Plasmid vector, method for detecting gene promoter activity, and assay kit
AssigneesKabushiki Kaisha Toshiba, Toshiba Medical Systems Corporation
InventorsEiichi Akahoshi, Mitsuko Ishihara
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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.
Claims19
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.
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.
Dependencies are inferred from the claim text.