ES2509350T3 - Novirhabdovirus recombinantes y usos de los mismos - Google Patents
Novirhabdovirus recombinantes y usos de los mismos Download PDFInfo
- Publication number
- ES2509350T3 ES2509350T3 ES07804957.4T ES07804957T ES2509350T3 ES 2509350 T3 ES2509350 T3 ES 2509350T3 ES 07804957 T ES07804957 T ES 07804957T ES 2509350 T3 ES2509350 T3 ES 2509350T3
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- ihnv
- novirhabdovirus
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- 241001112535 Novirhabdovirus Species 0.000 title abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 101150082239 G gene Proteins 0.000 abstract description 2
- 239000002773 nucleotide Substances 0.000 abstract 2
- 125000003729 nucleotide group Chemical group 0.000 abstract 2
- 108020004511 Recombinant DNA Proteins 0.000 abstract 1
- 230000008488 polyadenylation Effects 0.000 abstract 1
- 230000005026 transcription initiation Effects 0.000 abstract 1
- 230000005030 transcription termination Effects 0.000 abstract 1
- 230000009261 transgenic effect Effects 0.000 abstract 1
- 241000711804 Infectious hematopoietic necrosis virus Species 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 7
- 241000546112 Infectious salmon anemia virus Species 0.000 description 6
- 101710154606 Hemagglutinin Proteins 0.000 description 5
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 5
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 5
- 101710176177 Protein A56 Proteins 0.000 description 5
- 241000711825 Viral hemorrhagic septicemia virus Species 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000000185 hemagglutinin Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 241000710921 Infectious pancreatic necrosis virus Species 0.000 description 2
- 108010046722 Thrombospondin 1 Proteins 0.000 description 2
- 102100036034 Thrombospondin-1 Human genes 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 208000000230 African Trypanosomiasis Diseases 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 206010006811 Bursitis Diseases 0.000 description 1
- 101150034814 F gene Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 101150039660 HA gene Proteins 0.000 description 1
- 241000702626 Infectious bursal disease virus Species 0.000 description 1
- 101900217743 Infectious pancreatic necrosis virus Capsid protein VP2 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 101710081079 Minor spike protein H Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 241000277331 Salmonidae Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 208000029080 human African trypanosomiasis Diseases 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 208000019116 sleep disease Diseases 0.000 description 1
- 201000002612 sleeping sickness Diseases 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20041—Use of virus, viral particle or viral elements as a vector
- C12N2760/20043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Una construcción de ADN recombinante que comprende: a) una región que comprende una secuencia de terminación de la transcripción/poliadenilación del gen M de novirhabdovirus, estando definida dicha región por la siguiente secuencia: VHHAGAYAGAAAAAAA (SEC ID Nº: 4), en la que A, T, G, V, H e Y tienen su significado habitual en el código de nucleótidos de IUPAC; b) una región que comprende una secuencia de inicio de la transcripción del gen G de novirhabdovirus, definiéndose dicha región por la siguiente secuencia: GCACDWKWGTGY (SEC ID Nº: 5), en la que A, T, G, C, D, W, K e Y tienen su significado habitual en el código de nucleótidos de IUPAC; en la que dicha región a) está seguida o dicha región b) está precedida por un dinucleótido intergénico no transgénico de un novirhabdovirus, c) una región que comprende una fase abierta de lectura que codifica una proteína de interés; en la que las regiones a), b) y c) se disponen en un orden seleccionado del grupo que consiste en: a-b-c y b-c-a.
Description
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E07804957
01-10-2014
(continuación)
Leyenda de las tablas
SDV: Virus de la Enfermedad del Sueño ISAV: Virus de la Anemia Infecciosa del Salmón VHSV: Virus de la Septicemia Hemorrágica Viral IBDV: Virus de la Bursitis Infecciosa IPNV: Virus de la necrosis pancreática infecciosa 6KE1: gen de la glucoproteína E1 de SDV CAP: gen de la cápsida de SDV E3E2: gen de la glucoproteína E2 de SDV EGFP: Proteína verde fluorescente potenciada F5: gen F de ISAV GVHSV: gen de glucoproteína G de VHSV HA: gen de HA de ISAV IL1β: Interleucina 1 de la trucha arco iris LUCFF: Luciferasa de luciérnaga LUCRR: luciferasa de Renilla VP2: gen de VP2 de IPNV ΔG: supresión del gen G de IHNV
Las reservas virales de cada uno de los virus producidos están constituidas por pases sucesivos en cultivo de células (EPC) del sobrenadante tomado 7 días después de la transfección (sobrenadante PO). Las células se infectan con dilución 1/100 del sobrenadante clarificado. Después de 3 pases, se retiran los sobrenadantes cuando la capa de células se destruye por el efecto citopático del virus; esto sucede habitualmente de 3 a 6 días después de la infección. Las reservas virales se titulan después por dilución limitante.
La Figura 3 muestra ejemplos de título viral de diversos virus recombinantes en cultivo de células EPC. Aunque el título viral para todos los virus recombinantes está en el mismo intervalo (108 UFP/ml), los virus recombinantes que comprenden tres unidades de expresión adicionales tienen un crecimiento más lento en comparación con el virus de tipo silvestre. En general se tarda seis días en obtener un efecto citopático completo en células EPC en lugar de tres días para el IHNV de tipo silvestre.
Se produjeron proteínas de interés usando los IHNV recombinantes enumerados en las Tablas I, II y III.
IHNV recombinante que expresa las proteínas estructurales del virus de la enfermedad del sueño
Se infectan células EPC con IHNV de tipo silvestre, IHNV-6KE1, IHNV-CapE3E2 o IHNV-E3E26KE1 a una MOI (multiplicación de infección) de 0,02 (0,02 UFP/célula). Las células se lisan 48 horas después de la infección, y los lisados se analizan por transferencia de Western usando un anticuerpo monoclonal dirigido contra la glucoproteína E1 de SDV.
Los resultados se muestran en la Figura 4: Carril 1: IHNV-E3E26KE1; Carril 2: IHNV-CapE3E2; Carril 3: IHNV-6KE1; Carril 4: IHNV de tipo silvestre.
IHNV recombinante que expresa la glucoproteína hemaglutinina (HA) de virus de la anemia infecciosa del salmón (ISAV)
Se infectan células EPC con IHNV de tipo silvestre o IHNV-HAISAV, como se ha descrito anteriormente. Las células se lisan 2 días después de la infección, y los lisados se inmunoprecipitan con anticuerpo monoclonal anti-HA y se analizan por transferencia de Western usando un anticuerpo policlonal dirigido contra la glucoproteína HA de ISAV.
Los resultados se muestran en la Figura 5: Carril 1: IHNV de tipo silvestre; Carril 2: IHNV-ISAV HA.
INHV recombinante que expresa la glucoproteína G del virus de la septicemia hemorrágica viral (VHSV) fusionada con una proteína indicadora
Se infectan células EPC, como se ha descrito anteriormente, con IHNV de tipo silvestre, IHNV-GVHSV, IHNV-GVHSV/LUCRR, IHNVGVHSV/EGFP, o con el sobrenadante de células EPC no infectadas (infectadas con simulación). Las células se lisan 2 días después de la infección, y los lisados se analizan por transferencia de Western usando un anticuerpo monoclonal dirigido contra la glucoproteína G de VHSV.
9
Claims (1)
-
imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP06290982A EP1867725A1 (en) | 2006-06-16 | 2006-06-16 | Recombinant novirhabdoviruses and uses thereof |
| EP06290982 | 2006-06-16 | ||
| PCT/IB2007/002756 WO2007144773A2 (en) | 2006-06-16 | 2007-06-15 | Recombinant novirhabdoviruses and uses thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2509350T3 true ES2509350T3 (es) | 2014-10-17 |
Family
ID=37192589
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES07804957.4T Active ES2509350T3 (es) | 2006-06-16 | 2007-06-15 | Novirhabdovirus recombinantes y usos de los mismos |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US8859276B2 (es) |
| EP (2) | EP1867725A1 (es) |
| JP (2) | JP2009539396A (es) |
| AU (1) | AU2007258875B8 (es) |
| CA (1) | CA2655290C (es) |
| DK (1) | DK2032708T3 (es) |
| ES (1) | ES2509350T3 (es) |
| NO (1) | NO20085201L (es) |
| PL (1) | PL2032708T3 (es) |
| PT (1) | PT2032708E (es) |
| SI (1) | SI2032708T1 (es) |
| WO (1) | WO2007144773A2 (es) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2996856B1 (fr) | 2012-10-15 | 2015-06-05 | Agronomique Inst Nat Rech | Novirhabdovirus recombinant utilisable comme vecteur d'antigenes |
| CL2014003146A1 (es) | 2014-11-20 | 2015-06-12 | Univ Santiago Chile | Método para producir virus arn monocatenario negativo; plásmido recombinante funcional en células animales, que consta de un esqueleto pss-urg; método de obtención de partículas virales; y uso para expresar arn, proteínas autógenas o exógenas al virus. |
| EP4314305A4 (en) * | 2021-03-26 | 2025-07-30 | Univ North Carolina State | RECOMBINANT VIRAL EXPRESSION VECTORS AND METHODS OF USE |
-
2006
- 2006-06-16 EP EP06290982A patent/EP1867725A1/en not_active Withdrawn
-
2007
- 2007-06-15 SI SI200731530T patent/SI2032708T1/sl unknown
- 2007-06-15 EP EP07804957.4A patent/EP2032708B1/en active Active
- 2007-06-15 AU AU2007258875A patent/AU2007258875B8/en not_active Ceased
- 2007-06-15 CA CA2655290A patent/CA2655290C/en not_active Expired - Fee Related
- 2007-06-15 ES ES07804957.4T patent/ES2509350T3/es active Active
- 2007-06-15 JP JP2009514932A patent/JP2009539396A/ja active Pending
- 2007-06-15 WO PCT/IB2007/002756 patent/WO2007144773A2/en not_active Ceased
- 2007-06-15 DK DK07804957.4T patent/DK2032708T3/da active
- 2007-06-15 PL PL07804957T patent/PL2032708T3/pl unknown
- 2007-06-15 US US12/304,812 patent/US8859276B2/en active Active
- 2007-06-15 PT PT78049574T patent/PT2032708E/pt unknown
-
2008
- 2008-12-12 NO NO20085201A patent/NO20085201L/no unknown
-
2012
- 2012-10-10 JP JP2012224795A patent/JP2013055940A/ja active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU2007258875B8 (en) | 2013-08-29 |
| JP2009539396A (ja) | 2009-11-19 |
| US20090208529A1 (en) | 2009-08-20 |
| EP2032708B1 (en) | 2014-07-30 |
| WO2007144773A3 (en) | 2008-02-28 |
| EP2032708A2 (en) | 2009-03-11 |
| AU2007258875A1 (en) | 2007-12-21 |
| PT2032708E (pt) | 2014-10-28 |
| SI2032708T1 (sl) | 2014-11-28 |
| AU2007258875B2 (en) | 2013-07-11 |
| JP2013055940A (ja) | 2013-03-28 |
| DK2032708T3 (da) | 2014-10-20 |
| PL2032708T3 (pl) | 2014-12-31 |
| EP1867725A1 (en) | 2007-12-19 |
| CA2655290A1 (en) | 2007-12-21 |
| WO2007144773A2 (en) | 2007-12-21 |
| US8859276B2 (en) | 2014-10-14 |
| NO20085201L (no) | 2009-03-05 |
| CA2655290C (en) | 2016-04-19 |
| AU2007258875A8 (en) | 2013-08-29 |
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