公开(公告)号：US5720952A

公开(公告)日：1998-02-24

申请号：US472022

申请日：1995-06-06

申请人： Steven C. Clark ,  Randal J. Kaufman ,  Gordon C. Wong

发明人： Steven C. Clark ,  Randal J. Kaufman ,  Gordon C. Wong

IPC分类号： C12P21/02 ,  A61K38/00 ,  A61K38/17 ,  C07K14/535 ,  C12N15/27 ,  C12N15/70 ,  C12N15/81 ,  C12N15/85 ,  A61K38/19 ,  C07K14/52

CPC分类号： C07K14/535 ,  C12N15/70 ,  C12N15/81 ,  C12N15/85 ,  A61K38/00 ,  C12N2830/42 ,  C12N2840/105 ,  Y10S514/885

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 the RNA; preparing single stranded cDNA from the messenger RNA; converting the single stranded cDNA to double stranded cDNA; inserting the double stranded cDNA into transformation vectors and transforming bacteria with the 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.

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RNA制备聚腺苷酸化的信使RNA; 从信使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。