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    • 5. 发明授权
    • 재조합당단백호르몬의제조방법
    • KR100370942B1
    • 2003-12-24
    • KR1019980032997
    • 1998-08-14
    • 동아쏘시오홀딩스 주식회사
    • 박장현김현일서광석박세춘이성희
    • C12N15/85C12N15/19
    • PURPOSE: Preparation method of mass production of biologically active EPO from recombinant plasmid in CHO(chinese hamster ovary) cell is provided. CONSTITUTION: The invention is disclosed a mass production method from recombinant plasmid pDEP-173 consisted of zeocin resistance gene, EPO gene, isolated from human fetal liver cDNA library, under control of SRα promoter, AmvRNA4 enhancer, DHFR gene, and BGH polyadenylation signal. The plasmid is transfected into CHO cell and cultured with roller bottle in serum free medium. EPO is induced with n-butyric acid or methotrexate. Overexpressed EPO is purified with hydrophobic chromatography and anion exchange chromatography. The purified EPO only having N-acetyl neuraminic acid as N-terminal end of sialic acid has molecular weight of 34,000-47,000 and shows 7-8 isomer bands in isoelectric focusing around pH 3.7-5.2.
    • 目的:提供从CHO(中国仓鼠卵巢)细胞中重组质粒大量生产生物活性EPO的制备方法。 构成:本发明公开了一种由SRα-1控制下从人胎肝cDNA文库中分离的由zeocin抗性基因,EPO基因组成的重组质粒pDEP-173的大量生产方法。 启动子,AmvRNA4增强子,DHFR基因和BGH聚腺苷酸化信号。 将质粒转染CHO细胞并在无血清培养基中用滚瓶培养。 用正丁酸或甲氨蝶呤诱导EPO。 过度表达的EPO用疏水色谱和阴离子交换色谱纯化。 仅具有N-乙酰神经氨酸作为唾液酸N-末端的纯化EPO具有34,000-47,000的分子量,并且在等电聚焦约3.7-5.2的pH范围内显示出7-8个异构体带。
    • 6. 发明授权
    • 동물세포에서 재조합 당단백질의 연속식 대량생산 방법
    • 동물세포에서재조합당단백질의연속식대량생산방
    • KR100394474B1
    • 2003-08-09
    • KR1019990058878
    • 1999-12-18
    • 동아쏘시오홀딩스 주식회사
    • 나규흠박세춘김현일이태호서광석박장현김병문이성희김원배
    • C12N5/07
    • PURPOSE: Provided is a method for manufacturing recombinant glycoproteins in animal cells in a high yield. Thereby, the recombinant glycoprotein has high activity and cultured cells have high viability for a long time. CONSTITUTION: The method for manufacturing recombinant glycoproteins in animal cells in a high yield is comprised of the next steps of: i) cultivating and growing animal cells in medium including serum for 2-4 days; and ii) cultivating repeatedly the animal cells in medium having no serum for 2-3 days to obtain recombinant glycoproteins. And the recombinant glycoproteins include erythropoietin (EPO), tissue-type plasminogen activator (tPA), follicle stimulating hormone (FSH), luteinizing hormone (LH), interleukin-12 (IL-12), hCG (human chorionic gonadotropin), thrombopoietin (TPO), and Factor VIII.
    • 目的:提供一种以高产率在动物细胞中制造重组糖蛋白的方法。 由此,重组糖蛋白具有高活性,并且培养的细胞长时间具有高生存力。 构成:以高产率在动物细胞中制造重组糖蛋白的方法包括以下步骤:i)在包括血清的培养基中培养和生长动物细胞2-4天; 和ii)在不含血清的培养基中重复培养动物细胞2-3天以获得重组糖蛋白。 重组糖蛋白包括促红细胞生成素(EPO),组织型纤溶酶原激活剂(tPA),促卵泡激素(FSH),黄体生成素(LH),白介素-12(IL-12),hCG(人绒毛膜促性腺激素),血小板生成素 TPO)和因子VIII。
    • 8. 发明授权
    • 재조합 인간 에리트로포이에틴의 제조방법
    • 재조합인간에리트로포이에틴의제조방법
    • KR100369788B1
    • 2003-01-29
    • KR1019990037463
    • 1999-09-03
    • 동아쏘시오홀딩스 주식회사
    • 박장현나규흠김현일서광석박세춘이태호이성희김원배양재명
    • C12N15/12C12N15/63
    • PURPOSE: A method for mass-producing recombinant erythropoietine(EPO) is provided for the treatment of anemia by regulating the red blood cell generation. CONSTITUTION: The method for mass-producing recombinant erythropoietine(EPO) comprises the steps of: preparing a recombinant plasmid pDEP-521 harboring Zeocin-resistant gene, Rous Sarcoma virus (RSV) promoter, Avian myeloblastosis virus RNA4(AmvRNA4) leader sequence, dihydrofolate reductase gene, human EPO genome gene, and bovine growth hormone polyadenylation signal sequence; producing a transformant baby hamster kidney(BHK) cell line DBHE318(KCLRF-BR-00024) by the transfection of host cells with the recombinant plasmid pDEP-521; and incubating the transformant to produce EPO, in which the produced EPO is characterized by 34,000 to 40,000 dalton of molecular weight, positive in western blotting, 7 to 8 isomer bands around pH 3.8 to 5.2 in isoelectrofocusing analysis, and similar in vivo activity of facilitating the red blood cell making in comparison to that of the international standard liquid.
    • 目的:通过调节红细胞生成来提供大量生产重组促红细胞生成素(EPO)的方法用于治疗贫血。 构建:大量生产重组促红细胞生成素(EPO)的方法包括以下步骤:制备含有博莱霉素抗性基因,劳氏肉瘤病毒(RSV)启动子,禽成髓细胞瘤病毒RNA4(AmvRNA4)前导序列,二氢叶酸的重组质粒pDEP- 还原酶基因,人EPO基因组基因和牛生长激素聚腺苷酸化信号序列; 通过用重组质粒pDEP-521转染宿主细胞产生转化子幼仓鼠肾(BHK)细胞系DBHE318(KCLRF-BR-00024); 并且将转化体温育以产生EPO,其中产生的EPO的特征在于分子量为34,000至40,000道尔顿,Western印迹阳性,等电聚焦分析中pH为3.8至5.2的7至8个异构体带,以及类似的促进促进 红血球与国际标准液相比较。
    • 9. 发明公开
    • 재조합당단백호르몬의제조방법
    • 来自CHO细胞的重组蛋白(EPO)的制备方法
    • KR1020000013877A
    • 2000-03-06
    • KR1019980032997
    • 1998-08-14
    • 동아쏘시오홀딩스 주식회사
    • 박장현김현일서광석박세춘이성희
    • C12N15/85C12N15/19
    • C12N15/8509A61K38/1709C07K14/575
    • PURPOSE: Preparation method of mass production of biologically active EPO from recombinant plasmid in CHO(chinese hamster ovary) cell is provided. CONSTITUTION: The invention is disclosed a mass production method from recombinant plasmid pDEP-173 consisted of zeocin resistance gene, EPO gene, isolated from human fetal liver cDNA library, under control of SRα promoter, AmvRNA4 enhancer, DHFR gene, and BGH polyadenylation signal. The plasmid is transfected into CHO cell and cultured with roller bottle in serum free medium. EPO is induced with n-butyric acid or methotrexate. Overexpressed EPO is purified with hydrophobic chromatography and anion exchange chromatography. The purified EPO only having N-acetyl neuraminic acid as N-terminal end of sialic acid has molecular weight of 34,000-47,000 and shows 7-8 isomer bands in isoelectric focusing around pH 3.7-5.2.
    • 目的:提供CHO(中国仓鼠卵巢)细胞中重组质粒生物活性EPO的大规模生产制备方法。 构成:公开了在SRα启动子,AmvRNA4增强子,DHFR基因和BGH多聚腺苷酸化信号的控制下,从人胎肝cDNA文库中分离的zeocin抗性基因EPO基因的重组质粒pDEP-173的批量生产方法。 将质粒转染到CHO细胞中,并用滚筒瓶在无血清培养基中培养。 用正丁酸或甲氨蝶呤诱导EPO。 用疏水层析和阴离子交换色谱纯化过表达的EPO。 仅具有N-乙酰神经氨酸作为唾液酸N-末端的纯化EPO具有34,000-47,000的分子量,并且在pH 3.7-5.2附近显示7-8个等电聚焦异构体带。