WO1999025837A2 - Verfahren zur gewinnung von virusähnlichen partikeln auf der basis von polyomaviren - Google Patents
Verfahren zur gewinnung von virusähnlichen partikeln auf der basis von polyomaviren Download PDFInfo
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- WO1999025837A2 WO1999025837A2 PCT/DE1998/003329 DE9803329W WO9925837A2 WO 1999025837 A2 WO1999025837 A2 WO 1999025837A2 DE 9803329 W DE9803329 W DE 9803329W WO 9925837 A2 WO9925837 A2 WO 9925837A2
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- WIPO (PCT)
- Prior art keywords
- particles
- virus
- yeast
- yeast cells
- vpl
- Prior art date
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- 239000002245 particle Substances 0.000 title claims abstract description 30
- 241001505332 Polyomavirus sp. Species 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title abstract description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 18
- 210000005253 yeast cell Anatomy 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 5
- 101710197658 Capsid protein VP1 Proteins 0.000 claims abstract description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 17
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 241000829333 Mesocricetus auratus polyomavirus 1 Species 0.000 claims description 6
- 239000013613 expression plasmid Substances 0.000 claims description 5
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- 108010080698 Peptones Proteins 0.000 claims description 4
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
- C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
- C12N2710/22023—Virus like particles [VLP]
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
- C12N2710/22051—Methods of production or purification of viral material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to a method for obtaining virus-like particles based on polyomaviruses. Areas of application are medicine, u. a. Gene therapy, vaccine development and diagnostics, as well as veterinary medicine.
- virus-like particles Compared to such virus vectors, virus-like particles (VLPs) have the advantage that they are not infectious and have no tumorigenic potential.
- VLPs can be generated by expression of viral capsid and envelope proteins in various heterologous expression systems (overview in Ulrich et al., Adv. Virus Res. 50, 1997).
- Virus-like particles based on the main capsid protein VP1 ('virus protein 1') from polyomaviruses received special attention for gene transfer. So far, VP1 of various polyomaviruses have been expressed in insect cells.
- the recombinant VPI of non-human and human polyomaviruses spontaneously assembled into VLPs in the insect cells (Montross et al., 1991, J. Virol.
- capsomeres have been successful to express, which can be re-associated to VLPs in vitro (Salunke et al., 1986, Cell 46, 895-904).
- the aim of the invention is to develop a method for obtaining virus-like particles for gene therapy, diagnostics and vaccine development. This process is intended to enable the highly efficient production of non-infectious, endotoxin-free virus-like particles that are suitable for human applications.
- the essential component of the method according to the invention is a yeast expression system which allows the high expression of capsid-like particles of polyomaviruses. These particles can be isolated in large quantities from yeast cells and purified by simple centrifugation steps.
- the method involves PCR amplification and cloning of a polyomavirus VPI coding sequence.
- This DNA sequence is built into a yeast expression vector with a yeast-specific expression unit (promoter and transcription stop signal).
- the VP1 is expressed in yeast cells, preferably in the Saccharomyces cerevisiae strain AH 22 (leu2 his4).
- the virus-like particles are sedimented by ultracentrifugation (sucrose cushion).
- the particles are cleaned by subsequent ultracentrifugation in a cesium chloride gradient.
- the subsequent analysis of the particles is carried out by electron microscopy.
- the invention makes available a novel expression system for virus-like particles of polyomaviruses, which has significant advantages over the previously available systems in E. coli or insect cells.
- yeast expression systems allow the biotechnological production of antigens.
- yeasts have no toxins that would cause undesirable side effects in human or veterinary applications.
- yeast systems are already approved for human vaccine production: the recombinant HBV vaccine is based on yeast-expressed HBsAg.
- yeast cells synthesize the polyomavirus VP1 in large quantities.
- the VPl particles can be isolated and purified in large quantities using simple methods. You are u. a. can be used for the detection of infections with human polyomaviruses, as a vehicle for gene transfer, for a vaccine development against human polyomaviruses or against other viruses (eg by presenting foreign epitopes on HaPV-VPl capsids).
- HaPV virions are isolated from epitheliomas of HaPV-infected Z3 hamsters and purified by sucrose / cesium chloride gradient centrifugation.
- the virions are denatured by adding sample buffer, separated in the SDS polyacrylic gel and then transferred to a PVDF membrane.
- the VP1 protein band is cut out and sequenced using the Edman degradation at the N-terminus. According to the N-terminal protein sequence, molecular weight and predicted open reading frame the VPl-coding sequence is amplified by means of PCR.
- Viral DNA is used as a template and is obtained from HaPV virions by phenol-chloroform extraction and subsequent ethanol precipitation.
- oligonucleotide primers are used for the PCR, which contain the initiation codon (ATG) or termination codon (TAA; complementary sequence) and cleavage sites for the restriction endonuclease Xbal at the 5 'and 3' ends:
- VPl-Xba-N2 5'-A CGT CTA GAT ATG GCC CCA AAA AGA AAA AGC GGC-3 'VPl-Xba-C: 5'-TCT AGA_TTA GTT TGC TGG TTT TGC AGG GGG-3'
- pFR36 E. coli vector
- yeast-specific promoter (GALIO-PYKI hybrid promoter, which consists of a -138 to -511 nucleotide fragment of GAL10 with GAL1-10 UAS sequences and the -4 to -271 sequence of PYK1),
- the VPl-coding segment (with its own translation initiation and termination signal) is cut out of pFR36-VPl with Xbal and ligated into the yeast expression vector pFX7 linearized with Xbal. Recombinant plasmids are selected after transformation in E. coli K12 (XL-1 Blue) and characterized by restriction mapping. 3. Expression of the main capsid protein from polyomavirus in yeast
- the HaPV-VPl expression plasmid is transformed in yeast cells, preferably in Saccharomyces cerevisiae, strain AH22 (leu2 his4).
- the transformed yeast cells are grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) with 5 mM formaldehyde. After culturing for 30 minutes at 30 ° C., the cells are harvested, re-inoculated with formaldehyde in YEPG medium (1% yeast extract, 2% peptone, 3% galactose) and cultivated for a further 24 hours. An aliquot is taken for the expression control and denatured with sample buffer. After separation in the SDS polyacrylamide gel, the recombinant HaPV-VP1 is detected in a Western blot using a VP1-specific rabbit serum (Fig. 2).
- the yeast cells grown as described under 3 are resuspended after sedimentation in 1 ml of lysis buffer (20 mM sodium phosphate, pH 7.2; 100 mM NaCl; 2 M PMSF) and after adding 1 g of glass beads (0.5 mm) Vortexing for 4 minutes at 4 ° C.
- the resulting lysates are diluted with twice the volume of lysis buffer and centrifuged at 5000 g for 10 minutes.
- 8.5 ml of a 45% sucrose solution (w / v; in lysis buffer) are overlaid with 1.5 ml of the supernatant.
- the particles are sedated by centrifugation at 100,000 g for 4 hours in a Beckman Ti70.1 rotor.
- the sediment is resuspended in lysis buffer (20 mM sodium phosphate, pH 7.2; 100 mM NaCl and 2 mM PMSF).
- a previously layered cesium chloride gradient (1.38, 1.35, 1.32, 1.29, 1.26 and 1.23 g / ml in 50 mM Tris-HCl, pH 7.5) is loaded with this suspension and 15 Centrifuged for hours at 100,000 g in the Beckman Ti70.1 rotor. Fractions are collected and analyzed by SDS-polyacylamide gel electrophoresis and Western blot. The formation of HaPV-VPl particles is demonstrated by negative staining with 1% uranyl acetate and electron microscopic analysis (Fig. 3). Fractions containing particles are dialyzed against lysis buffer.
- HaPV-VPl serum (b) or a serum of a HaPV-infected
- Lanes B and C HaPV-VPl expressing yeast cells
- Lane A yeast cells without VPI expression plasmid
- the VPl protein band is marked by arrows.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98965067A EP1030923B1 (de) | 1997-11-13 | 1998-11-13 | Verfahren zur gewinnung von virusähnlichen partikeln auf der basis von polyomaviren |
AT98965067T ATE288961T1 (de) | 1997-11-13 | 1998-11-13 | Verfahren zur gewinnung von virusähnlichen partikeln auf der basis von polyomaviren |
DE59812566T DE59812566D1 (de) | 1997-11-13 | 1998-11-13 | Verfahren zur gewinnung von virusähnlichen partikeln auf der basis von polyomaviren |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19750220.2 | 1997-11-13 | ||
DE19750220A DE19750220A1 (de) | 1997-11-13 | 1997-11-13 | Verfahren zur Gewinnung von virusähnlichen Partikeln auf der Basis von Polyomaviren |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1999025837A2 true WO1999025837A2 (de) | 1999-05-27 |
WO1999025837A3 WO1999025837A3 (de) | 1999-07-15 |
Family
ID=7848557
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE1998/003329 WO1999025837A2 (de) | 1997-11-13 | 1998-11-13 | Verfahren zur gewinnung von virusähnlichen partikeln auf der basis von polyomaviren |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1030923B1 (de) |
AT (1) | ATE288961T1 (de) |
DE (2) | DE19750220A1 (de) |
WO (1) | WO1999025837A2 (de) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10055886A1 (de) * | 2000-11-08 | 2002-05-29 | Humboldt Uni Zu Berlin Univers | Impfstoffe, die rekombinante Hantavirusproteine enthalten, Verfahren zu iher Herstellung und ihre Verwendung |
LT5414B (lt) | 2005-04-13 | 2007-04-25 | Biotechnologijos Institutas | Monokloninių antikūnų gavimo būdas, hibridomos, gautos šiuo būdu ir rekombinantinės chimerinės į virusus panašios dalelės su įterptais kitų baltymų fragmentais kaip imunogenai hibridomų gavimui šiuo būdu |
IL176377A0 (en) | 2006-06-18 | 2006-10-05 | Ariella Oppenheim | Viral capsid proteins and any peptides or compositions thereof for the treatment of pathologic disorders |
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1997
- 1997-11-13 DE DE19750220A patent/DE19750220A1/de not_active Withdrawn
-
1998
- 1998-11-13 DE DE59812566T patent/DE59812566D1/de not_active Expired - Lifetime
- 1998-11-13 WO PCT/DE1998/003329 patent/WO1999025837A2/de active IP Right Grant
- 1998-11-13 EP EP98965067A patent/EP1030923B1/de not_active Expired - Lifetime
- 1998-11-13 AT AT98965067T patent/ATE288961T1/de not_active IP Right Cessation
Non-Patent Citations (15)
Title |
---|
D. CHANG ET AL.: "Self-assembl of the JC virus major capsid protein, VP1, expressed in insect cells" J. GENERAL VIROLOGY, Bd. 78, Nr. 6, Juni 1997, Seiten 1435-1439, XP002100677 READING, BERKS, GB in der Anmeldung erw{hnt * |
D.M. SALUNKE ET AL.: "Polymorphism in the assembly of polyomavirus capsid protein VP1" BIOPHYSICAL JOURNAL, Bd. 56, November 1989, Seiten 887-900, XP002100674 * |
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DE19750220A1 (de) | 1999-05-20 |
WO1999025837A3 (de) | 1999-07-15 |
EP1030923B1 (de) | 2005-02-09 |
DE59812566D1 (de) | 2005-03-17 |
EP1030923A2 (de) | 2000-08-30 |
ATE288961T1 (de) | 2005-02-15 |
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