复制起始区,OGF II为bOGF-II cDNA。
图7表示His6-bOGF-II的成骨细胞增殖促进活性。
图中第一栏为对照,第二栏为添加10%His6-bOGF-II溶液时的活性。
实施发明的最佳方案
〔实施例〕
以下列举实施例更详细地说明本发明。然而,它们仅仅是示例,本发明并不受其限制。
实施例1天然型bOGF-II的采取
(1)人成纤维细胞IMR-90培养液的配制
人胎儿肺成纤维细胞IMR-90(ATCC-CCL186)在转瓶(490cm2,110×171cm,コ-ニング公司制)中,附着于80g的铝陶瓷片(铝99.5%,东芝陶瓷公司制)上进行培养。培养中使用60个转瓶,每个转瓶中用添加500ml 10mM HEPES缓冲液的DMEM培养基(Gibco公司制),缓冲液中加有5%小牛血清,在5%CO2存在下于30℃培养7—10天。培养后回收培养液,添加新的培养基,每次培养得到30升IMR-90培养液。所得的培养液作为试样1。
(2)成骨细胞促进活性的测定方法
本发明的蛋白性成骨细胞增殖因子OGF活性测定,按对小鼠成骨细胞株MC3T3-E1(明海大学口腔学科久米川正好教授赠)的DNA合成促进活性测定来进行。即,在96孔板上加入用含0.2%BSA、不含核酸的α-MEM培养基(Gibco公司制)稀释的样品50μl,用2×103个细胞/50μlα-MEM培养基接种MC 3T3-E1细胞,在5%CO2、37℃下培养15—20小时。培养后,每孔加入用磷酸缓冲生理盐水稀释成0.1mCi/ml的3H-胸腺嘧啶(TRK 686,Amersham公司制)10μl,再培养2小时后,用β计数器(パッ力-ド公司制)测定细胞上集聚的3H-胸腺嘧啶的放射活性。所得的放射活性(cpm)作为OGF活性。
(3)bOGF-II的精制
i)用肝素葡聚糖6B(Heparin Sepharose L-6B)精制
将约90升IMR-90培养液(试样1)用0.22μm滤膜(亲水性ミリデイスク,2000cm2,Millipore公司制)过滤后,分3次上80ml肝素葡聚糖6B(5×4.1cm)柱,该柱用含0.3M NaCl的10mMTris-HCl(pH7.5)平衡过。以流速500ml/小时,10mM Tris-HCl(pH7.5)洗涤后,用10mM Tris-HCl/2M NaCl(pH7.5)洗脱,得肝素葡聚糖6B吸附部分900ml,所得组分作为试样2。
ii)用HiLoad-Q/FF精制
将肝素葡聚糖吸附部分(试样2)对10mM Tris-HCl(pH7.5)进行透析后,加CHAPS使成1%,4℃放置一夜,分2次上用50mMTris-HCl/0.1%CHAPS(pH7.5)平衡过的阴离子交换柱(HiLoad-Q/FF,2.6×10cm,Pharmacia公司制),得非吸附部分1000ml。所得部分作为试样3。
iii)用HiLoad-S/HP精制
将HiLoad-Q非吸附部分(试样3)上以50mM Tris-HCl/0.1%CHAPS(pH7.5)平衡过的阳离子交换柱(HiLoad-S/HP,2.6×10cm,Pharmacia公司制)。用50mM Tris-HCl/0.1%CHAPS(pH7.5)洗涤后,在100分钟内,将NaCl O-1M线性梯度,以8ml/分流速进行洗脱,按12ml/级分进行收集。用各10μl级分按上述(2)的测定法测定OGF活性。OGF活性见于3个峰(峰1:bOGF-I;峰2:bOGF-II;峰3:bOGF-III)。结果见图1。
峰3的部分所见的OGF活性,70℃热处理10分钟失活,以约1.8M NaCl从肝素柱洗脱,可用人bOGF抗体中和,因此推测为bOGF。
iv)用亲和层析(肝素-5PW)精制
将峰2(bOGF-II)部分120ml用240ml 50mM Tris-HCl/0.1CHAPS(pH7.5)稀释后,上以50mM Tris-HCl/0.1%CHAPS(pH7.5)平衡后的亲和层析柱(肝素-5PW,0.8×7.Scm,ト-ソ公司制)。用50mM Tris-HCl/0.1 CHAPS(pH7.5)洗涤后,在60分钟内,以NaClO-2M线性梯度,0.5ml/分流速进行洗脱,按0.5ml/级分进行分取。各级分用2μl测定OGF活性,得到约1.0-1.2MNaCl洗脱的OGF活性部分,将所得部分作为试样4。
将5ml试样4用10ml 50mM Tris-HCl/0.1%CHAPS(pH7.5)稀释后,上以50mM Tris-HCl/0.1%CHAPS(pH7.5)平衡过的亲和层析柱(肝素-5PW,0.8×7.5cm,ト-ソ-公司制)。用50mMTris-HCl/0.1%CHAPS(pH7.5)洗涤后,在60分钟内,以NaClO-2M线性梯度,0.5ml/分流速进行洗脱,接0.5ml/级分进行分取。各级分用2μl测定OGF活性,得到用约1.0-1.2M NaCl洗脱的OGF活性部分5ml,所得部分作为试样5。
将5ml试样5用10ml 50mM Tris-HCl/0.1%CHAPS(pH7.5)稀释后,上以50mM Tris-HCl/0.1%CHAPS(pH7.5)平衡过的亲和层析柱(肝素-5PW,0.5×7.Scm,ト-ソ-公司制)。用50mMTris-HCl/0.1%CHAPS(pH7.5)洗涤后,在60分钟内,以NaCl2M线性梯度,0.5ml/分流速进行洗脱,按0.5ml/级分进行分取。各级分用4μl测定OGF活性。结果见图2。
(4)bOGF-II分子量测定
将所得的OGF活性部分分成5个部分,各2ml(图2、部分A-E),用各部分100μl在非还原条件下进行SDS-聚丙烯酰胺凝胶电泳。即,将所得的洗脱部分A-E各100μl对水透析后,冷冻干燥,溶解于1.5μl10mM Tris-HCl(pH8)/1mM EDTA/2.5%SDS/0.01%溴酚兰中。37℃下放置一夜后,将此样品1μl用SDS-聚丙烯酰胺凝胶电泳进行分析。电泳使用8-25%丙烯酰胺的梯度凝胶(Pharmacia公司制),用电泳仪(Fast System,Pharmacia公司制)进行,用碳酸脂酶(94KD)、血清白蛋白(67KD)、卵清白蛋白(43KD)、碳酸酐酶(30KD)、胰蛋白酶抑制剂(20.1KD)α-乳清白蛋白(14. 4KD)作为分子量标记。电泳结束后,按Pharmacia公司的操作规程进行银染色。结果见图3。
结果,在15KD附近检出与OGF活性强度成比例的蛋白质区带,而对于部分D和E,未检出15KD以外的蛋白质区带。且,此活性蛋白质在还原条件下进行SDS-聚丙烯酰胺凝胶电泳,仅检出与非还原条件大致相同分子量的一条蛋白带。
(5)N-末端氨基酸序列的测定
将所得的洗脱级分D500μl上以0.1%三氟乙酸(TFA)/10%乙腈平衡过的反相层析柱(BU-200,C4,2.1×220mm,Amerite公司制),以50分钟内的乙腈10%—60%的线性梯度,以0.2ml/分流速进行洗脱,脱盐、浓缩后,用蛋白质测序仪(477-120A,アブライド公司制)进行N-末端氨基酸序列的分析。将所得多肽的氨基酸序列表示为序列表中的序列号1。序列中-Xaa-为未确定的氨基酸。
(6)蛋白质氨基酸序列的测定
将洗脱的级分C2000μl浓缩后,加入含1mg二巯基苏糖醇的300μl 0.5M Tris-HCl(pH8.5)/10mM EDTA/7M盐酸胍,室温下放置4小时还原后,加入2μl 4-乙烯基吡啶,于室温下暗处放置一夜,进行吡啶基乙基(PE)化。在这样的样品中加入3μl 25%TFA,上以10%乙腈/0.1%TFA平衡过的反相层析柱(BU-300,C4,4.6×30mm,アプライド公司制),以50分钟内乙腈浓度0-50%线性梯度,1ml/分流速,进行洗脱,得还原PE化bOGF-II。将所得还原PE化b GF-II的1/4用蛋白质测序仪(477A-120A,アブライド公社制)进行N-末端氨基酸序列分析。所得的氨基酸序列表示为序列表中序列号2。
将剩下的3/4还原PE化bOGF-II在50μl 50mM磷酸缓冲液(pH8.5)/1M尿素中,用蛋白酶Asp-N(Boehringer公司制)于37℃下消化15分钟,上以0.1%TFA平衡过的反相层析柱(OD-300,C18,2.1×220mm,アブライド公司制),以乙腈0%-40%线性梯度、0.2ml/分流速进行洗脱,分部收集。洗脱图型见图4。对于检出的5个峰,用蛋白质测序仪(477A-120A,アブライド公司制)进行N-末端氨基酸序列分析。所得的5种多肽的氨基酸序列分别表示为序列表中序列号3-7号。
(7)bOGF-II的容量依赖性成骨细胞增殖促进活性
洗脱部分D的蛋白质浓度以BSA(牛血清白蛋白)为标准,用Lowry法进行测定。用此样品,从100ng/ml开始,作1/2稀释,按实施例1(2)成骨细胞增殖促进活性测定方法测定OGF活性。结果见图5。
实施例2
用遗传工程方法采取b GF-II
(1)bOGF-II基因的克隆化
1)由PCR法对bOGF-IIcDNA片断决定克隆值的bOGF-II的氨基酸序列,以该序列为基础,制得基因扩增用的引物。即,从N末端氨基酸序列的Glu-Thr-Glu-Tyr-Gly-Pro-Cys推断对其编码的DNA序列,合成具有5’-GA(A/G)ACNGA(A/G)TA(T/C)GGNCCNTG-3’序列的混合引物。这里,A/G表示A或G,T/C表示T或C,N表示A,G,C或T。又,从作为中间氨基酸序列的Asp-Lys-Lys-Gly-Phe Tyr-Lys中推定对其编码的DNA序列,合成具有其互补链5’-TT(A/G)TA(A/G)AANCC(T/C)TT(T/C)TT(A/G)TC-3’序列的混合引物。引物的合成使用了アプライド公司的DNA合成机394,使用该二种引物(各200p-mol)和使用来自胎儿肺成纤维细胞的Imp90聚ARAA的单链cD-NA(1μg)作为模板DNA,进行PCR法(poly merase chminreaction)反应,酶用EX Taq(宝酒造公司制),反应溶液使用10XExTaq缓冲液5μl,2.5mM的DNTP 4μl,cDNA溶液1μl Ex29.75μl引物(各40μM)5μl。其最终容量设为50μl,反应条件为:在95℃下3分钟保温后,作30个循环的95℃30秒、50℃30秒、70℃2分钟的三个阶段的保温,然后,在75℃保温5分钟的条件下进行反应。反应完毕后,将反应液8μl供至4%的琼脂糖凝胶电泳,检出含约120bp的片断的数个区带。混合该反应液4.5μl,pCRII(original TAcloning kit Invitrogen)公司克隆量0.5μl,及DNA连结盒(version2)液(宝酒造公司)5μl,16℃下保温一夜。使用连结反应液5μl,转化大肠杆菌DH52(BRL公司)。由PCR方法推定所得到的对氨苄青霉素具抗异性的细菌中的质粒的插入片断的长度,分离得到4株具有约120bp的插入片断的菌株。反应使用了上述二种引物,从分离的4株菌株中精制质粒DNA,决定插入片断的DNA序列。分析编码了这些碱基序列的氨基酸序列,存在着各个克隆皆与由bOGF-II蛋白质决定的氨基酸序列一致的阅读框架,其中之一种(克隆1#)供作以下的实验。
2)bOGF-II全长cDNA的克隆化
精制克隆1#的质粒,用限制酶EcoRI(宝酒造公司)将其切断,作琼脂糖凝胶电泳后,单个分离出约120bp的bOGF CDNA片断。
用Megaprime试剂盒(Amersham公司)及α32P-dCTP对此片断作32P标记,作为以下实验用的探针,从胎儿肺成纤维细胞IMR90聚ARNA5μg中,按Great Lengths cDNA Synthesis Kit(Clontech公司)的说明书所载方法,合成双链cDNA。聚A RNA用FastTrack(Stratagene公司)配制。以下简单说明双链cDNA的合成方法。
混合poly A RNA 5μg和Oligo(dT)2s(dN)引物,加入蒸馏水,最终容量作成12.5μl,在70℃下保温3分钟后,在冰中冷却2分钟。对此溶液再加入蒸馏水3.2μl,5×标准缓冲液(First-strandbuffer)5μl DTT(二硫代苏糖醇)0.5μl,dNTP(各20mM)1.3μl、MMLV(RNaseH-)2.5μl(500单位)在,42℃下保温90分钟。再加入蒸馏水123.5μl、5×第二条链缓冲液(second-s-frand buffer)40μl,dNTP(各20mM)1.5μl、第二条链缓冲液混合物(Second-strand enszyme cocktail)10μl,在16℃下保温2小时。对该反应液加入聚合酶15单位,再在16℃下保温30分钟后添加0.2M的ED-TA10μl,使反应停止,作氯仿、异戊醇处理后,乙醇沉淀。在该双链cDNA的末端加上EcoRI-Salt-NotI接头后,插入预先以限制酶EcoRI切断及作CIAP(牛精巢碱性磷酸酯酶)处理的ZAP表达的噬菌体(Stratagene公司)DNA中。包装所得组换噬菌体DNA,使其感染大肠杆菌XLI-Blue MRF’(Stratagent公司),在NZY琼脂培养基(0.5%NaCl,0.2%MgSO4·7H2O,0.5%酶提取物,1%NZ胺,pH7.5,1.5%琼脂)上形成菌落。包装使用了Gigapack II Goldpackaging extract(stratagene公司)。转印至Hybond-N(Amersham公司)上,固定噬菌体DNA。将所得的膜浸入含100μg/ml的鲑鱼精子DNA的杂交缓冲液(Amersham公司)中,在65℃处理4小时后,浸于含热变性的上述32P标记DNA探针(2.5×105cpm/ml)的上述缓冲液中,在65℃下保温一夜。洗净膜,可从约100万个噬菌体中选出带有bOGF-IICDNA的三个克隆。再进行二次筛选,纯化该三个克隆。使纯化的噬菌体感染大肠杆菌XLI-Blue MRF’之后,以春福寿草噬菌体ExAssist(Stratagene公司)进行多层感染,以其培养液上清液感染大肠杆菌XLOLR(Stratagene公司),得到对卡那霉素具抗异性的大肠杆菌。分析这些大肠杆菌中的质粒DNA的结构,决定插入片断的碱基序列。该序列示于序列表中第8号序列。又,据此,将bOGF-II的氨基酸序列示于序列表中序列编号9。将该氨基酸序列和天然型bOGF-II的氨基酸序列对比,可以看到,C末端的氨基酸(第85个的氨基酸)Lys多了一个。因此,从IMR-90培养液提纯的天然型bOGF-II也可能是C末端的赖氨酸残基)被羧肽酶切断的蛋白质。其结果,可以明白,三个克隆皆含有可对从bOGF-IIN末端Glu-Thr-Glu-Tyr中,由85个氨基酸组成的阅读框架进行编码的DNA,阅读框架由DNA序列中最初的停止密码的位置及来自bOGF-II蛋白质的氨基酸序列决定。所得质粒中的一个命名为pBK-CMV OGF-II(3)。
(2)bOGF-II cDNA的大肠杆菌中的表达
用PCR扩增,分离OGF-IIcDNA全长,设计所用的引物,使在用Q30F 5’…GGGGATCCGAGACAGAATATGGTC-3’Q30R5’-CCAAGCTTCTACTTGCTCTGCATACT-3’扩增后,可用限制酶Bam HI和HindIII切断。以pBK-CMV OGF-II(3)20mg作为模板,使用引物(Q30F和Q30R)进行PCR扩增、分离。反应溶液各由10XExTaq缓冲液10μl、25mM的dNTP8μl、Ex Taq 0.5μl,DNA溶液9.5μl、蒸馏水70μl、引物(各100μM)1μl作成最终容量为100μl。反应条件为:在95℃下作3分钟的保温后,反复进行96℃30秒、55℃30秒、72℃3分钟的循环反应25次,然后,在75℃、保温5分钟的条件下进行,扩增后,由Microcon 100(Amycon)去除引物,用限制酶BamHI和HindIII切断,再与预先以BamHI和HindIII切断的pQE30(QIAGEN公司)20g混合,供给连结反应。使用连结反应液转化大肠杆菌XL2-Blue(Stratagene公司)。从所得的对氨苄青霉素具抗异性的菌株中以限制酶切断图、碱基序列等进行分析,分离出带有目的物插入片断的克隆。将该克隆中的质粒命名为pQE30-OGF-II。具有所述质粒的大肠杆菌XL2-Blue(pQE30-OGF-II)寄存于日本通商产业省工业技术院生命工程工业技术研究所。寄存的大肠杆菌所具的质粒的结构示于图6。将该菌以Supper medi-um(2.5%的蛋白胨bactotryptone),1.5%的酵母抽提物(bacto-yeast extracti),0.5%NaCl,50μg/ml的氨苄青霉素)作振荡培养,在OD600nm为0.8时,添加IPTG(Isopronyl β-D-Thio-Galac-topylanoside),使其最终浓度为0.5mM,再继续进行20小时的振荡培养,以大肠杆菌产生出bOGF-II。培养完毕后,收集细菌,作SDS-PAGE凝胶电泳分析,确认分子量约为15KD的主要区带。该分子量与从IMR90的培养液上清液中所得到的bOGF-II的分子量非常相近。
(3)His6-bOGF-II的精制
为了对翻译bOGF-II基因而得的蛋白质的成骨细胞的增殖促进活性进行试验,试用QIAexpress kit(QIAGEN制),进行表达。该系统为在表达具有组氨酸6聚体的蛋白质后,使用具有与该组氨酸6聚体高的亲和性的镍螯合-氰基三乙酸树脂柱,进行精制的系统。该系统为通常用于更有效地提纯表达的蛋白质的系统。使用该系统,则可以在N-末端上具有组氨酸6个的区域的形式(His6-bOGF-II)翻译并检出bOGF-II。对以IPTG加以诱导的大肠杆菌菌体0.35g加入含8M脲和0.1M磷酸钠的10mM Tris-HCl,pH8.0,1.75ml,搅拌至均匀状态,然后,在室温下再搅拌1小时。搅拌后,在室温下以12000的转速离心15分钟,收集上清液。将该上清液加于用8M脲和0.1M pH8.0的磷酸钠的10mM Tris-HCl平衡的镍螯合一硝基三乙酸树脂(nickel-chelating nitrilo triacetic acidresin,QIAGEN制)的50%的悬浊液8ml中,在室温下搅拌45分钟。将该树脂移入内径1.6cm的柱内,用含20ml的8M脲和0.1M的磷酸钠的10mM的pH8.0的Tris-HCl,流速约0.5ml/分洗净。其次,用含8M脲和0.1M磷酸钠的10mM的、pH6.3的Tris-HCl,流速为约0.5ml/分再次洗净所述树脂柱。最后,以含250mM的咪唑、8M的脲及0.1M的磷酸钠的10mm,pH6.3的Tris-HCl,流速约0.5ml/分溶出目的物的His6-bOGF-II。提纯的His6-bOGF-II成分透析于磷酸盐缓冲液生理食盐水中。
(4)His6-bOGF-II的成骨细胞增殖促进活性
用实施例1(2)的成骨细胞增殖促进活性的测定方法测定His6-bOGF-II的成骨细胞增殖促进活性。即,作为试样,添加上述的提纯的His6-bOGF-II成分至含量达10%,进行3H-胸腺定对小鼠成骨细胞株MC 3T3-E1细胞的参入试验。其结果示于图7。从该结果可以确认,本发明者们鉴定为bOGF-II基因的基因的翻译产物的蛋白质具有与天然型bOGF-II相同的成骨细胞增殖促进活性。
根据本发明,提供了一种具有成骨细胞增殖活性的新颖的蛋白质及其有效的制造方法,由于本发明的蛋白质具有成骨细胞增殖活性,可用于治疗骨质疏松症等各种骨质减少性疾病的治疗剂,或者,用于上述疾病的免疫学的诊断。
关于寄存的微生物的说明
寄存本发明微生物的寄存单位名称及地址:
日本通商产业省工业技术院生命工程工业技术研究所
日本茨城县筑波市东1丁目1番3号
寄存日:
1995年5月26日
寄存号:
FERM BP-5139
上述微生物于1995年6月19日根据布达佩斯条约移存(寄存号:微工研菌FERM P-14942号)。
序列号:1序列长度:25序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Glu Thr Glu Tyr Gly Pro Xaa Arg Arg Glu Met Glu Asp Thr Leu Asn1 5 10 15His Leu Lys Phe Leu Asn Val Leu Ser
20 25序列号:2序列长度:40序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn1 5 10 15His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val Xaa Ile Pro
20 25 30Asn Cys Xaa Lys Lys Gly Phe Tyr
35 40序列号:3序列长度:9序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Asp Val His Cys Tyr Ser Met Gln Ser1 5序列号:4序列长度:12序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu1 5 10序列号:5序列长度:16序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu1 5 10 15序列号:6序列长度:25序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly1 5 10 15Arg Lys Arg Gly Phe Cys Trp Cys Val
20 25序列号:7序列长度:22序列类型:氨基酸链的数目:1同源性:直链状序列的种类:肽序列Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly1 5 10 15Val His Ile Pro Asn Cys
20序列号:8序列长度:258序列类型:核酸链的数目:2同源性:直链状序列的种类:cDNA→mRNA来源:人序列的特征表示特征的符号:存在位置:1.258确定特征的方法:E序列GAG ACA GAA TAT GGT CCC TGC CGT AGA GAA ATG GAA GAC ACA CTG AAT 48CAC CTG AAG TTC CTC AAT GTG CTG AGT CCC AGG GGT GTA CAC ATT CCC 96AAC TGT GAC AAG AAG GGA TTT TAT AAG AAA AAG CAG TGT CGC CCT TCC 144AAA GGC AGG AAG CGG GGC TTC TGC TGG TGT GTG GAT AAG TAT GGG CAG 192CCT CTC CCA GGC TAC ACC ACC AAG GGG AAG GAG GAC GTG CAC TGC TAC 240AGC ATG CAG AGC AAG TAG 258序列号:9序列长度:85序列类型:氨基酸链的数目:1同源性:直链状序列的种类:蛋白质序列Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn1 5 10 15His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro
20 25 30Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser
35 40 45Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln
50 55 60Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr65 70 75 80Ser Met Gln Ser Lys ***
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Claims (16)
1.具有如下特征的蛋白质:
(a)在还原和非还原条件下,在十二烷基磺酸钠-聚丙烯酰胺凝胶电泳中分子量为约15KD;
(b)对阳离子交换体和肝素具有强的亲和性;
(c)具有成骨细胞增殖活性;
(d)70℃加热处理10分钟,成骨细胞增殖活性降低,90℃加热处理10分钟则失活。
2.按权利要求1所述的蛋白质,其来源为人成纤维细胞。
3.按权利要求1或2所述的蛋白质,其N-末端氨基酸序列特定为序列表中序列号1。
4.按权利要求1或2所述的蛋白质,其N-末端氨基酸序列特定为序列表中序列号2。
5.一种cDNA,其特征在于编码特定为序列表中序列号9的氨基酸序列。
6.一种蛋白质,其特征在于全氨基酸序列特定为序列表中序列号9,并具有成骨细胞增殖活性。
7.一种蛋白质,其特征在于含有序列表中以序列号9特定的氨基酸序列作为部分序列,并具有成骨细胞增殖活性。
8.一种蛋白质,其特征在于与序列表中以序列号9特定的氨基酸序列具有80%以上同源性,并具有成骨细胞增殖活性。
9.一种cDNA,其特征在于编码与序列表中以序列号9特定的氨基酸序列有80%以上同源性并具有成骨细胞增殖活性的蛋白质的氨基酸序列。
10.具有如下特征的蛋白质的制备方法,
(a)在还原和非还原条件下,在十二烷基磺酸钠-聚丙烯酰胺凝胶电泳中分子量为约15KD;
(b)对阳离子交换体和肝素具有强的亲和性;
(c)具有成骨细胞增殖活性;
(d)70℃加热处理10分钟,成骨细胞增殖活性降低,90℃加热处理10分钟则失活,
该方法的特征在于:培养人成纤维细胞,将培养液作肝素柱层析处理,洗脱吸附部分,将此部分上阳离子交换柱,再用肝素层析柱处理。
11.按权利要求10所述的蛋白质的制备方法,其所得蛋白质为与序列表中以序列号9特定的氨基酸序列具有80%以上同源性,并具有成骨细胞增殖活性的蛋白质。
12.一种宿主细胞,其特征在于:用含有编码序列表中以序列号9特定的氨基酸序列或与其有80%以上同源性、具有成骨细胞增殖活性的蛋白质的氨基酸序列的cDNA的表达质粒转化。
13.按权利要求12所述的宿主细胞,其特征还在于是来自哺乳动物、细菌、酵母或昆虫的细胞。
14.按权利要求13所述的宿主细胞,其特征在于该宿主细胞为大肠杆菌。
15.按权利要求14所述的宿主细胞,其特征还在于大肠杆菌为pQE30-OGFII(保藏号FERM BP-5139)。
16.蛋白质的制备方法,其特征在于:用含有编码序列表中以序列号9特定的氨基酸序列或与其有80%以上同源性、具有成骨细胞增殖活性的蛋白质的氨基酸序列的cDNA的表达质粒转化宿主细胞,培养宿主细胞,从培养物中提取权利要求1、3、4、6、7和8记载的任一蛋白质。
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CA2422625A1 (en) * | 2000-09-19 | 2002-03-28 | Bioexpertise, Llc | Method for use of igf-binding protein for selective sensitization of target cells in vivo |
US20050107350A1 (en) * | 2003-08-22 | 2005-05-19 | Pharmacia Corporation | Method for the treatment or prevention of bone disorders with a cyclooxygenase-2 inhibitor alone and in combination with a bone disorder treatment agent and compositions therewith |
US20220259265A1 (en) * | 2019-07-30 | 2022-08-18 | The University Of Sydney | Inhibitors and Use Thereof in Cancer Treatment |
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US5200509A (en) * | 1987-04-06 | 1993-04-06 | Celtrix Pharmaceuticals, Inc. | Human somatomedin carrier protein subunits and process for producing them; recombinant DNA molecules, hosts, processes and human somatomedin carrier protein-like polypeptides |
US5258287A (en) * | 1988-03-22 | 1993-11-02 | Genentech, Inc. | DNA encoding and methods of production of insulin-like growth factor binding protein BP53 |
-
1995
- 1995-06-26 EP EP95922759A patent/EP0725080A1/en not_active Withdrawn
- 1995-06-26 HU HU9600395A patent/HU220130B/hu not_active IP Right Cessation
- 1995-06-26 CN CN95190587A patent/CN1129944A/zh active Pending
- 1995-06-26 AU AU27535/95A patent/AU2753595A/en not_active Abandoned
- 1995-06-26 NZ NZ288352A patent/NZ288352A/en unknown
- 1995-06-26 CA CA002169953A patent/CA2169953A1/en not_active Abandoned
- 1995-06-26 US US08/604,965 patent/US6046033A/en not_active Expired - Fee Related
- 1995-06-26 WO PCT/JP1995/001270 patent/WO1996000240A1/ja not_active Application Discontinuation
- 1995-06-26 KR KR1019960700946A patent/KR960703943A/ko not_active Application Discontinuation
- 1995-06-27 ZA ZA955319A patent/ZA955319B/xx unknown
- 1995-06-27 IL IL11435695A patent/IL114356A0/xx unknown
-
1996
- 1996-02-16 FI FI960706A patent/FI960706A/fi unknown
- 1996-02-26 NO NO960766A patent/NO960766L/no not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
WO1996000240A1 (fr) | 1996-01-04 |
EP0725080A1 (en) | 1996-08-07 |
AU2753595A (en) | 1996-01-19 |
FI960706A (fi) | 1996-04-12 |
KR960703943A (ko) | 1996-08-31 |
FI960706A0 (fi) | 1996-02-16 |
HU220130B (hu) | 2001-11-28 |
MX9600765A (es) | 1997-07-31 |
NO960766L (no) | 1996-04-24 |
HUT76748A (en) | 1997-11-28 |
ZA955319B (en) | 1996-12-27 |
HU9600395D0 (en) | 1996-04-29 |
IL114356A0 (en) | 1995-10-31 |
NZ288352A (en) | 1997-12-19 |
US6046033A (en) | 2000-04-04 |
CA2169953A1 (en) | 1996-01-04 |
NO960766D0 (no) | 1996-02-26 |
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