Rescue of mumps virus from cDNA

A kind of mumps virus, nucleic acid molecule technology, applied in the field of producing as attenuated and/or infectious mumps virus

Inactive Publication Date: 2002-12-11
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The recombinant virus was then collected

Method used

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  • Rescue of mumps virus from cDNA
  • Rescue of mumps virus from cDNA
  • Rescue of mumps virus from cDNA

Examples

Experimental program
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Embodiment 1

[0102] Materials and methods

[0103] cells and viruses. Primary chicken embryo fibroblast (CEF) cells were obtained from SPAFAS Inc., Preston, CT and cultured on Eagle's basal medium (BME) with 5% fetal bovine serum. Hep2 cells, 293 cells, A549 and Vero cells were obtained from the American Type Culture Collection (ATCC) and cultured on Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. The Jeryl Lynn strain of mumps virus was cultured directly on CEF cells in miniature tubes from Mumpsvax(R), lot numbers 0089E, 0656J and 1159H (Merck and Co., Inc., West Point, PA). A recombinant poxvirus Ankara (MVA-T7) expressing bacteriophage T7 RNA polymerase was obtained from B. Moss [(National Institute of Health, Bethsda, MD), see Wyatt et al., 1995].

[0104] 1. Generation of A. mumps virus Jeryl Lynn strain consensus sequence

[0105] Growth of the Jeryl Lynn strain of mumps virus. Directly on primary chicken embryo fibroblasts (CEF, Spafas, Inc.), on...

Embodiment 2

[0219] Rescue reporter gene activity from transfected cells. To help define a system that allows rescue of infectious mumps virus from cDNA, a mumps virus minireplicon containing a CAT reporter gene was assembled. The designed construct is capable of synthesizing negatively polarized RNA minigenomes under the control of the T7 RNA polymerase promoter. The three terminal G residues of the T7 promoter were omitted in the construction of the minireplicon to provide a transcription initiation site from the exact 5' nucleotide of the MUV genome. The HDV ribozyme included in the minireplicon construct allows cleavage of the T7 RNA polymerase transcript to generate a true MUV-specific 3' end. The total number of nucleotides (966) in the MUVCAT small replicon RNA is divided by 6, consistent with the Rule of Six (Calain and Roux, 1993), which states that unless the genome length is a multiple of 6, full replication will not occur. Expression of the CAT gene is under the control of a ...

Embodiment 3

[0223] Full-length mumps virus was recovered from transfected cells. Assemble the full-length MUV cDNA, under the control of the T7 RNA polymerase promoter, synthesize the exact 15,384nt sense RNA copies of the viral genome. Together with the MUVCAT minireplicon, the T7 RNA polymerase promoter sequence was modified to remove the terminal 3 G residues to provide a transcription initiation site from the exact MUV terminal nucleotide. The exact MUV 3' terminal nucleotides of the sense genomic transcripts were generated using HDV ribozymes.

[0224] To recover MUV from cDNA, A549 cells were infected with MVA-T7 expressing T7 RNA polymerase and then transfected with pMUVFL and plasmids expressing MUV NP, P and L. The results of rescue of reporter gene activity from the MUVCAT small replicon and similar work against the related rubella virus SV5 (He et al., 1997; Murphy and Parks, 1997) suggest that the MUV NP, P and L proteins are essential for packaging, replication and then Tra...

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Abstract

The present invention relates to a method of recombining and producing immunogenic composition by rescuing non-segmented antisense single-stranded RNA virus-mumps virus. Other embodiments relate to methods of making attenuated and / or infectious mumps viruses. Prepare recombinant viruses from cDNA clones to make specific changes, including deletions, insertions, substitutions and rearrangements of nucleotides / polynucleotides within the genome.

Description

field of invention [0001] The present invention relates to a method for generating non-segmented antisense single-stranded RNA virus-mumps virus and its immunogenic composition through recombination. Other embodiments relate to methods of producing mumps viruses that are attenuated and / or infectious. Recombinant viruses are prepared from cDNA clones so that they acquire specific changes in the genome. The present invention also relates to the use of the recombinant virus as a carrier to express exogenous genetic information, such as the application of exogenous genes, including the immunogenicity or pharmaceutical composition of pathogens other than mumps, gene therapy and cell targeting. Background of the invention [0002] Enveloped antisense single-stranded RNA viruses are unique in their composition and expression. The genomic RNA of the antisense single-stranded virus serves two template roles in the nucleocapsid: as a template for the synthesis of messenger RNA (mRNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/00A61K39/165A61P31/14C07K14/12C12N1/15C12N1/19C12N1/21C12N5/10C12N7/04C12N15/86
CPCA61K39/00A61K39/165A61K2039/5254C07K14/005C12N7/00C12N15/86C12N2760/18722C12N2760/18734C12N2760/18743C12N2760/18751C12N2760/18761C12N2840/20C12N2840/203A61K39/12A61K2039/70A61P31/14
Inventor D·K·克拉克E·J·约翰逊M·S·西迪S·A·乌登
Owner WYETH LLC
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