Hybrid vector having a cytomegalovirus enhancer and myeloproliferative sarcoma virus promoter

Abstract

An expression vector capable of expressing high levels of heterologous proteins having a cytomegalovirus (CMV) enhancer 5′ upstream from a myeloproliferative sarcoma virus (MPSV) promoter.

1 . A non-retroviral expression vector comprising a cytomegalovirus (CMV) enhancer and a myeloproliferative sarcoma virus (MPSV) promoter.  
     
     
         2 . The vector of  claim 1  wherein the CMV enhancer is located upstream from the 5′ end of the MPSV promoter.  
     
     
         3 . The vector of  claim 2  wherein the CMV enhancer and MPSV promoter comprises the polynucleotide sequence of SEQ ID NO:1.  
     
     
         4 . The vector of  claim 1  that further comprises at least one additional element selected from the group consisting of a consensus Ig intron, a tPA pre-proleader sequence, a polio IRES, a Δ CD8 selection marker, and a human growth hormone polyA signal sequence.  
     
     
         5 . The vector of  claim 1  that further comprises a consensus Ig intron, a tPA pre-porleader sequence, and a polio IRES.  
     
     
         6 . The vector of  claim 2  that further comprises a consensus Ig intron, a tPA pre-proleader sequence, and a polio IRES.  
     
     
         7 . The vector of  claim 3  that further comprises a consensus Ig intron, a tPA pre-proleader sequence, and a polio IRES.  
     
     
         8 . The vector of  claim 7  further comprising a structural gene such that the gene is operably linked to the CMV enhancer and MPSV promoter.  
     
     
         9 . The vector pZMP21 as deposited with the ATCC, having the reference number ATCC PTA-5266.  
     
     
         10 . A mammalian cell transfected with the vector of  claim 1 .  
     
     
         11 . The mammalian cell of  claim 10  wherein the CMV enhancer and the MPSV promoter comprises the polynucleotide sequence of SEQ ID NO: 1.  
     
     
         12 . The mammalian cell of  claim 11  wherein the cell is a CHO cell.  
     
     
         13 . The mammalian cell of  claim 12  wherein the CHO cell is of strain DXB11.  
     
     
         14 . A method of producing a recombinant protein comprising 
 a. transfecting a mammalian host cell with the vector of  claim 1;     b. growing the cells under conditions that selectively propagates those cells that have integrated the vector of  claim 1  into its genome;    c. growing the cells of step b) under conditions that cause the recombinant protein to be secreted into the cell medium;    d. isolating the recombinant protein from the cell medium.    
     
     
         15 . The method of  claim 14  wherein the transfection occurs by electroporation.  
     
     
         16 . The method of  claim 14  wherein the conditions that selectively propagates cells that have integrated the vector of  claim 1  into its genome comprises growing the cells in the presence of methotrexate.  
     
     
         17 . A method of producing a recombinant protein comprising 
 a. randomly integrating the vector of  claim 8  into the genome of CHO cells;    b. growing the cells in the presence of increasing concentrations of methotrexate;    c. isolating cells from step b) and growing under conditions such that the CHO cells produce the recombinant protein into the culture medium;    d. isolating the recombinant protein from the culture medium.    
     
     
         18 . The method of  claim 17  wherein the CHO cells are of the strain DXB11.