Nodavirus-like DNA expression vector and uses therefor
AssigneeThe UAB Research Foundation
InventorsLaurence A. Ball, Kyle L. Johnson, Karen N. Johnson, B. Duane Price
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Abstract
The present invention describes the production of a nodavirus-based DNA vector that drives abundant expression of foreign genes in a wide variety of cell types. The DNA plasmid is initially transcribed by a host-cell RNA polymerase to produce primary transcripts from which a nodaviral RNA-dependent RNA polymerase (RNA replicase) is translated. These primary transcripts are then amplified by the RNA replicase in an autonomous, cytoplasmic RNA replication. Such a vector is a useful addition to the current arsenal of expression vectors, and well suited to laboratory-scale and larger-scale expression of transcripts and/or proteins in eukaryotic cells.
Claims28
An isolated cDNA encoding a Nodamura virus RNA2 genome segment and having the sequence shown in SEQ ID No. 2.
An isolated cDNA encoding a Pariacoto virus RNA1 genome segment and having the sequence shown in SEQ ID No. 3.
An isolated cDNA encoding a Pariacoto virus RNA2 genome segment and having the sequence shown in SEQ ID No. 4.
A nodavirus-based DNA expression vector, wherein said expression vector comprises:
- i) a DNA-dependent RNA polymerase promoter operably linked to a cDNA encoding a nodavirus RNA-dependent RNA polymerase (RNA replicase), wherein said nodavirus is selected from the group consisting of flock house virus, Nodamura virus, and Pariacoto virus
- ii) a heterologous gene inserted at the 5′ end of a cDNA encoding a nodavirus RNA2 transcript, wherein said nodavirus is selected from the group consisting of flock house virus Nodamura virus, and Pariacoto virus;
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- iii) a cDNA encoding a ribozyme, wherein upon transcription of said expression vector, said ribozyme cleaves the transcripts of said expression vector and generates a 3′ end recognized by said RNA replicase, and wherein said recognition results in replication of said transcripts.
Dependencies are inferred from the claim text.