Gene coded for interleukin-2 polypeptide, recombinant DNA carrying the said gene, a living cell line possessing the recombinant DNA, and method for producing interleukin-2 using the said cell

Abstract

A gene coded for a polypeptide which possesses interleukin-2 is isolated, and connected with a vector DNA which is capable of replicating in a procaryotic or eucaryotic cell at a position downstream of a promoter gene in the vector obtaining a recombant DNA, with which the cell is transformed to produce interleukin-2.

What is claimed as new and desired to be secured by letters patent of the united states is:  
     
         1 . A cloned gene coded for a polypeptide possessing the activity of interleukin-2.  
     
     
         2 . The gene of    claim 1   , which is prepared from a messenger RNA produced by an interleukin-2 producing mammalian cell line.  
     
     
         3 . The gene of    claim 2   , wherein said messenger RNA is obtainable as a sediment of 11 to 12S of sucrose density gradient centrifugation.  
     
     
         4 . The gene of    claim 2   , wherein said mammalian cell line is a human T-lymphocyte, a transformed human T-lymphocyte or a human T-cell hybridoma.  
     
     
         5 . The gene of    claim 1   , which has sites cleaved with restriction endonuclease in the order of Bst NI, Xba I and Bst NI from 5′-terminus of the coding sequence.  
     
     
         6 . The gene of    claim 1   , which has sites cleaved with restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from 5′-terminus of the coding sequence.  
     
     
         7 . The gene of    claim 1   , which has the base sequence shown in FIG. 2 ( a ).  
     
     
         8 . The gene of    claim 1   , of which the base sequence initiates from ATG sequence at position 48 to 50 and has the sequential bases following the ATG sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         9 . The gene of    claim 1   , of which the base sequence initiates from GCA sequence at position 108 to 110 and has the sequential bases following the GCA sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         10 . The gene of    claim 1   , of which the base sequence initiates from CCT sequence at position 111 to 113 and has the sequential bases following the CCT sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         11 . The gene of    claim 1   , of which the base sequence initiates from A at position 1 and has the sequential bases following the A ending at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         12 . The gene of    claim 8   , of which the base sequence ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         13 . The gene of    claim 9   , of which the base sequence ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         14 . The gene of    claim 10   , of which the base sequence ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         15 . The gene of    claim 1   , of which the base sequence initiates from A at position 1 and has the sequential bases following the A ending at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         16 . The gene of    claim 8   , of which the base sequence ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         17 . The gene of    claim 9   , of which the base sequence ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         18 . The gene of    claim 10   , of which the base sequence ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         19 . The gene of    claim 1   , of which the base sequence initiates from A at position 1 and has the sequential bases following the A ending at C at position 801 in FIG. 2 ( a ).  
     
     
         20 . The gene of    claim 8   , of which the base sequence ends at C at position 801 in FIG. 2 ( a ).  
     
     
         21 . The gene of    claim 9   , of which the base sequence ends at C at position 801 in FIG. 2 ( a ).  
     
     
         22 . The gene of    claim 10   , of which the base sequence ends at C at position 801 in FIG. 2 ( a ).  
     
     
         23 . The gene of    claim 8   , of which the base sequence ends at poly (A) in FIG. 2 ( a ).  
     
     
         24 . The gene of    claim 9   , of which the base sequence ends at poly (A) in FIG. 2 ( a ).  
     
     
         25 . The gene of    claim 10   , of which the base sequence ends at poly (A) in FIG. 2 ( a ).  
     
     
         26 . The gene of    claim 1   , which has the base sequence corresponding to Amino Acid Sequence I in FIG. 2 ( b ).  
     
     
         27 . The gene of    claim 1   , which has the base sequence corresponding to Amino Acid Sequence II in FIG. 2 ( b ).  
     
     
         28 . The gene of    claim 1   , which has the base sequence corresponding to Amino Acid Sequence III in FIG. 2 ( b ).  
     
     
         29 . A DNA, prepared recombinantly which comprises a gene coded for a polypeptide which possesses the activity, of inter-leukin-2, and a vector DNA capable of propagating in a procaryotic or eucaryotic the coding sequence of said gene being located at a position downstream of a promoter sequence.  
     
     
         30 . The recombinant DNA of    claim 29   , wherein said gene is prepared with a messenger RNA-produced by an interleukin-2 producing mammalian cell line.  
     
     
         31 . The recombinant DNA of    claim 30   , wherein said mammalian cell is a human T-lymphocyte, a transformed human T-lymphocyte or a human T-cell hybridoma.  
     
     
         32 . The recombinant DNA of    claim 30   , wherein said messenger RNA is obtainable as a sediment of 11 to 12S of sucrose density gradient centrifugation.  
     
     
         33 . The recombinant DNA of    claim 29   , wherein said gene has sites cleaved with restriction endonuclease in the order of Bst NI, Xba I and Bst NI from 5′-terminus of the coding sequence.  
     
     
         34 . The recombinant DNA of    claim 29   , wherein said gene has sites cleaved with restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from 5′-terminus of the coding sequence.  
     
     
         35 . The recombinant DNA of    claim 29   , wherein said gene has the base sequence shown in FIG. 2 ( a ).  
     
     
         36 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene initiates from ATG sequence at position 48 to 50 and has the sequential bases following the ATG sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         37 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene initiates from GCA sequence at position 108 to 110 and has the sequential bases following the GCA sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         38 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene initiates from CCT sequence at position 111 to 113 and has the sequential bases following the CCT sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         39 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         40 . The recombinant DNA of    claim 36   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         41 . The recombinant DNA of    claim 37   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         42 . The recombinant DNA of    claim 38   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         43 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at TGA sequence at position 507 to 509 in FIG. 2.  
     
     
         44 . The recombinant DNA of    claim 36   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         45 . The recombinant DNA of    claim 37   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         46 . The recombinant DNA of    claim 38   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         47 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at C at position 801 in FIG. 2 ( a ).  
     
     
         48 . The recombinant DNA of    claim 36   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         49 . The recombinant DNA of    claim 37   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         50 . The recombinant DNA of    claim 38   , wherein the base sequence of said gene ends at C at position 501 in FIG. 2 ( a ).  
     
     
         51 . The recombinant DNA of clam  36 , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         52 . The recombinant DNA of    claim 37   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         53 . The recombinant DNA of    claim 38   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         54 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene corresponds to Amino Acid Sequence I in FIG. 2 ( b ).  
     
     
         55 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene corresponds to Amino Acid Sequence II in FIG. 2 ( b ).  
     
     
         56 . The recombinant DNA of    claim 29   , wherein the base sequence of said gene corresponds to Amino Acid Sequence III in FIG. 2 ( b ).  
     
     
         57 . The recombinant DNA of    claim 29   , wherein said procaryotic cell line belongs to the genus Escherichia.  
     
     
         58 . The recombinant DNA of    claim 29   , wherein said procaryotic cell line belongs to  Escherichia coli.    
     
     
         59 . The recombinant DNA of    claim 29   , wherein said eucaryotic cell line belongs to the genus Saccharomyces.  
     
     
         60 . The recombinant DNA of    claim 29   , wherein said eucaryotic cell line belongs to the genus  Saccharomyces cerevicea.    
     
     
         61 . The recombinant DNA of    claim 29   , wherein said eucaryotic cell line is a monkey cell transformed with SV-40 constitutively expressing large T antigen.  
     
     
         62 . A cell of eucarlote or procaryote transformed with a recombinant DNA comprising a gene coding for a polypeptide possessing the activity of interleukin-2 and a vector DNA capable of propagating in said cell, and the coding sequence of said gene being located at a position downstream of a promoter sequence.  
     
     
         63 . The cell of    claim 62   , wherein said gene is prepared with a messenger RNA produced by an interleukin-2 producing mammalian cell.  
     
     
         64 . The cell of    claim 63   , wherein said mammalian cell is a human T-lymphocyte, a transformed human T-lymphocyte or a T-cell hybridoma.  
     
     
         65 . The cell of    claim 63   , wherein said messenger RNA is obtainable as a sediment of 11 to 12S of sucrose density gradient centrifugation.  
     
     
         66 . The cell of    claim 63   , wherein said gene has sites cleaved with restriction endonuclease in the order of Bst NI, Xba I and Bst NI from 5′-terminus of the coding sequence.  
     
     
         67 . The cell of    claim 62   , wherein said gene has sites cleaved with restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from 5′-terminus of the coding sequence.  
     
     
         68 . The cell of    claim 62   , wherein said gene has the base sequence shown in FIG. 2 ( a ).  
     
     
         69 . The cell of    claim 62   , wherein the base sequence of said gene initiates from ATG sequence at position 48 to 50 and has the sequential bases following the ATG codon up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         70 . The cell of    claim 62   , wherein the base sequence of said gene initiates from GCA sequence at position 108 to 110 and has the sequential bases following the CCA sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         71 . The cell of    claim 62   , wherein the base sequence of said gene initiates from CCT sequence at position 111 to 113 and has the sequential bases following the CCT sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         72 . The cell of    claim 62   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         73 . The cell of    claim 69   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         74 . The cell of    claim 70   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         75 . The cell of    claim 71   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         76 . The cell of    claim 62   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         77 . The cell of    claim 69   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         78 . The cell of    claim 70   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         79 . The cell of    claim 71   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         80 . The cell of    claim 62   , wherein the base sequence of said gene initiates from A at position I and has the sequential bases following the A ending at C at position 801 in FIG. 2 ( a ).  
     
     
         81 . The cell of    claim 69   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         82 . The cell of    claim 70   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         83 . The cell of    claim 71   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         84 . The cell of    claim 69   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         85 . The cell of    claim 70   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         86 . The cell of    claim 71   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         87 . The cell of    claim 65   , wherein the base sequence of said gene corresponds to Amino Acid Sequence I in FIG. 2 ( b ).  
     
     
         88 . The cell of    claim 65   , wherein the base sequence of said gene corresponds to Amino Acid-Sequence II in FIG. 2 ( b ).  
     
     
         89 . The cell of    claim 65   , wherein the base sequence of said gene corresponds to Amino Acid Sequence III in FIG. 2 ( b ).  
     
     
         90 . The cell of    claim 62   , wherein said procaryotic cell belongs to the genus Escherichia.  
     
     
         91 . The cell of    claim 62   , wherein said procaryotic cell belongs to  Escherichia Coli.    
     
     
         92 . The cell of    claim 62   , wherein said eucaryotic cell belongs to the genus Saccharomyces.  
     
     
         93 . The cell of    claim 62   , wherein said eucaryotic cell belongs to the genus  Saccharomyces cerevicea.    
     
     
         94 . The cell of    claim 62   , wherein said eucaryotic cell is a monkey cell transformed with SV-40 constitutively expressing large T antigen.  
     
     
         95 . A method for producing interleukin-2 which comprises culturing aleucaryotic or procaryotic cell culture medium, transformed with a recombinant DNA to produce interleukin-2 and recovering the produced interleukin-2; said recombinant DNA comprising a gene coded for a polypeptide which posesses the activity of interleukin-2 and a vector DNA which is capable of replicating in said cell, and the coding sequence of said gene being located at a position downstream of a promoter sequence.  
     
     
         96 . The method of    claim 95   , wherein said gene is prepared with a messenger RNA produced by an interleukin-2 producing mammalian cell line.  
     
     
         97 . The method of    claim 96   , wherein said mammalian cell is a human T-lymphocyte, a transformed human lymphocyte or a T-cell hybridoma.  
     
     
         98 . The method of    claim 96   , wherein said messenger RNA is obtainable as a sediment of 11 to 12S of sucrose density gradient centrifugation.  
     
     
         99 . The cell of    claim 95   , wherein said gene has sites cleaved with restriction endonuclease in the order of Bst NI, Xba I and Bst NI from 5′-terminus of the coding sequence.  
     
     
         100 . The method of    claim 95   , wherein said gene has sites cleaved with restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from 5′-terminus of the coding sequence.  
     
     
         101 . The method of    claim 95   , wherein said gene has the base sequence shown in FIG. 2 ( a ).  
     
     
         102 . The method of    claim 95   , wherein the base sequence of said gene initiates from ATG sequence at position 48 to 50 and has the sequential bases following the ATG sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         103 . The method of    claim 95   , wherein the base sequence of said gene initiates from GCA sequence at position 103 to 110 and has the sequential bases following the GCA sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         104 . The method of    claim 95   , wherein the base sequence of said gene initiates from CCT sequence at position 111 to 113 and has the sequential bases following the CCT sequence up to at least ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         105 . The method of    claim 95   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         106 . The method of    claim 102   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         107 . The method of    claim 103   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         108 . The method of    claim 104   , wherein the base sequence of said gene ends at ACT sequence at position 504 to 506 in FIG. 2 ( a ).  
     
     
         109 . The method of    claim 95   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         110 . The method of    claim 102   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         111 . The method of    claim 103   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         112 . The method of    claim 104   , wherein the base sequence of said gene ends at TGA sequence at position 507 to 509 in FIG. 2 ( a ).  
     
     
         113 . The method of    claim 95   , wherein the base sequence of said gene initiates from A at position 1 and has the sequential bases following the A ending at C at position 801 in FIG. 2 ( a ).  
     
     
         114 . The method of    claim 102   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         115 . The method of    claim 103   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         116 . The method of    claim 104   , wherein the base sequence of said gene ends at C at position 801 in FIG. 2 ( a ).  
     
     
         117 . The method of    claim 102   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         118 . The method of    claim 103   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         119 . The method of    claim 104   , wherein the base sequence of said gene ends at poly (A) in FIG. 2 ( a ).  
     
     
         120 . The method of    claim 101   , wherein the base sequence said gene corresponds to Amino Acid Sequence I in FIG. 2 ( b ).  
     
     
         121 . The method of    claim 101   , wherein the base sequence of said gene corresponds to Amino Acid Sequence II in FIG. 2 ( b ).  
     
     
         122 . The method of    claim 101   , wherein the base sequence of said gene corresponds to Amino Acid Sequence III in FIG. 2 ( b ).  
     
     
         123 . The method of    claim 95   , wherein said procaryotic cell belongs to the genus Escherichia.  
     
     
         124 . The method of    claim 95   , wherein said procaryotic belongs to  Escherichia coli.    
     
     
         125 . The method of    claim 95   , wherein said eucaryotic cell belongs to the genus Saccharomyces.  
     
     
         126 . The method of    claim 95   , wherein said eucaryotic cell belongs to the genus  Saccharomyces cerevicea.    
     
     
         127 . The method of    claim 95   , wherein said eucaryotic cell is a monkey cell transformed with SV-40 constitutively expressing large T antigen.