CN87102823A - 生产肽的方法,用于生产肽的重组质粒和用该重组质粒转化的动物细胞 - Google Patents
生产肽的方法,用于生产肽的重组质粒和用该重组质粒转化的动物细胞 Download PDFInfo
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Abstract
一种用转化哺乳动物细胞和培养转化细胞生产所需的肽的方法可以通过用骨髓瘤细胞作为哺乳动物细胞和/或用具有SV40早期启动区顺序的载体予以改进,该启动区顺序位于所需的肽编码基因的5′末端上行链上和3′末端下行链上。
Description
本发明涉及应用遗传工程技术生产肽的方法、用于生产肽的重组质粒和用该重组质粒转化的动物细胞。
关于用转化动物细胞和培养转移基因接受体来生产肽已经作了各种尝试。其中的实例与近似的计算产率一并阐述如下。
1.BPV(牛乳头状瘤病毒)小鼠C127系统:人体IFN-γ基因(cDNA),SV40早期启动区;3×105单位/毫升(R.Fukunaga等,Proc.Nat1.Acad.Sci.USA.81,5086(1984));
2.SV40-CV-1系统:人体IFN-β基因(cDNA),SV40早期启动区;2×104单位/毫升(D.Gheysen等,J.Mol.Appl.Genetics,1,305(1982));
3.SV40-COS系统:人体胰岛素基因(cDNA),SV40早期启动区(O.Laud等,J.Biol.Chem.,258,6043(1983));
4.Eco-gpt-CHO系统:人体IFN-γ基因(cDNA),SV40早期启动区;1×104单位/毫升(T.Kadotani等,Seikagaku,56,915(1984));和
5.dhfr-CHO系统:人体IFN-γ基因(cDNA),SV40早期启动区;1×105单位/毫升(S.Scahill等,Proc.Natl.Acad.Sci.USA,80,4654(1983))。
尽管这些方法中每个方法都有其特点,但每个方法仍然都不能令人满意,特别是在所需要的肽的产率方面更是如此。
在生产单克隆抗体肽方面,骨髓瘤已用作产生抗体的细胞,然而将他们用于生产其他肽方面至今仍无所知。
本发明涉及用转化的动物细胞,特别是骨髓瘤细胞生产肽的方法,包括转化细胞和培养转化细胞。在一个实施例中,本发明尤其涉及生产所需肽的方法,包括用重组体质粒作为载体转化哺乳动物的细胞,该重组体质粒在所需的肽的编码基因的5′末端上行链和3′末端下行链两者上都具有SV40早期启动区顺序,然后培养由此转化的哺乳动物细胞。
在另一实施例中,本发明涉及大量生产所需肽的方法,包括用重组体质粒作为载体转化骨髓瘤细胞,该重组体质粒在用于所需要的肽编码基因的5′末端上行链和3′末端下行链两者上都具有SV40早期启动区顺序。
骨髓瘤细胞的优点在于,他们具有在体内和体外迅速生长的能力,他们具有高度合成蛋白质的能力(相对于IgG而言);他们能够使细胞密度增至很高倍数。作为该实施例的一个显著特点,本发明通过将上述骨髓瘤细胞的有利特征与SV40启动区和强化因子强有力地表达出的增强效应相结合,可以成功地产生大量的肽。
此外,本发明涉及一种重组体质粒,该质粒具有处于用于合成所需要的肽的编码基因的5′末端上行链及3′末端下行链上的SV40早期启动区顺序,并涉及用该重组体质粒转化的动物细胞。
附图说明:
图1和图2:载体构造图。
图3和图4:显示DNA片段的限制图。
图5~12:显示所用各个质粒的粗糙型限制图。
将引入动物细胞的质粒加入到动物染色体DNA中并保持其稳定。基因产物即肽稳定地由转化细胞产生,而转化的细胞又能够长久地存活着。
不产生骨髓瘤细胞或不分泌(就IgG而言)骨髓瘤细胞的用途是能够避免IgG的污染,因此有利于所需的肽的纯化。此外,由于骨髓瘤细胞能够在动物,例如在小鼠的腹膜腔内增殖,所以基因产物(肽)预计可以作为副产品大量产生。
本发明的另一个特点就是使用带有位于所需肽(如IFN)编码异源基因的5′末端上行链和3′末端下行链两者上的SV40启动区,使基因产物的产率增加到大约10倍。
用作宿主细胞的细胞可以是上述的各种骨髓瘤细胞,例如小鼠、大鼠和人体的骨髓瘤细胞,其中从产品纯化的角度来看,非IgG产生的或非IgG分泌的细胞较为适宜。P3-X63-Ag8-U1(不分泌型)HGPRT、X63-Ag8-6.5.3(不产生型)HGPRT和SP2/O-Ag14(不产生型)HGPRT等细胞系都是大鼠骨髓瘤细胞系的例子,并已为大家熟知(Munemura编:《细胞培养手册》,Kodansha,东京,1982年):Iwasaki等,《单克隆抗体》,Kodanrha,东京,1982年)。
准备应用的载体最好具有能在动物细胞内产生基因表达的有效的启动区(如SV40启动区、BPV启动区、金属硫因(metallothionein)启动区、dhfr启动区、各种长度末端的还原病毒的重复或已为公众所熟知的所有LTRS)和选择的标志。选择的标志可以是如上所述的,例如,Eco-gpt、tk和dhfr所有这些都是人们熟悉的。其他熟知的选择标志也可以应用。
SV40启动区的碱基顺序已公诸于《科学》杂志(Seience,200,495~498,(1978))。一种载体(在其中将SV40启动区与合成多肽所需的基因相结合)的建造可轻易地由一位熟悉该领域的作业人员来完成。例如可参阅下面所提供的实施例。用于载体建造所需的DNA片段的切割、合成、分析、分离和其他处理都可用常规技术(Am.J.Hum.Genet,31,531(1979);T.Maniatis等:“分子的克隆”,冷泉实验室(1982))。
原生质体融合方法和磷酸钙方法等都能用于通过将基因引入动物细胞的重组质粒来转化动物细胞。(Mol.Cell.Biol.,1(8),743~753(1981);Proc.Natl.Acad.Sci.USA,80,825~829(1983);Ogawara:Wakariyasui Idenshi Kumikae(可理解的基因重组),144~165页,Hirokawa Shoten,Tokyo(1985))。
在体外或体内都能有效地进行细胞增殖。
适宜地选择细胞增殖系统决定于高效地产生所需的肽的条件和目的(Iwasaki等:Tankuron Kotai(单克隆抗体),Kodansha,Tokyo(1982))。
用于体外培养,可以用一般用于体外培养骨髓瘤细胞的培养基:如加有10%(体积/体积)牛犊胎盘血清(FCS)的RPMI-1640培养基或加有10%(体积/体积)FCS或人造血清的RDF培养基(RPMI-1640∶DMEM∶F12=8∶1∶1)。(添加物第27号,Tanpakushitsu,Kakusan,Koso(蛋白质,核酸和酶),453~455(1984))。
在体外培养可用培替氏培养皿和旋转瓶。使用空心丝的高密度培养法也是颇有效益的。体外培养可以用各种熟知的方法处理(Munemura编:Saibo Baiyo Manual(细胞培养手册),Kosansha,Tokyo(1982)和Fukui等编:Saibo Baiyo Gijutsu(细胞培养技术),Kodansha,Tokyo,1985)。因此,举例而言,体外细胞培养是在5%二氧化碳存在下进行的。
体内骨髓瘤细胞的增殖可以在本领域熟知的动物的腹膜腔内进行。动物,特别是上面已经提及的小鼠品种Balb/c nu/nu或Balb/c,有可能将骨髓瘤细胞移植在其腹膜腔内。
要产生的肽对本发明而言其要求并不严格。这些肽的实例包括:淋巴激活素,如干扰素、肿瘤坏死因子和中间白细胞素(Interleukin);激素,如胰岛素和生长激素;抗原蛋白,如肝炎疫苗和流感疫苗;组织血纤维蛋白溶酶原激活因子和促生长因子。
产生的肽可用已知方法分离和纯化。
下面所述的以使用γ干扰素(γ-IFN)作为使用肽的实例的实施例,将进一步说明本发明,但并不是限制本发明。
实施例
Ⅰ.载体的建造(图1)
a.γ-IFN基因插入基本载体:
用克列诺片段(klenow fragment)在最接近SV40早期启动区后面的Bgl Ⅱ位点上切割质粒pKSV-10(Pharmacia P-L Biochemicals;见图5,该质粒的Kpn Ⅰ位点-BamHI位点片段的限制图,见图5)切成钝端,尔后插入一个Sma Ⅰ联结子(Takara Shuzo)。由此得到的质粒pKSV-10-Sma Ⅰ(见图6)在大肠杆菌HB101中繁殖。
另外,用Bam Ⅲ和BamHI处理含有人体γ-IFN基因的质粒pMK-5(以大肠杆菌MK-5形态保藏在发酵研究所,保藏号:FERM P-8736,见图11)。如此切割的γ-IFN基因(基因组)用克列诺片段使之成为钝端,并在Sma Ⅰ位点插入到pKSV-10-Sma Ⅰ中,得到pKSV-10-γ(见图7)。
b.选择标志Eco-gpt基因插入pKSV-10-γ:
用Pvu Ⅱ和EcoRⅠ处理质粒pSV-2-gpt(Bethesda研究室(见图8)或ATCC No.37145)。如此切割的Eco-gpt片段与通过用Sma Ⅰ和EcoRⅠ处理质粒pUC13(Pharmacia P-L Biochemicals)得到的片段连接,得到质粒pUC13-sv-gpt(见图9)。用BamHⅠ从所述pUC13-SV-gpt切割Eco-gpt基因(SV40启动区+Eco-gpt+SV终止区)。将得到的片段(其限制图示于图4)在BamHⅠ位点插入pKSV-10-γ(见图7)(在γ-IFN基因3′末端链上的SV40终止区末端上具有BamHⅠ位点)得到pKSV-10-γ-gpt(见图10)。
由于上述Eco-gpt基因在5′末端下行链上具有SV40早期启动区,所以在上述建造的质粒中的γ-IFN基因处于两个SV40启动区之间的夹层。这个质粒可用于转化。
c.选择标志tk引入pKSV-10-γ(见图2):
用BamHⅠ从质粒pFG-5(Proc.Natl.Acad.Sci.USA,76,3757(1979))切割tk基因(tk启动区+tk+tk终止区)并在BamHⅠ位点插入到pKSV-10-γ(见图7),得到质粒pKSV-10-γ-tk(见图12)。这个质粒只在γ-IFN基因的5′末端链上具有一个SV40早期启动区,可用于转化。
Ⅱ.转化骨髓瘤品系的建立
a.通过原生质体融合引入质粒:
(1)原生质体的制备
将带有载体pKSV-10-γ-gpt(见图10)的大肠杆菌在37℃条件下培养在含有葡萄糖(最终浓度为1(重量/体积)%)和氨苄青霉素(最终浓度为50微克/毫升)(最终体积为50毫升)的L肉汤(LB)中,至在600nm时浊度(光密度)为0.5,在4℃条件下进行连续处理。培养肉汤在3,000rpm离心15分钟,沉淀物用含有20(重量/体积)%蔗糖的0.05摩尔三羟甲基氨基甲烷缓冲液(pH8)冲洗,加入1.25毫升相同的缓冲液,搅拌混合物。加入0.25毫升溶菌酶(Sigma)(溶于0.25摩尔三羟甲基氨基甲烷缓冲液(pH8)至5毫克/毫升),生成的混合物可静置5分钟。在另外再加0.5毫升的0.25摩尔EDTA后将混合物静置5分钟。
将0.5毫升的0.05摩尔三羟甲基氨基甲烷缓冲液(pH8)加入上述混合物中,温度升至37℃,将混合物静置15分钟。再加入10毫升添加了蔗糖(最终浓度为10(重量/体积)%)和氯化镁(最终浓度为10毫升摩尔)的DME培养基。得到的混合物可在37℃下静置10分钟。
(2)原生质体与骨髓瘤细胞的融合(转化细胞的制备)
将骨髓瘤细胞(1×107细胞;X63-Ag8-6,5,3(HGPRT-))(戴尼邦医药有限公司-流动实验室公司)加到(1)所得到的培养物中。混合物在1500rpm离心机上离心5分钟。弃去上清液,将沉淀物搅松,再缓慢地加入0.5毫升聚乙二醇溶液(将磷酸缓冲生理食盐水(PBS)加到9克PEG400(Sigma)中配制成20毫升),使沉淀物均匀悬浮。静置于1分钟后,在37℃下将DME培养基分8次加入悬浮液,每次1毫升,每次相隔30秒钟。在整个混合物经离心后,将沉淀物悬浮在含有下述组分的RPMI-XHT培养基中。得到的细胞悬浮液分配在3块96-槽平板中,保温48小时。
RPMT-XHT培养基组分:在RPMI-1640(Gibco;1升)中加入36毫克卡那霉素,120毫克链霉素,250毫克黄嘌呤,13.6毫克次黄嘌呤,3.87毫克胸腺嘧啶核苷,3.5微克2-巯基乙醇,2.0克碳酸氢钠。配制成总容积为1升,然后再加100毫升FCS(小牛胎儿血清)。上述培养另加霉酚酸至6毫克/升(RPMI-XHMT),在其中通过培养进行转化细胞的选择。培养2周后,完成选择,在此期间每隔3天用新鲜培养基换去一半培养基。
用磷酸钙方法将pKSV-10-γ-gpt和pKSV-10-γ-tk引入L(tk-)细胞中并用以上同一方法分别在RPMI-XHMT培养基和RPMI-HAT培养基上进行选择。
Ⅲ.γ-IFN的表达
用含有下述组分的培养基将pKSV-10-γ-gpt转化骨髓瘤细胞X63-Ag8-6,5,3(HGPRT-)调节至5×104细胞/毫升。把20毫升悬浮液置于直径100毫微米的皿中,并在37℃保温箱内培养4天。收集的细胞置于旋转的瓶内,细胞密度为8×10细胞/毫升,培养物体积为100毫升,在37℃恒温室转速为50rpm中培养。
培养基成分:在8体积RPMI-1640(Sigma;1升),1体积DME(Sigma)和1体积F-12(Sigma)的混合物中,溶有25毫摩尔HEPES(Sigma),4克葡萄糖,2克碳酸氢钠,3,5微克2-巯基乙醇,36毫克卡那霉素,150毫克链霉素,250毫克黄嘌呤,13.6克次黄嘌呤和3.87毫克胸腺嘧啶核苷,配制成总体积为1升。再加入霉酚酸6毫克/升。生成的混合物再加100毫升Nu血清(合作研究)。
第5天起,每天用新鲜培养基置换一半培养基。在保持旋转瓶内细胞密度为1.0×106~1.5×106/毫升的同时进行连续培养。按下述方法,测定每天回收的培养物上清液。
将Ⅱ中所得转化L细胞置于60毫米的几个园皿中按常规方法培养,并按下述方法测定每份上清液。
另外,用0.5毫升姥鲛烷(2,6,10,14-四甲基-十五烷;瓦科纯化学工业社)对7周龄Balb/c nu/nu裸鼠进行腹腔内接种,一周后再用pKSV-10-γ-gpt转化的1×107骨髓瘤细胞X63-Ag8-6,5,3(HGPRT-)进行腹腔内接种。连续20天喂养后,按下述方法收集和测定腹水液。
Ⅳ.γ-IFN的测定
用50%细胞病效应(CPE)抑制测定法,在96-槽微滴定板上按菲利浦等人(酶学方法,78,389~394,1981)报告方法,用人体羊膜衍生FL细胞和Shindbis病毒(SV)与由NIH得到的标准αIFN和由杰奈梯奇(Genentech)得到的γ-IFN共同测定培养物上清液和腹水液样品的γ-IFN。
得到的测定结果如下。
体外培养:
载体 宿主 γ-IFN滴定率
(单位/毫升)
pKSV-10-γ-gpt X63-Ag8-6,5,3 4×105
(HGPRT)
pKSV-10-γ-gpt L(tk) 3×103
pKSV-10-γ-tk L(tk) 3×102
在Balb/c nu/nu 小鼠腹腔内培养:
pKSV-10-γ-gpt X63-Ag8-6,5,3 7×105
(HGPRT-)
在旋转培养连续20天期间,培养开始后第5天起每天回收一半培养基中的产率几乎保持不变。50毫升培养基/100毫升旋转瓶/天表达出4×105单位/毫升,这是非常高的产率。据估计,用空心丝进行的高密度培养可以得到5×107单位/毫升的表达。因此,本发明提供了一个生产肽的极佳方法。
用于代替γ-IFN基因的α-IFN基因的产率在pKSV-10-γ-gpt-L(tk-)和pKSV-10-γ-tk-L(tk-)组合中表达出得到的滴定率分别为1×104单位/毫升和2×103单位/毫升。
当在pKSV-10-γ-gpt和X63-Ag8-6,5,3组合中应用无血清培养基旋转培养(不加FSC胰岛素,而将铁传递蛋白,硒,乙醇胺和牛血清白蛋白(BSA)加入其中进行表达时,得到的滴定率为1×106单位/毫升。
Claims (11)
1、一种产生所需肽的方法,包括:
(1)用具有SV40早期启动区顺序的载体转化哺乳动物细胞,该SV40早期启动区顺序位于所需的肽编码基因的5′末端上行链上和3′末端下行链上,
(2)培养转化细胞,和
(3)纯化所需的肽。
2、一种产生所需的肽的方法,包括:
(1)用具有SV40早期启动区顺序的载体转化骨髓瘤细胞,该SV40早期启动区顺序位于所需的肽编码基因的5′末端上行链上和3′末端下行链上,
(2)培养转化细的胞,和
(3)纯化所需的肽。
3、一种具有SV40早期启动区顺序的载体,该启动区顺序位于所需的肽编码基因的5′末端上行链上和3′末端下行链上。
4、一种用具有SV40早期启动区顺序的一种载体转化的哺乳动物细胞,该启动区顺序位于所需的肽编码基因的5′末端上行链上和3′末端下行链上。
5、一种如在权利要求4中所要求专利保护的哺乳动物细胞,其中所述哺乳动物细胞是一种骨髓瘤细胞。
6、一种如在权利要求2中所要求专利保护的方法,其中所述骨髓瘤细胞选自由大鼠骨髓瘤细胞,小鼠骨髓瘤细胞和人体骨髓瘤细胞组成的一组细胞。
7、一种如在权利要求6中所要求专利保护的方法,其中所述小鼠骨髓瘤细胞选自由P3-X63-Ag8-U1 HGPRT-,X63-Ag8-6,5,3HGPRT-和SP2/O-Ag14 HGPRT-组成的一组细胞。
8、一种如在权利要求1中所要求专利保护的方法,其中所述的所需的肽选自由淋巴激活素、激素、抗原蛋白、组织血纤维蛋白溶酶原激活因子和促生长因子组成的一组肽。
9、一种如在权利要求2中所要求专利保护的方法,其中所述的所需的肽选自由淋巴激活素、激素、抗原蛋白、组织血纤维蛋白溶酶原激活因子和促生长因子组成的一组肽。
10、一种如在权利要求3中所要求专利保护的方法,其中所述的所需的肽选自由淋巴激活素、激素、抗原蛋白、组织血纤维蛋白溶酶原激活因子和促生长因子组成的一组肽。
11、一种如在权利要求4中所要求专利保护的哺乳动物细胞,其中所述的所需的肽选自由淋巴激活素、激素、抗原蛋白、组织血纤维蛋白溶酶原激活因子和促生长因子组成的一组肽。
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CN87102823A Expired - Fee Related CN1034427C (zh) | 1986-04-14 | 1987-04-14 | 用载体转化骨髓瘤细胞生产肽的方法 |
CN94116755A Pending CN1107513A (zh) | 1986-04-14 | 1994-09-28 | 制备转化的骨髓瘤细胞的方法 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN94116755A Pending CN1107513A (zh) | 1986-04-14 | 1994-09-28 | 制备转化的骨髓瘤细胞的方法 |
Country Status (15)
Country | Link |
---|---|
US (1) | US5019499A (zh) |
EP (1) | EP0244677B1 (zh) |
JP (1) | JP2573215B2 (zh) |
KR (1) | KR960007195B1 (zh) |
CN (2) | CN1034427C (zh) |
AT (1) | ATE109827T1 (zh) |
AU (1) | AU600481B2 (zh) |
CA (1) | CA1297434C (zh) |
DE (1) | DE3750348T2 (zh) |
DK (1) | DK189987A (zh) |
FI (1) | FI97550C (zh) |
IE (1) | IE65500B1 (zh) |
IL (1) | IL82207A (zh) |
NO (1) | NO175872C (zh) |
PH (1) | PH26343A (zh) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU622870B2 (en) * | 1987-08-07 | 1992-04-30 | Medical Research Council, The | Vector for integration site independent gene expression in mammalian host cells |
GB8809129D0 (en) * | 1988-04-18 | 1988-05-18 | Celltech Ltd | Recombinant dna methods vectors and host cells |
US5879936A (en) * | 1988-04-18 | 1999-03-09 | Aluguisse Holding A.G. | Recombinant DNA methods, vectors and host cells |
US5573937A (en) * | 1989-12-07 | 1996-11-12 | Snow Brand Milk Products Co., Ltd. | Serum free culture medium |
US5891693A (en) * | 1990-01-25 | 1999-04-06 | Alusuisse Holdings A.G. | Recombinant DNA methods vectors and host cells |
GB9022545D0 (en) | 1990-10-17 | 1990-11-28 | Wellcome Found | Culture medium |
GB9022543D0 (en) * | 1990-10-17 | 1990-11-28 | Wellcome Found | Antibody production |
EP0798379A3 (en) * | 1996-03-29 | 1999-10-06 | Tosoh Corporation | Human glutamic acid decarbocylase-expressing myeloma cell line |
UY26317A1 (es) * | 2000-08-30 | 2000-10-31 | Alfonso Cayota Carlos Pritsch | Sistema de produccion de trombopoyetina humana por celulas de mamiferos en cultivo |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4713339A (en) * | 1983-01-19 | 1987-12-15 | Genentech, Inc. | Polycistronic expression vector construction |
AU572108B2 (en) * | 1983-01-19 | 1988-05-05 | Genentech Inc. | Human tpa production using vectors coding for dhfr protein |
US4740461A (en) * | 1983-12-27 | 1988-04-26 | Genetics Institute, Inc. | Vectors and methods for transformation of eucaryotic cells |
US4663281A (en) * | 1984-03-22 | 1987-05-05 | Mass Institute Of Technology | Enhanced production of proteinaceous materials in eucaryotic cells |
JPS6147500A (ja) * | 1984-08-15 | 1986-03-07 | Res Dev Corp Of Japan | キメラモノクロ−ナル抗体及びその製造法 |
JPS61134325A (ja) * | 1984-12-04 | 1986-06-21 | Teijin Ltd | ハイブリツド抗体遺伝子の発現方法 |
JPS61134324A (ja) * | 1984-12-04 | 1986-06-21 | Teijin Ltd | ヒト抗体h鎖遺伝子の発現方法 |
JP2532858B2 (ja) * | 1985-04-01 | 1996-09-11 | セルテツク リミテツド | 形質転換したミエロ―マ細胞系 |
JPH0714341B2 (ja) * | 1985-11-20 | 1995-02-22 | 鐘淵化学工業株式会社 | 蛋白質の高生産株の取得方法 |
JP2581668B2 (ja) * | 1985-11-27 | 1997-02-12 | 三井東圧化学株式会社 | ヒト正常細胞由来のヒト組織プラスミノ−ゲン活性化因子をコ−ドする新しいdna配列とそれを含むベクタ−及び細胞 |
GB8607679D0 (en) * | 1986-03-27 | 1986-04-30 | Winter G P | Recombinant dna product |
-
1987
- 1987-04-10 CA CA000534474A patent/CA1297434C/en not_active Expired - Lifetime
- 1987-04-13 FI FI871607A patent/FI97550C/fi not_active IP Right Cessation
- 1987-04-13 IE IE95287A patent/IE65500B1/en not_active IP Right Cessation
- 1987-04-13 IL IL82207A patent/IL82207A/xx unknown
- 1987-04-13 DK DK189987A patent/DK189987A/da not_active Application Discontinuation
- 1987-04-13 PH PH35127A patent/PH26343A/en unknown
- 1987-04-14 NO NO871573A patent/NO175872C/no unknown
- 1987-04-14 KR KR1019870003544A patent/KR960007195B1/ko not_active IP Right Cessation
- 1987-04-14 CN CN87102823A patent/CN1034427C/zh not_active Expired - Fee Related
- 1987-04-14 US US07/038,177 patent/US5019499A/en not_active Expired - Fee Related
- 1987-04-14 EP EP87105559A patent/EP0244677B1/en not_active Expired - Lifetime
- 1987-04-14 JP JP62091446A patent/JP2573215B2/ja not_active Expired - Fee Related
- 1987-04-14 DE DE3750348T patent/DE3750348T2/de not_active Expired - Fee Related
- 1987-04-14 AT AT87105559T patent/ATE109827T1/de not_active IP Right Cessation
- 1987-04-14 AU AU71516/87A patent/AU600481B2/en not_active Ceased
-
1994
- 1994-09-28 CN CN94116755A patent/CN1107513A/zh active Pending
Also Published As
Publication number | Publication date |
---|---|
KR870010190A (ko) | 1987-11-30 |
IE870952L (en) | 1987-10-14 |
IL82207A0 (en) | 1987-10-30 |
KR960007195B1 (ko) | 1996-05-29 |
CN1107513A (zh) | 1995-08-30 |
AU7151687A (en) | 1987-10-15 |
CA1297434C (en) | 1992-03-17 |
FI97550C (fi) | 1997-01-10 |
EP0244677A3 (en) | 1988-08-03 |
FI871607A0 (fi) | 1987-04-13 |
NO175872C (no) | 1994-12-21 |
DK189987D0 (da) | 1987-04-13 |
DE3750348T2 (de) | 1994-12-15 |
NO871573L (no) | 1987-10-15 |
JP2573215B2 (ja) | 1997-01-22 |
ATE109827T1 (de) | 1994-08-15 |
EP0244677A2 (en) | 1987-11-11 |
EP0244677B1 (en) | 1994-08-10 |
IL82207A (en) | 1992-03-29 |
FI97550B (fi) | 1996-09-30 |
NO175872B (zh) | 1994-09-12 |
CN1034427C (zh) | 1997-04-02 |
IE65500B1 (en) | 1995-11-01 |
DE3750348D1 (de) | 1994-09-15 |
FI871607A (fi) | 1987-10-15 |
US5019499A (en) | 1991-05-28 |
PH26343A (en) | 1992-04-29 |
DK189987A (da) | 1987-10-15 |
AU600481B2 (en) | 1990-08-16 |
NO871573D0 (no) | 1987-04-14 |
JPS6344897A (ja) | 1988-02-25 |
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