Megakaryocyte differentiation factor

Abstract

A novel megakaryocyte differentiation factor, for example, consisting essentially of SEQ ID NO: 30; DNA coding for the megakaryocyte differentiation factor, an expression vector comprising the DNA, a host transformed with the expression vector, and a process for production of the megakaryocyte differentiation factor using the host. The megakaryocyte differentiation factor accelerates differentiation of megakaryocytes in the presence of IL-3, and acts as a thrombopoietin, and therefore an effective medicament to various diseases involving a decrease in platelete.

1 . A megakaryocyte differentiation factor having the following properties; 
 (1) stimulating differentiation of megakaryocytes;    (2) exhibiting a molecular weight of 55 to 57 kD as determined by gel filtration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and having no intermolecular disulfide linkage;    (3) exhibiting an isoelectric point of 6.5±0.5; and    (4) having at least one of the amino acid sequences shown in SEQ ID NO: 1 to 9 in the Sequence Listing.    
     
     
         2 . A megakaryocyte differentiation factor according to  claim 1 , produced by human cells.  
     
     
         3 . A megakaryocyte differentiation factor according to  claim 2 , produced by human cancer cells.  
     
     
         4 . A megakaryocyte differentiation factor according to  claim 3 , produced by cells derived from human epidermoid carcinoma cell A431.  
     
     
         5 . A megakaryocyte differentiation factor according to  claim 4 , produced by cells derived from human epidermoid carcinoma cell A431, cultured in a protein-free medium.  
     
     
         6 . A megakaryocyte differentiation factor having an amino acid sequence the same as the native amino acid sequence of a megakaryocyte differentiation factor according to  claim 1 , an amino acid sequence wherein one or more than one amino acid residue in said native amino acid sequence is deleted, an amino acid sequence wherein one or more than one amino acid residue in said native amino acid sequence is replaced with other amino acids, an amino acid sequence wherein one or more than one amino acid is added to said native amino acid sequence, or an amino acid sequence including a combination of said amino acid modifications.  
     
     
         7 . A megakaryocyte differentiation factor essentially consisting of the amino acid sequence shown in SEQ ID NO: 30.  
     
     
         8 . A megakaryocyte differentiation factor having an amino acid sequence wherein one or more than one amino acid residue in the amino acid sequence shown in SEQ ID NO: 30 is deleted, an amino acid sequence wherein one or more than one amino acid residue in the amino acid sequence shown in SEQ ID NO: 30 is replaced with other amino acids, or an amino acid sequence wherein one or more than one amino acid is added to the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence including a combination of said amino acid modification.  
     
     
         9 . A megakaryocyte differentiation factor according to  claim 6 , which is glycosylated.  
     
     
         10 . A megakaryocyte differentiation factor according to  claim 7 , which is glycosylated.  
     
     
         11 . A megakaryocyte differentiation factor according to  claim 8 , which is glycosylated.  
     
     
         12 . A megakaryocyte differentiation factor obtainable from a culture of transformed cells constructed by a gene recombination technique using a polynucleotide hybridizable with a polynucleotide coding for at least one of the amino acid sequences shown in SEQ ID NO: 1 to 9.  
     
     
         13 . A megakaryocyte differentiation factor according to  claim 6 , wherein the N-terminus of the factor is biochemically or chemically modified, preferably the first methionine is deleted and the second alanine is acethylated.  
     
     
         14 . A megakaryocyte differentiation factor according to  claim 8 , wherein the N-terminus of the factor is biochemically or chemically modified, preferably the first methionine is deleted and the second alanine is acethylated.  
     
     
         15 . A megakaryocyte differentiation factor according to  claim 12 , wherein the N-terminus of the factor is biochemically or chemically modified, preferably the first methionine is deleted and the second alanine is acethylated.  
     
     
         16 . An isolated DNA coding for a megakaryocyte differentiation factor according to  claim 1 .  
     
     
         17 . An isolated DNA coding for a megakaryocyte differentiation factor according to  claim 6 .  
     
     
         18 . An expression vector comprising a DNA according to  claim 16 .  
     
     
         19 . An expression vector comprising a DNA according to  claim 17 .  
     
     
         20 . A host transformed with an expression vector according to  claim 18 .  
     
     
         21 . A host transformed with an expression vector according to  claim 19 .  
     
     
         22 . A pharmaceutical composition comprising a megakaryocyte differentiation factor according to  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         23 . A pharmaceutical composition comprising a megakaryocyte differentiation factor according to  claim 6  and a pharmaceutically acceptable carrier.  
     
     
         24 . A pharmaceutical composition according to  claim 22 , for treating thrombo cytopenia.  
     
     
         25 . A pharmaceutical composition according to  claim 23 , for treating thrombo cytopenia.  
     
     
         26 . An antibody to megakaryocyte differentiation factor according to  claim 1 .  
     
     
         27 . An antibody to megakaryocyte differentiation factor according to  claim 6 .  
     
     
         28 . A process for production of a megakaryocyte differentiation factor according to  claim 1 , comprising the steps of culturing a host according to  claim 20 , and recovering the megakaryocyte differentiation factor.  
     
     
         29 . A process according to  claim 28 , wherein the host is silkworm  Bombyx mori.