Expression vector

Patent metadata

Field	Value
Publication number	US-9657310-B2
Application number	US-201314396929-A
Country	US
Kind code	B2
Filing date	Apr 25, 2013
Priority date	Apr 27, 2012
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

Disclosed are a novel expression vector for efficient expression of recombinant proteins in mammalian cells, a mammalian cell transformed with the vector, and a method for production of the mammalian cell. The expression vector is an expression vector for expression of a mammalian protein and includes a gene expression regulatory site, and a gene encoding the protein downstream thereof, and an internal ribosome entry site further downstream thereof, and a gene encoding a glutamine synthetase further downstream thereof, and a dihydrofolate reductase gene downstream of either the same gene expression regulatory site or another gene expression regulatory site in addition to the former.

First claim

The invention claimed is:

1. An expression vector for expression of a protein, comprising a gene expression regulatory site (A), and a gene encoding the protein downstream thereof, an internal ribosome entry site further downstream thereof, a gene encoding a glutamine synthetase still further downstream thereof, and additionally a dihydrofolate reductase gene downstream of another gene expression regulatory site (B) in addition to the former,
wherein the internal ribosome entry site is derived from the 5′ untranslated region of mouse encephalomyocarditis virus,
wherein the internal ribosome entry site is that in which the 2nd or 3rd start codon from the 5′ end has been destroyed by introducing one or more mutations into the nucleotide sequence of a wild-type internal ribosome entry site, and
wherein the internal ribosome entry site comprises the nucleotide sequence set forth as SEQ ID NO:4.

2. The expression vector according to claim 1 , wherein the gene expression regulatory site (A) is an elongation factor 1 promoter, and the gene expression regulatory site (B) is an SV40 early promoter.

3. The expression vector according to claim 1 , further comprising, either in the region between the gene encoding the protein and the internal ribosome entry site or in the region downstream of the gene encoding the glutamine synthetase, another internal ribosome entry site in addition to the former internal ribosome entry site, and a drug resistance gene downstream thereof.

4. The expression vector according to claim 1 , wherein the expression vector, in addition to the gene expression regulatory site (A) and the gene expression regulatory site (B), further comprises another gene expression regulatory site (C) and a drug resistance gene downstream thereof.

5. The expression vector according to claim 3 , wherein the drug resistance gene is a neomycin resistance gene.

6. The expression vector according to claim 1 , wherein the gene encoding the protein is a human-derived gene.

7. The expression vector according to claim 6 , wherein the human-derived gene is selected from the group consisting of the genes encoding lysosomal enzymes, tissue plasminogen activator (t-PA), blood coagulation factors, erythropoietin, interferon, thrombomodulin, thyroid stimulating hormone (TSH), follicle-stimulating hormone, granulocyte colony-stimulating factor (G-CSF), and antibodies.

8. The expression vector according to claim 6 , wherein the human-derived gene is a gene encoding a lysosomal enzyme.

9. The expression vector according to claim 8 , wherein the lysosomal enzyme is selected from the group consisting of α-galactosidase A, iduronate-2-sulfatase, glucocerebrosidase, galsulfase, α-L-iduronidase, and acid α-glucosidase.

10. The expression vector according to claim 6 , wherein the human-derived gene is a gene encoding erythropoietin.

11. An isolated mammalian cell transformed with the expression vector according to claim 1 .

12. The isolated mammalian cell according to claim 11 , wherein the cell lacks the intrinsic dihydrofolate reductase gene.

13. The isolated mammalian cell according to claim 11 , wherein the cell is a CHO cell.

14. A method for production of a transformed cell expressing a gene encoding the protein comprising the steps of:
(a) introducing the expression vector according to claim 1 into an isolated mammalian cell,
(b) subjecting the mammalian cell containing the introduced expression vector to a selective culture in the presence of an inhibitor of dihydrofolate reductase, and
(c) subjecting the cells selected through the selective culture to a further selective culture in the presence of an inhibitor of glutamine synthetase.

15. A method for production of a transformed cell expressing a gene encoding the protein comprising the steps of:
(a) introducing the expression vector according to claim 3 into an isolated mammalian cell,
(b) subjecting the mammalian cell containing the introduced expression vector to a selective culture in the presence of an inhibitor of dihydrofolate reductase, and
(c) subjecting the cells selected through the selective culture to a further selective culture in the presence of an inhibitor of glutamine synthetase,
further comprising the step of subjecting the mammalian cell containing the introduced expression vector to a selective culture in the presence of a drug corresponding to the drug resistance gene.

16. The expression vector according to claim 4 , wherein the drug resistance gene is a neomycin resistance gene.

Assignees

• Japan Chem Res

Inventors

• Takahashi Kenichi
• Kakimoto Shinji

Classifications

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N2800/107

  for mammalian · CPC title

• C12N2830/50

  regulating RNA stability, not being an intron, e.g. poly A signal · CPC title

• C12N2840/60

  from viruses · CPC title

• C07K16/2887

  against CD20 · CPC title