| 主权项 |
1. A method selected from the group consisting of (I), (II) and (III):
(I) a method for producing a protein of interest, comprising the steps of:
(1) introducing an expression vector into a mammalian cell adapted to suspension culture, wherein the expression vector comprises a DNA sequence encoding a protein of interest and a selectable marker, and wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals;(2) integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell to obtain a mammalian cell capable of expressing the protein of interest; and(3) culturing the mammalian cell to produce the protein of interest, (II) a method for producing a protein of interest, comprising the steps of:
(1) simultaneously introducing expression vector (a) and expression vector (b) into a mammalian cell adapted to suspension culture, wherein expression vector (a) comprises a DNA sequence encoding a protein of interest and a selectable marker, wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals, and wherein expression vector (b) comprises a DNA sequence encoding a transposase which recognizes the transposon sequences, and wherein the transposase has an activity of integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell;(2) transiently expressing the transposase encoded by expression vector (b) to integrate the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell; and(3) culturing the mammalian cell to produce the protein of interest, and (III) a method for obtaining a mammalian cell capable of expressing a protein of interest, comprising the steps of:
(1) introducing an expression vector into a mammalian cell adapted to suspension culture, wherein the expression vector comprises a DNA sequence encoding a protein of interest and a selectable marker, and wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals; and(2) integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell, wherein in the methods of (I)-(III), the transposon sequences are nucleotide sequences derived from a pair of Tol1 transposons or derived from a pair of Tol2 transposons, and the mammalian cell adapted to suspension culture is selected from the group consisting of: a CHO cell adapted to suspension-culture; a PER.C6 cell; a rat myeloma YB2/0 cell; and a mouse myeloma NS0 cell adapted to suspension-culture. |
