公开(公告)号：EP0337359A3

公开(公告)日：1990-02-07

申请号：EP89106339.8

申请日：1985-07-04

申请人： SANDOZ AG ,  SANDOZ-PATENT-GMBH ,  SANDOZ-ERFINDUNGEN Verwaltungsgesellschaft m.b.H.

发明人： Clark, Steven C. ,  Kaufman, Randal J. ,  Wong, Gordon G. ,  Wang, Elisabeth A.

IPC分类号： C12N15/00 ,  C12N5/00 ,  C07H21/04 ,  C12N1/20 ,  C12N1/16 ,  C12P21/02 ,  A61K37/02 ,  C07K3/00

CPC分类号： C12N15/85 ,  A61K38/00 ,  C07K14/535 ,  C12N15/70 ,  C12N15/81 ,  C12N2840/44

摘要： A method for preparing and isolating a transformation vector containing CSF/cDNA is described. The method comprises:
preparing RNA from a cell that produces CSF;
preparing polyadenylated messenger RNA from said RNA;
preparing single stranded cDNA from said messenger RNA;
converting the single stranded cDNA to double stranded cDNA;
inserting the double stranded cDNA into transformation vectors and transforming bacteria with said vector to form colonies;
picking pools of 200 to 500 colonies each and isolating plasmid DNA from each pool;
transfecting the plasmid DNA into suitable host cells for expressing CSF protein;
culturing the transfected cells and assaying the supernatant for CSF activity; and
selecting CSF positive pools and screening the colonies used to make the pool to identify a colony having CSF activity. Also described are a cDNA coding for a protein having CSF activity (i.e. CSF/cDNA), a microorganism or cell line transformed with a recombinant vector containing such CSF/cDNA, and a method for producing CSF protein by expressing said CSF/cDNA by culturing a microorganism or cell line. The invention also provides a method of purifying the CSF proteins and the purified proteins so produced.

公开(公告)号：EP0337359A2

公开(公告)日：1989-10-18

申请号：EP89106339.8

申请日：1985-07-04

申请人： SANDOZ AG ,  SANDOZ-PATENT-GMBH ,  SANDOZ-ERFINDUNGEN Verwaltungsgesellschaft m.b.H.

发明人： Clark, Steven C. ,  Kaufman, Randal J. ,  Wong, Gordon G. ,  Wang, Elisabeth A.

IPC分类号： C12N15/00 ,  C12N5/00 ,  C07H21/04 ,  C12N1/20 ,  C12N1/16 ,  C12P21/02 ,  A61K37/02 ,  C07K3/00

CPC分类号： C12N15/85 ,  A61K38/00 ,  C07K14/535 ,  C12N15/70 ,  C12N15/81 ,  C12N2840/44

摘要： A method for preparing and isolating a transformation vector containing CSF/cDNA is described. The method comprises:
preparing RNA from a cell that produces CSF;
preparing polyadenylated messenger RNA from said RNA;
preparing single stranded cDNA from said messenger RNA;
converting the single stranded cDNA to double stranded cDNA;
inserting the double stranded cDNA into transformation vectors and transforming bacteria with said vector to form colonies;
picking pools of 200 to 500 colonies each and isolating plasmid DNA from each pool;
transfecting the plasmid DNA into suitable host cells for expressing CSF protein;
culturing the transfected cells and assaying the supernatant for CSF activity; and
selecting CSF positive pools and screening the colonies used to make the pool to identify a colony having CSF activity.
Also described are a cDNA coding for a protein having CSF activity (i.e. CSF/cDNA), a microorganism or cell line transformed with a recombinant vector containing such CSF/cDNA, and a method for producing CSF protein by expressing said CSF/cDNA by culturing a microorganism or cell line.
The invention also provides a method of purifying the CSF proteins and the purified proteins so produced.

摘要翻译： 描述了制备和分离含有CSF / cDNA的转化载体的方法。 该方法包括：从产生CSF的细胞制备RNA; 从所述RNA制备聚腺苷酸化的信使RNA; 从所述信使RNA制备单链cDNA; 将单链cDNA转化为双链cDNA; 将双链cDNA插入转化载体并用所述载体转化细菌以形成菌落; 分别取200至500个菌落的池，每个池中分离出质粒DNA; 将质粒DNA转染到合适的宿主细胞中以表达CSF蛋白; 培养转染的细胞并测定上清液的CSF活性; 并选择CSF阳性池并筛选用于制备池的菌落以鉴定具有CSF活性的菌落。 还描述了编码具有CSF活性的蛋白质（即CSF / cDNA）的cDNA，用含有这种CSF / cDNA的重组载体转化的微生物或细胞系，以及通过培养表达所述CSF / cDNA来产生CSF蛋白的方法 微生物或细胞系。 本发明还提供了一种纯化CSF蛋白质和如此制备的纯化蛋白质的方法。