CN101041819A - 人红血球生成素基因在稳定转染的哺乳动物细胞中的高水平表达 - Google Patents
人红血球生成素基因在稳定转染的哺乳动物细胞中的高水平表达 Download PDFInfo
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Abstract
一种表达重组人红血球生成素的方法,包括用主要含有人红血球生成素基因的2.4kb Apa I限制片段的DNA、RNA或核苷酸序列转染宿主细胞,使转染细胞与培养基相接触,表达并回收红血球生成素。以及一种通过与保温的培养基接触的细胞系表达重组人红血球生成素的方法,包括在所说的方法中加入能在保温培养基中产生红血球生成素的细胞系,所说的细胞系通过用主要含有人红血球生成素基因的2.4kb Apa I限制片段的DNA、RNA或核苷酸序列转染宿主细胞系产生。
Description
本申请是申请号为98115963.X,申请日为87年6月26日,发明名称为“制备相当于人红血球生成素基因片段的核苷酸序列的方法”的发明专利申请的分案申请。
本发明一般地涉及基因工程领域,具体说与重组基因的糖蛋白产物表达有关,更具体地说与从稳定转染的细胞中高水平地表达有生物活力的人红血球生成素有关。
红血球生成素这一激素在调节红血球生成、红血细胞的形成以及由贫血导致的红血球生成素缺乏中起着重要作用。由于缺少纯的材料,对这个激素做详细研究以及试图用它进行替补治疗一直都很困难。
人红血细胞的产生,需要肾脏分泌成熟糖蛋白形式的红血球生成素。在恒态时,这个激素在循环血液中浓度为每毫升10至18毫单位(128-230微微克),在严重的组织供氧不足(缺氧)刺激下,它的水平可以增加一千倍。提高激素水平可触发骨髓中感受干细胞群的增生和分化,激活成熟红细胞中血红蛋白的合成,加速红细胞从骨髓释放进入循环,从而增加红细胞量,改善供氧不足的局面。缺乏红血球生成素的病人如慢性肾脏病人,常患有严重的贫血症。
红血球生成素是一个34-38千道尔顿的糖蛋白,分子量的约40%是糖类提供的。至少一个二硫桥是活力所需的,对此激素的结构所知甚少,其合成的详情也未全清楚。最近分离到的CDNA和基因组克隆,提供了分析红血球生成素产生的调节控制的机会,但是,还没有表达足够数量的有生物活力的人红血球生成素以用于替补治疗。
按照本发明公开的内容,有生物活力的人红血球生成素可以由稳定转染的哺乳动物细胞系进行高水平表达(每升上清液超过二百万名义滴度单位),这就为临床应用纯的人红血球生成素提供了巨大的来源。红血球生成素的这一惊人的高水平表达,是通过用人红血球生成素基因的Apa I限制片段转染宿主细胞系实现的。Apa I限制片段的意义链有图1所示的核苷酸序列。
图1表示含有人红血球生成素基因序列的2426个碱基对的Apa I限制片段;
图2绘出了含有2426个碱基对的Apa I限制片段的有代表性的质粒表达载体(pD11-Ep);
图3描绘的是另一个带有Apa I限制片段的表达载体(pBD-Ep)。
在较好的实施方案中,如图1所示的通过基因工程构建的Apa I限制片段被插入如图2和图3所示的哺乳动物表达载体中,然后将表达载体引入哺乳动物细胞系以发展稳定转染的细胞,产生大量有生物活力的人红血球生成素。所选择的人红血球生成素基因的Apa I限制片段要能最高效率地转录红血球生成素信使RNA,有效地转译RNA和进行转译后的修饰,以生成具有生物活力的成熟红血球生成素糖蛋白。具体地说,重要的是将红血球生成素基因5′端干扰序列除去,而保留增强子序列。为了保存有潜在价值的增强子序列,Apa I限制片段中的内含子也被保留。在基因的3′端某些3′非转译序列也被保留,使推测的调控序列最适化。由Apa I片段提供的增强表达为以下实例所证实,在下述实例中,该片段与两个启动子和两个细胞系合用均得到稳定的高水平表达的红血球生成素。
提出下列实例是用以阐述发明的优越性,并帮助普通的专门技术人员掌握和应用本发明。这些实例无论如何都不是企图限制本发明公开的范围,或本专利证书所提供的保护。
实例1.基因克隆的提取
在入噬菌体中构建的人基因组文库(Cell,15:1157-1174,1978)用低严紧度杂交条件及在Cell,38:287-297,1984(特此引证)中所述的寡核苷酸探针混合物进行筛选
寡核苷酸混合物系用Applied Biosystems合成仪制备,用32P-ATP及T4多核苷酸激酶作末端标记。设计的合成寡核苷酸相应于氨基末端部分的氨基酸序列:
H2N-Ala-Pro-?-Arg-Leu-Ile-Leu-Asp-Ser-Arg-Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-?-Ile-Thr-Asp-Gly-Gly-Ala
24此序列得自Yanagawa等人(J.Biol.Chem.259:2707-2710,1984)从再生障碍贫血病人尿中纯化的人蛋白质。为了减少这段氨基酸序列密码子的简并程度,采用了Grantham等(Nu-cleic Acids Research,8:43-59,1981)和Jaye等(Nucleic Acids Research,11:2325-2335,1983)的密码子使用规律。这些规律考虑到了脊椎动物DNA中二核苷酸CpG相对地稀少,和适当地避免A:G潜在错配。第24位氨基酸最可能是门冬酰胺(J.Biol.Chem 259:2707-2710,1984)。对于氨基酸序列谷-丙-赖-谷-丙-谷-门冬酰胺,2组各72种相应于预期的密码子序列被合成,第一组是20核苷酸探针,为TT(c/t)TC(a/g/t)GC(c/t)TC(c/t)TT(a/g/t)-GCTTC,第二组在第18位将T换为C。对于氨基酸序列谷-门冬酰胺-异亮-苏-门冬-甘,构建一组序列为AGC TCC TCC ATC AGT ATT ATT T(c/t)的23核苷酸探针。
用寡核苷酸探针杂交到的噬菌斑,于低密度下重筛选直到纯化。在起始的阳性噬菌体克隆通过噬菌斑纯化后,Jacobs等发表了红血球生成素基因的进一步的序列资料(Nature,313:806-810,1985)。根据这一资料构建了寡核苷酸,并用于验证阳性克隆,用EcoRI限制性酶酶解阳性克隆,用凝胶电泳纯化其插入片段DNA,再用标准技术连结入预先用EcoRI限制性酶解的pUC13质粒中。借助双脱氧核苷酸链终止法测定DNA序列(Proc.Natl.Acad.Sci.USA,74:5463-5467,1977),此测定使用dATP(α-35S)和通用的17核苷酸引物,或在选定的区域使用特殊的寡核苷酸引物进行。32P-ATP从ICN获得,酶从New EnglandBiolabs或Bethesda Research Laboratories获得。
大约4.8×106个噬菌体被双份硝酸纤维素滤膜杂交法筛选。三个不同的克隆在噬菌斑纯化过程中保持阳性,其DNA插入片段作限制图谱和部分双脱氧序列测定。三个克隆中有两个显然含有红血球生成素基因的全部信息。这些克隆的限制图谱及其2426个碱基对的Apa I片段的序列(见图1)与最近Jacobs等发表的人红血球生成素基因序列(Nature,313:806-810,1985)基本相同。在这个增殖的文库中提出的含有红血球生成素基因的噬菌体频度之低-约2×106个噬菌体中有1个-与红血球生成素基因在人染色体基因组中以单拷贝存在的说法相符。用红血球生成素基因的Apa I片段或其它限制片段与总人染色体DNA作Southern吸印杂交,只显现单一杂交区带,没有其它高度同源的DNA区域。
实例2.ApaI限制片段的选择
为构建表达载体,红血球生成素基因的Apa I限制片段按Proc.Natl.Acad.Sci.USA 74:5463-5467,1977所述的技术(特此引证)制备得到。简言之,提取的噬菌体DNA用限制酶Apa I(Bethesda Research Laboratories)酶解,然后在1%琼脂糖凝胶上分离,电泳洗脱,酚抽提和乙醇沉淀。此片段通过如实例1中的部分测序法验证。
参照图1,插入的Apa I限制片段含有58个碱基对的5′非翻译序列(0001-0058核苷酸),后面是推测的27个氨基酸的信号肽编码序列、成熟蛋白、四个推测的插入序列和222个碱基对的3′非编码DNA序列。在基因的5′端,Apa I位点是在蛋白序列的起始密码子ATG(0058)上游58个碱基对处。选择这个5′端位点(0001)是为了避免紧靠Apa I限制位点上游的一个假起始点,同时又保存了被认为是对核糖体有效地结合和加工很重要的调控序列。选择3′端的Apa I位点(2426),以在大多数3′非翻译区保留推测的加工信号,并除去更下游的序列。使用完整的红血球生成素基因,包括其内含子序列,是为了保留在内含子中潜在的调节和增强子序列,它们可能在红血球生成素基因的表达或蛋白质的修饰和分泌方面发挥作用。
实例3.带有Apa I限制片段的表达质粒的构建
包含完整的人红血球生成素基因的2426碱基对的Apa I限制片段被插入两个表达载体pD11和pBD,二者分别基于不同的哺乳动物启动子。
质粒表达载体pD11是从前述的质粒衍生的(Nucleic AcidsResearch,13:841-857,1985,此处特予引证),含有猴肾病毒40(SV 40)的增强子序列和复制起始区,以及腺病毒2的主要晚期启动子和三重引导序列。人红血球生成素基因组序列的Apa I片段(图1)为凝胶纯化,单链末端用T4 DNA聚合酶补齐,用Bam HI连接子连接二个平末端,将组建物插入pD11的单一Bam HI限制位点,以从一个强启动子直接转录红血球生成素的基因。
组建的带有Apa I片段(Ep)的表达质粒pD11-Ep的结构绘如图2。质粒pD11是由PML的EcoRI(RI)位点插入以下成分所构成,即:含有350碱基对的腺病毒左侧末端(0-1),SV-40复制起始区和增强子序列(E),腺病毒的主要晚期启动子(MLP),腺病毒2的三重引导序列(L1-3),第三个引导序列的5′拼接位点(5′SS),免疫球蛋白的3′拼接位点(3′SS),以及SV-40晚期多聚腺苷酸信号(pA)。重组的质粒在E.Coli HB101中克隆,氯化铯等密度离心纯化,表达质粒pD11-Ep大约为6500碱基对,构建产物为限制图谱及部分双脱氧测序法验证。
pBD含有金属硫蛋白I启动子序列(MT-1,Glanville,Durham and Palmiter,Nature,292:267-269,1981特此引证)和带有二氢叶酸还原酶(DHFR)选择标记序列的pUC质粒。参照图3,此2426碱基对的Apa I限制片段(Ep)插入pBD的唯一SmaI限制位点,形成表达质粒pBD-Ep。pBD-EP的DHFR序列与SV-40的复制起始序列和增强子序列以及肝炎病毒B表面抗原聚腺苷酸序列相连。pBD-Ep的构建基本如上述的pD11-Ep。
虽然在这些确证实验中使用质粒载体,但也设想了改用病毒或逆病毒表达载体,以相似的方式将Apa I红血球生成素基因片段引入寄主细胞系。适合此目的的DNA病毒包括腺病毒或BPV(牛乳头状瘤病毒),而合适的逆病毒也是已知的和现成的。很清楚这样的逆病毒(RNA)载体将携带Apa I限制片段的反义链,即相应于图1的意义链的RNA转录产物序列,或其等价物。逆病毒载体亦将包括将病毒基因组反转录及将病毒DNA整合入寄主基因组所需的跨染色体作用因子的序列。
实例4.哺乳动物细胞的转染
二种哺乳动物细胞系,各用pD11-Ep和pBD-Ep转染。哺乳动物细胞系COS-7(猴肾)和BHK(幼大鼠肾)保存在含10%胎牛血清的改良Dul becco基本培养基中。细胞传代至50-70%融合时,用磷酸钙法转染(Virology,52:456-467,1973)。BHK系从肾上皮细胞而来,一般认为它们是最可能在哺乳动物贫血或缺氧时产生出天然状态红血球生成素的,因此,这些细胞有可能识别红血球生成素Apa I片段上关键性的调控序列,有效地加工和产生出红血球生成素糖蛋白。
为做细胞的临时表达,在100毫米培养皿中加入20微克总DNA,其中10微克带有红血球生成素基因的pD11-Ep质粒,及10微克载体鲑鱼精子DNA,48小时后,收集上清液,400g离心10分钟,除去细胞及颗粒,于-20℃冰冻,细胞亦分别收集,单独用pD11-EP转染临时表达结果见表1,数据是每型细胞各三次实验而来。
表1
哺乳动物细胞 | 每毫升培养物中红血球生成素 | |
蛋白(微克) | 单位(体外生物检测) | |
BHKCOS-7 | 3.4±0.23.2±0.4 | 270±16255±32 |
观察到的哺乳动物细胞系COS-7或BHK分泌到上清液中的红血球生成素,比以前报导的编码红血球生成素cDNA或用整个红血球生成素基因作的临时表达都要高约80倍。
为建立产生高水平红血球生成素的稳定细胞系,COS-7或BHK细胞用pD11-EP和pDHFR-1a质粒同时转染(后者为包含二氢叶酸还原酶cDNA的相似的哺乳动物表达载体)。转染步骤改为用5微克pD11-EP质粒,5微克pDHFR-1a质粒和10微克载体DNA做共同转染,继续保温18-24小时,然后将不同浓度的氨甲蝶呤(10毫微摩尔/升至1毫摩尔/升)加入培养基。掺入DHFR基因的细胞在此选择培养基上成活。当保温数天后,分离出抗氨甲蝶呤的集落,传代及筛选上清液中红血球生成素活力,被检测的抗氨甲蝶呤的集落中,大约一半分泌有可测得的红血球生成素活力。
为用表达载体pBD-EP建立稳定的细胞系,用磷酸钙法转染BHK细胞,18-24小时后,培养基中加入1微摩尔/升至1毫摩尔/升的氨甲蝶呤,培养数天后,分离出在较高氨甲蝶呤中存活的集落,传代和筛选,在这一系列中,所有被检测的抗氨甲蝶呤的集落都分泌有可测得的红血球生成素的活力。
为了使含有红血球生成素基因和DHFR基因的转录单位表达最适化,将含有pBD-Ep或pD11-EP的分泌高水平红血球生成素的各BHK细胞系在浓度递增的氨甲蝶呤中传代数次(Nature,316:271-273,1985)。然而,不是通过氨甲蝶呤浓度的小间隔逐步增加对细胞系施加选择压力,而是使这些细胞立即受到浓度非常高的氨甲蝶呤(即1毫摩尔/升)的挑战。仅仅极少数细胞存活下来,但这些细胞中质粒组建物掺入到DNA中特别有利于表达的部位(所谓热点)和/或拥有许多组建的转录单位的复本。因此,一步就可以选择到最高产的细胞系,假若在缺少氨甲蝶呤选择压力下传15代以上,仍保持红血球生成素的高产,这些细胞系(包括在表2所列的F7.2和S5.2)可以看做是稳定的了。
不能在细胞颗粒中定量红血球生成素的活力,因为细胞抽提物中有许多抑制检测的因子。所以表2所列的结果只是细胞系产生和分泌到上清液中的红血球生成素蛋白,没有分析胞内的红血球生成素的水平。
表2
从稳定转染的BHK细胞系中
表达的重组红血球生成素
细胞系pBD-EP | 每毫升上清液中的红血球生成素 | |
蛋白(微克) | 单位(体外生物检测) | |
F1.1F3.4F6.1F7.2 | 12.432.079.684.1 | 970250062106728 |
表2(续)
细胞系pD11-EP | 每毫升上清液中的红血球生成素 | |
蛋白(微克) | 单位(体外生物检测) | |
S1.2S2.4S5.2 | 6.464.282.1 | 50050006400 |
观察到的红血球生成素分泌相当于高达每升七百万单位的名义产率。这一名义产率是将观察到的每毫升产量乘一千倍得到的,以给出预期的大规模生产时每升的产量。
使用Apa I片段所观察到的红血球生成素产量,比早先报道的使用不同的人红血球生成素基因片段稳定转化的CHO细胞系得到的产量(Lin等,Proc.Natl.Acad.Sci.USA,82:7580-7584,Nov.1985)高三百倍以上。
这些转染物测定中的对照实验,包括非转染细胞的上清液,和用编码其它蛋白质—包括细菌的氯霉素转乙酰酶和人凝血因子IX-的DNA转染的细胞平行培养物的上清液,这些对照培养物,模拟的转染或用其它基因转染的培养细胞,均无可测得的红血球生成素活力。也须指出,上述各红血球生成素基因的实验中,从选择出的细胞系获得的表达水平,与伴随红血球生成素基因的选择性标记是与之共同感染,还是在感染前先插入含Apa I片段的质粒,并无关系。
适于实用于此发明的其它有代表性的感染方法,包括DEAE-葡聚糖中介的转染技术,溶菌酶融合法或红细胞融合法,刮削法,直接摄入法,渗透休克或葡糖休克法,直接微注射或间接微注射法,诸如红细胞中介的技术和/或把宿主细胞置于电流中等方法。转染是指将遗传信息,具体地说是将人红血球生成素基因Apa I限制片段的编码信息,利用提取的DNA、RNA或合成的核苷酸多聚物转移入细胞内,上面所列的转染技术并不能算已经完善,因为把遗传信息导入细胞的其它手段无疑将会发展出来。在进行转染之前,典型地说,Apa I限制片段通常可以接于(连接到)其它的核酸的序列,诸如启动子,增强子和聚腺苷酸序列。虽然叙述了哺乳动物来源的各宿主细胞系,并特别提到了肾脏上皮细胞,但也考虑到了本发明可以使用其它的真核和原核(细菌或酵母)宿主细胞系。最近将哺乳动物基因引入植物细胞的成功,提供了利用植物和藻类细胞的潜在可能性。
实例5.从转染的细胞系表达红血球生成素
分泌到转染的细胞系上清液中的红血球生成素是有生物活力的,分泌出大量的激素高达每毫升七千单位。
红血球生成素活力的体外测定是根据小鼠骨髓细胞在血浆凝块培养基上红色集落的形成(CFU-E,红色集落形成细胞)(BloodCells,4:89-103,1978)这个测定的灵敏度约为5毫单位/毫升。作为测定标准的红血球生成素是由贫血绵羊血浆部分纯化得到的制剂(Connaught,第三步的EP,批号3026),用于测定的是在不含氨甲蝶呤的新鲜培养基上生长24小时的传代细胞系的上清液,每毫升含2×105个骨髓细胞、10%加柠檬酸盐处理的牛血清、20%胎牛血清、1%牛血清白蛋白和1.6%胎牛提取物(Gibco)的测定培养基中,加入1至10微升用培养基按1∶200稀释的上清液,培养36至48小时后,血浆凝块在载玻片上固定,用联苯胺做血红蛋白染色,数红细胞集落的数目。当不加红血球生成素时,测不到由CFU-E产生的集落。通常,每毫升培养物用50毫单位的红血球生成素(0.64毫微克)能够观察到最合适的集落生长(每2×104个骨髓细胞可测得100-150个CFU-E)。如表1和表2所示,大量的红血球生成素激素分泌到转染细胞系上清液中,高达每毫升7000单位,假设重组的红血球生成素的比活力与天然红血球生成素等价(每毫克蛋白质78000单位),此生物测定结果即相当于每毫升大约80微克的红血球生成素蛋白质。
此外,使用多价的抗人红血球生成素兔抗血清(J.Cell.physiol.118:87-96,1984)做竞争性放射免疫测定,以检测选择出的细胞系上清液中有免疫反应活力的红血球生成素。用放射免疫测定检测到的蛋白量,与生物检测估计的蛋白水平相等。这些数据说明转染细胞系表达和分泌的红血球生成素蛋白约98%以上是有活力的。
对转染的细胞产生的重组红血球生成素做进一步的检查,以证实这些细胞分泌的确是天然的激素,选择出的细胞系被用在重度缺氧引起红细胞增生的小鼠中做红血球生成素的体内检测(Nature,191:1069-1087,1961)。细胞系分泌的上清液用重度缺氧引起红细胞增生的小鼠检测,有着强烈的体内生物活力。在应用部分纯化的天然红血球生成素做的实验中,早就注意到,当在整体动物中检测时,唾液酸酶的处理可以完全消除红血球生成素的活力(J.Biol.Chem.247:5159-5160,1958)。此活力的丧失推测是由于肝脏对去唾液酸激素的清除增强的结果,因为唾液酸酶处理的红血球生成素在体外检测可保有其全部活力。所观察到的体内的强烈的生物活力,说明了转染的哺乳动物细胞系在转译后修饰阶段,在红血球生成素蛋白上添加上了适当的糖和末端唾液酸。
在分开的实验中,体外生物检测到的红血球生成素的活力,亦为加到培养基中的抗人红血球生成素抗体所中和。
那些有代表性的转染细胞系分泌到上清液中的红血球生成素,也检测了它们对其它骨髓祖代细胞的增生效应。检测重组的红血球生成素对人和小鼠骨髓来源的各种祖代细胞的效应,包括:红色群落生成细胞(CFU-E)、红色突发形成细胞(erythroid burst-forvming cells,EFU-E)、粒细胞—巨噬细胞前体(CFU-GM)以及混合细胞群落生成细胞(CFU-Mix)(J.Cell.Physiol.Suppl.1:79-85,I982;J.Cell.Phy-siol.118:87-96,1984)。红色干细胞对重组红血球生成素显示的增生效应,与对天然红血球生成素显示的效应有平行的剂量关系。由对于CFU-GM和CFU-MiX,重组红血球生成素的浓度增加到每毫升检测细胞培养物10个单位,都不显示任何增生效应。
在还原的或非还原条件下做SDS-PAGE分析时,纯化的重组红血球生成素与再生障碍贫血病人尿中纯化的红血球生成素电泳情形相同。这些蛋白显示出同样的微观不均一性,其主要成分都在分子量34千道尔顿处。
尽管本发明与提出的实施例一起叙述,普通专门技术人员在阅读了上述实例后将能够做出各种修改,使用等价物取代,或对上面提到的组分和方法做其它的更改。因此,我们打算把这一专利证书的保护范围仅仅由所附的权利要求及其等价部分所定义的内容加以限制。
Claims (17)
1.基本上由人基因组红细胞生成素基因Apa I 2.4kb限制片段组成的重组DNA。
2.权利要求1的重组DNA,具有图1A和1B所示的核苷酸序列。
3.含有基本上由人基因组红细胞生成素基因Apa I 2.4kb限制片段组成的插入片段的重组DNA构建体。
4.由权利要求3的重组DNA构建体转化的真核宿主细胞。
5.权利要求4的真核宿主细胞,其为哺乳动物细胞。
6.权利要求5的真核宿主细胞,其为幼仓鼠肾细胞。
7.基本上由相应于人红细胞生成素基因Apa I限制片段的核苷酸序列组成的基本上纯化的DNA或RNA。
8.权利要求7的DNA或RNA,其中Apa I限制片段基本上由图1所示核苷酸序列的有义链或其互补RNA序列组成。
9.与能够实现其表达的第二核酸序列有效连接的权利要求7的DNA或RNA序列。
10.权利要求9的DNA或RNA,其中第二核酸序列选自以下一个或多个:启动子序列、增强子序列、聚腺苷酰化序列、可选择标记序列、质粒、病毒和逆转录病毒表达载体和逆转录病毒反式作用因子。
11.含有权利要求9的DNA或RNA的细胞。
12.权利要求11的细胞,其中细胞用所述DNA或RNA稳定转染。
13.权利要求12的细胞,其中细胞选自真核细胞和细菌。
14.权利要求13的细胞,其中真核细胞是酵母细胞。
15.权利要求13的细胞,其中真核细胞是肾来源的。
16.权利要求15的细胞,其中肾细胞是上皮细胞。
17.由权利要求1的方法制备的、具有红细胞生成调节活性的糖蛋白。
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-
1987
- 1987-06-17 DK DK198703093A patent/DK173067B1/da not_active IP Right Cessation
- 1987-06-22 CA CA000616544A patent/CA1341361C/en not_active Expired - Fee Related
- 1987-06-23 WO PCT/US1987/001459 patent/WO1988000241A1/en active IP Right Grant
- 1987-06-25 AT AT87305672T patent/ATE76431T1/de not_active IP Right Cessation
- 1987-06-25 ES ES198787305672T patent/ES2037083T3/es not_active Expired - Lifetime
- 1987-06-25 EP EP87305672A patent/EP0255231B1/en not_active Expired - Lifetime
- 1987-06-25 DE DE8787305672T patent/DE3779206D1/de not_active Expired - Lifetime
- 1987-06-26 BR BR8703269A patent/BR8703269A/pt not_active Application Discontinuation
- 1987-06-26 CN CN87104424A patent/CN1044133C/zh not_active Expired - Lifetime
- 1987-06-26 AU AU74757/87A patent/AU611088B2/en not_active Expired
- 1987-06-26 CN CNA2006101006871A patent/CN101041819A/zh active Pending
- 1987-06-26 PT PT85193A patent/PT85193B/pt not_active IP Right Cessation
- 1987-06-27 KR KR1019870006561A patent/KR970009935B1/ko not_active IP Right Cessation
- 1987-06-27 JP JP62160799A patent/JPS63126488A/ja active Pending
-
1988
- 1988-02-26 NO NO880863A patent/NO303398B1/no not_active IP Right Cessation
- 1988-02-26 FI FI880899A patent/FI95393C/fi not_active IP Right Cessation
-
1992
- 1992-05-26 GR GR920401043T patent/GR3004707T3/el unknown
-
1993
- 1993-10-06 US US08/132,489 patent/US5688679A/en not_active Expired - Lifetime
-
2001
- 2001-10-10 US US09/975,063 patent/US6867020B2/en not_active Expired - Fee Related
- 2001-11-05 US US10/011,858 patent/US6682910B2/en not_active Expired - Fee Related
Also Published As
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---|---|
FI95393C (fi) | 1996-01-25 |
PT85193A (en) | 1987-07-01 |
AU611088B2 (en) | 1991-06-06 |
DE3779206D1 (de) | 1992-06-25 |
EP0255231A1 (en) | 1988-02-03 |
US6867020B2 (en) | 2005-03-15 |
KR880007726A (ko) | 1988-08-29 |
CA1341361C (en) | 2002-05-21 |
ES2037083T3 (es) | 1993-06-16 |
DK309387D0 (da) | 1987-06-17 |
DK309387A (da) | 1987-12-28 |
FI880899A (fi) | 1988-02-26 |
EP0255231B1 (en) | 1992-05-20 |
CN87104424A (zh) | 1988-04-27 |
DK173067B1 (da) | 1999-12-13 |
WO1988000241A1 (en) | 1988-01-14 |
GR3004707T3 (zh) | 1993-04-28 |
KR970009935B1 (ko) | 1997-06-19 |
US6682910B2 (en) | 2004-01-27 |
NO880863L (no) | 1988-04-26 |
ATE76431T1 (de) | 1992-06-15 |
NO303398B1 (no) | 1998-07-06 |
JPS63126488A (ja) | 1988-05-30 |
BR8703269A (pt) | 1988-03-15 |
FI95393B (fi) | 1995-10-13 |
CN1044133C (zh) | 1999-07-14 |
US20020045255A1 (en) | 2002-04-18 |
FI880899A0 (fi) | 1988-02-26 |
US5688679A (en) | 1997-11-18 |
PT85193B (pt) | 1990-08-31 |
NO880863D0 (no) | 1988-02-26 |
AU7475787A (en) | 1988-01-07 |
US20020137145A1 (en) | 2002-09-26 |
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