# US 9,040,294 B2 · Plasmid vector, method for detecting gene promoter activity, and assay kit

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- Number: US 9,040,294 B2
- Document ID: US9040294B2
- Status: Granted
- Kind: B2
- Application number: 13/593,912
- Filed: 2012-08-24
- Published: 2013-09-05
- Granted: 2015-05-26
- Earliest claimed: 2011-08-25
- Assignees: Kabushiki Kaisha Toshiba; Toshiba Medical Systems Corporation
- Applicants: Eiichi Akahoshi; Mitsuko Ishihara
- Inventors: Eiichi Akahoshi; Mitsuko Ishihara
- Web page: https://patentgrep.com/patent/US9040294B2

## CPC

- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/69: Increasing the copy number of the vector
- C12N 15/85: for animal cells
- C12Q 1/66: involving luciferase
- C12Q 1/6897: involving reporter genes operably linked to promoters
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.

## Claims

### Claim 1

A method for detecting the activity of a gene promoter, comprising:
introducing a circular plasmid vector into a cell,
culturing the cell under a condition in which the cell divides and proliferates so that the plasmid vector can be amplified in accordance with the activity of a gene promoter included in the plasmid vector, and
measuring the amount of expression of the reporter gene included in the plasmid vector in the cultured cell,
wherein the circular plasmid vector comprises:
a gene promoter, which is activated in a host cell that satisfies an activation condition of the gene promoter,
a first base sequence of a first gene that encodes a reporter protein for visualizing the activity of the gene promoter, the first base sequence functionally linked downstream of the gene promoter,
an internal ribosome entry site that is linked downstream of the first gene,
a second base sequence of a second gene that encodes a replication origin-binding protein, the second base sequence linked downstream of the internal ribosome entry site, wherein the second gene is a large T-antigen gene of Simian Virus 40 has the base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4,
a transcription termination signal sequence for encoding a signal for terminating the transcription of the first base sequence and second base sequence, the transcription termination signal sequence linked downstream of the second base sequence, and
a replication origin sequence that is recognized by the replication origin-binding protein, linked downstream of the transcription termination signal sequence.

### Claim 2 (depends on 1)

The detection method according to claim 1, wherein the cell is a cell derived from a human or a simian.

### Claim 3 (depends on 1)

The detection method according to claim 1, wherein the cell is a cell derived from a mouse or a rat.

### Claim 4 (depends on 1)

The detection method according to claim 1, wherein the reporter gene is a luciferase gene, a β-galactosidase gene, a blue fluorescent protein gene, a green fluorescent protein gene or a red fluorescent protein gene, and
the measuring comprises measuring the amount of light emission or the amount of fluorescence of a translation product of the reporter gene as the amount of expression by utilizing an optical detection apparatus.

### Claim 5 (depends on 1)

The detection method according to claim 1, wherein the reporter gene is a nitric monoxide synthase gene or a xanthine oxidase gene, and
the measuring comprises measuring the amount of active oxygen generated by a translation product of the reporter gene as the amount of expression by utilizing an electron spin resonance apparatus.

### Claim 6 (depends on 1)

The detection method according to claim 1, wherein the reporter gene is a heavy metal-binding protein, and
the measuring comprises measuring the amount of a heavy metal bound to a translation product of the reporter gene as the amount of expression by utilizing a magnetic resonance imaging apparatus, a nuclear medicine diagnostic apparatus or an X-ray computed tomographic apparatus.

### Claim 7 (depends on 1)

The detection method according to claim 1, wherein the plasmid vector is self-amplified by binding of a replication origin-binding protein that is synthesized by the expression of the second gene included in the plasmid vector to a replication origin sequence included in the plasmid vector.

### Claim 8 (depends on 1)

The detection method according to claim 1, wherein the cell is a cell in a pre-disease state at an early stage of a disease.

### Claim 9 (depends on 1)

The detection method according to claim 1, wherein the gene promoter can be activated under a condition in which the cell divides and proliferates.

### Claim 10

An assay kit, comprising:
a circular plasmid vector and
a reagent for detecting the expression of a gene that encodes a reporter protein included in the cyclic plasmid vector in a cell;
wherein the circular plasmid vector comprises:
a gene promoter, which is activated in a host cell that satisfies an activation condition of the gene promoter,
a first base sequence of a first gene that encodes a reporter protein for visualizing the activity of the gene promoter, the first base sequence functionally linked downstream of the gene promoter,
an internal ribosome entry site that is linked downstream of the first gene,
a second base sequence of a second gene that encodes a replication origin-binding protein, the second base sequence linked downstream of the internal ribosome entry site, wherein the second gene is a large T-antigen gene of Simian Virus 40 has the base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4,
a transcription termination signal sequence for encoding a signal for terminating the transcription of the first base sequence and second base sequence, the transcription termination signal sequence linked downstream of the second base sequence, and
a replication origin sequence that is recognized by the replication origin-binding protein, linked downstream of the transcription termination signal sequence.

### Claim 11 (depends on 10)

The assay kit according to claim 10, which further comprises a reagent for introducing the circular plasmid vector into the cell.

### Claim 12 (depends on 10)

The assay kit according to claim 10, which further comprises a reagent for extracting the reporter protein from the cell.

### Claim 13 (depends on 10)

The assay kit according to claim 10, wherein the first gene is a reporter gene selected from the group consisting of a luciferase gene, a β-galactosidase gene, a nitric monoxide synthase gene, a xanthine oxidase gene, a blue fluorescent protein gene, a green fluorescent protein gene, a red fluorescent protein gene, and a heavy metal-binding protein gene.

### Claim 14 (depends on 10)

The assay kit according to claim 10, wherein the replication origin sequence is a replication origin sequence of Simian Virus 40.

### Claim 15 (depends on 10)

The assay kit according to claim 10, wherein the large T-antigen gene has the base sequence of SEQ ID NO: 1.

### Claim 16 (depends on 10)

The assay kit according to claim 10, wherein the large T-antigen gene has the base sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

### Claim 17 (depends on 10)

The assay kit according to claim 10, wherein the second gene is an EBNA-1 gene of Epstein-Barr virus, and the replication origin sequence is a replication origin sequence of Epstein-Barr virus.

### Claim 18 (depends on 10)

The assay kit according to claim 10, wherein the replication origin sequence is a replication origin sequence of Mouse polyomavirus.

### Claim 19 (depends on 10)

The assay kit according to claim 10, wherein the gene promoter is a gene promoter that is activated at an early stage of a disease, or a gene promoter that is activated in response to an environmental stimulus.