Plasmid system for multigene expression
View Patent ↗The present invention provides a plasmid system which facilitates the construction of a single amplifiable plasmid that, having the potential to accommodate many independent expression cassettes, has the ability to express multi-subunit complex proteins such as antibodies and receptors.
1. An isolated host cell comprising a recombined amplifiable vector which is the product of a method comprising:
(a) introducing one or more expression cassettes into a first multiple cloning site of a first universal transfer vector wherein said first universal transfer vector comprises the plasmid map set forth in FIG. 2 ; wherein the first multiple cloning site comprises nucleotides 620-766 of the nucleotide sequence set forth in SEQ ID NO: 2; and wherein said first multiple cloning site is in said orientation or in a reverse orientation;
(b) introducing one or more expression cassettes, different from the cassettes introduced into said first universal transfer vector, into a second multiple cloning site of a second universal transfer vector which second multiple cloning site comprises nucleotides 620-772 of the nucleotide sequence set forth in SEQ ID NO: 1; wherein said second multiple cloning site is in said orientation or in a reverse orientation; and
(c) introducing said cassettes from steps (a) and (b) into a third multiple cloning site of an amplifiable vector; wherein said amplifiable vector comprises the plasmid map set forth in FIG. 3 ; wherein said third multiple cloning site comprises nucleotides 5037-5183 of the nucleotide sequence set forth in SEQ ID NO: 3; wherein said third multiple cloning site is in said orientation or in a reverse orientation; and
(d) introducing said amplifiable vector comprising said cassettes from steps (a) and (b) into a host cell.
2. An isolated host cell comprising a recombined amplifiable vector which comprises the nucleotide sequence set forth in SEQ ID NO: 26 which is the product of a method comprising:
(a) introducing one or more expression cassettes into a first multiple cloning site of a first universal transfer vector which first multiple cloning site comprises nucleotides 620-766 of the nucleotide sequence set forth in SEQ ID NO: 2; wherein said first multiple cloning site is in said orientation or in a reverse orientation;
(b) introducing one or more expression cassettes, different from the cassettes introduced into said first universal transfer vector, into a second multiple cloning site of a second universal transfer vector which second multiple cloning site comprises nucleotides 620-772 of the nucleotide sequence set forth in SEQ ID NO: 1; wherein said second multiple cloning site is in said orientation or in a reverse orientation; and
(c) introducing said cassettes from steps (a) and (b) into a third multiple cloning site of an amplifiable vector wherein said third multiple cloning site comprises nucleotides 5037-5183 of the nucleotide sequence set forth in SEQ ID NO: 3; wherein said third multiple cloning site is in said orientation or in a reverse orientation; and
(d) introducing said amplifiable vector comprising said cassettes from steps (a) and (b) into a host cell.