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WO1999012948A2 - Acides polyribonucleiques enveloppes de proteines, procede de production correspondant et leur utilisation - Google Patents

Acides polyribonucleiques enveloppes de proteines, procede de production correspondant et leur utilisation Download PDF

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Publication number
WO1999012948A2
WO1999012948A2 PCT/EP1998/005258 EP9805258W WO9912948A2 WO 1999012948 A2 WO1999012948 A2 WO 1999012948A2 EP 9805258 W EP9805258 W EP 9805258W WO 9912948 A2 WO9912948 A2 WO 9912948A2
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protein
rna
sequence
phage
coated
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PCT/EP1998/005258
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German (de)
English (en)
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WO1999012948A3 (fr
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Olfert Landt
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Olfert Landt
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    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Protein-coated polyribonucleic acids a process for their preparation and their use
  • the invention relates to protein-coated polyribonucleic acids containing a naturally occurring RNA virus or RNA bacteriophage, the natural nucleic acid sequence of which is varied, a process for their preparation and their use, preferably in diagnostic processes.
  • PCR polymerase chain reaction
  • RCR repair chain reaction
  • LCR ligase chain reaction
  • isothermal amplification techniques such as strand displacement amplification, SDA, transcription-based amplification) System
  • TAS Self-sustained Sequence Replication
  • 3SR Nucleic Acid Sequence-based Ampiification
  • nucleic acids are quantification of specific ribonucleic acids in cells (messenger RNA, mRNA). Measuring the amount of mRNA transcription products can shed light on Give clinical pictures or a disease status and can be used to assess the efficacy or mechanisms of action of pharmaceuticals on patients, in cell culture and in basic research.
  • Prominent examples are the quantification of cytokines (growth factors) that play a role in inflammatory processes and cancer, or of cytochromes, which are a sensitive parameter to environmental pollution [B ecker-Andre, M, 1991, Methods Mol Cell Biol 2, 189-201]
  • RNA detection or quantity determination of RNA is carried out by direct hybridization or after its transcription (reverse transcription, RT) in cDNA by means of amplification.
  • the cDNA is then quantified by means of a comparative method, that is to say the detection of a known amount of nucleic acid in addition to the target nucleic acid , the amount of which is unknown. Examples of such methods are the competitive PCR and the competitive NASBA.
  • the sequence of the nucleic acid, the amount of which is known should be as similar as possible to the nucleic acid sequence which is to be quantified. However, it always contains sequence regions by means of which it or its amplification product can be distinguished from the nucleic acid to be quantified.
  • Quantity standards carry deletions, insertions or substitutions, so that they can be differentiated by means of differential hybridization, nucleotide sequence analysis, restriction fragment analysis or their length [Reischl , U and Kochanowski, B 1995, Mol Biotechnol 3, 55-71]
  • the nucleic acids to be detected can consist of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA is very stable and can be used as a Serving Reference for Diagnostic Methods Usually synthetic or cloned DNA fragments of the desired sequence are used [Lebo RV et al, 1990, AmJ.Hum.Genet 47, 583-590; Mudd, J.L., et al, 1989, Crime Lab Dig. 16, 41-59, TWGDAM (Technical Working Group on DNA Analysis Methods, U S A)]
  • RNA is extremely sensitive to degradation. It can be caused, for example, by the action of heavy and transition metal ions, by alkali and by a large number of enzymes, in particular by the omnipresent ribonucleases RNA can therefore only be cut and degraded with precautionary measures, that is to say using high-purity water and, if possible, in the presence of ribonuclease inhibitors [Dobbeling, U et al, 1997, BioTechniques 22, 88-90] Rapidly degraded RNA synthesized or RNA transcribed in vitro cannot be added to a sample containing RNA degrading components without loss, and is not suitable for tracking and assessing the processing and transcription of RNA into DNA
  • RNA from a sample and its reverse transcription When isolating the RNA from a sample and its reverse transcription, part of the sample is lost in particular due to enzymatic degradation and the low efficiency of the reverse transcription.
  • a reference sample For a direct comparison, a reference sample must be processed together with the sample to be determined in order to avoid these losses
  • a synthetically or enzymatically produced reference ribonucleic acid which is not additionally protected, for example, by proteins which envelop it or bind to it, is degraded before the start of the workup if it becomes an untreated one
  • Sample material is added so that the measurement values are falsified.
  • a corresponding DNA sequence is usually used as the standard.
  • a DNA standard after the digestion of the sample is not subject to the same degradation as the RNA sample to be determined of ribonucleic acids and the efficiency of their reverse transcription are not documented
  • Ribonucleic acid standards can be prepared chemically synthetically or by in vitro transcription [Piatak M et al, 1993, BioTechniques, 14, 70-81] RNA sequences can be replicated in vitro by means of the replicase from Q-beta if they contain appropriate recognition sequences the genome of the phage Q-beta [Lizardi et al, 1988,
  • RNA sequences can be used as a quantitative standard for RT-PCR [Fille et al, BioTechniques 23 (1997) 34-36)] They are not protected against degradation
  • Standards for ribonucleic acids can also consist of the material to be detected.
  • viruses that are pathogenic to humans for example when using a blood or serum sample, they can be infectious, fall under epidemic provisions and laws and are therefore restricted in their handling.
  • Standards from a natural isolate are difficult to produce on a large scale. Blood samples are not homogeneous and aliquots virus or titers can have very different virus titers. Natural isolates cannot be used as a competitive standard because they do not necessarily contain sequence variations
  • Retroviral packaging systems based on human and monkey viruses have been developed which allow the packaging of ribonucleic acids with sequence variations in virus particles, for example in order to investigate the effect of certain gene sequences or to introduce certain RNA sequences for gene therapy in cells in the future. These systems are based on virus particles similar to the AIDS virus.
  • the virus particles are produced in cell culture from which novel infectious viruses can develop [Flamant, F, Cosset, F-L and Samarut, J, J Mol Med 73 (1995) 181-187] Constructs have also been reported that were genetically unstable and have lost all or part of the varied sequences [Olsen et al, J Virol 64 (1990) 5475-5484).
  • RNA phages in bacteria which are divided into several families.They consist of a polyribonucleic acid which is encased by a large number of protein molecules, the so-called 'coat protein'.
  • the phage nucleic acid encodes a polymerase, its coat protein and further protein factors.
  • the phages are also dependent on host factors. Some phages can only attack 'male' bacteria, i.e. bacteria with an F-particle (F +).
  • the phage Q-beta codes for a 'Maturation' protein a2, a 'Coat' protein, a 'Readthrough' protein al, which as Following a read-through event via a stop codon, a C-terminally extended coat protein is present, and an RNA polymerase, the so-called replicase.
  • the read-through protein is involved in host infection by the Q-beta phage [Weissmann, C, FEBS Letters 40 (1974) S10-S18] It is known that a failure of each individual protein as a result of variations in the respective coding nucleic acid sequences in the case of the phage Q-beta can be complemented by converting the protein concerned into trans, i.e.
  • the phage genome forms a distinctive secondary structure. Changes in the genome of the phage are usually not genetically stable and are reversed within a few phage generations or neutralized by further changes in the genome A stable folding pattern is obviously generated again [Arora R et al., J Mol Biol.
  • RNA phage Q-beta has been used for the production of recombinant proteins or peptides which form artificial extensions of the 'Coat' protein [Kozlovska, TM et al, I-ntervirology 39 (1996) 9-15]
  • the 'Coat' protein forms extremely stable particles, the 'Coat' proteins are linked to each other by disulfide bridging [ Golmohammadi, R et al, ⁇ Structure 4 (1996) 543-554]
  • polyribonucleic acids can be produced with freely definable sequence fractions which contain a desired sequence and which of a
  • Protein sheath that protects them against degradation.
  • the detection limits of qualitative nucleic acid detections can be checked, and standards can also be produced which, after addition to the sample to be determined, are degraded to the same extent as the substance to be determined and can thus be used as a quantity standard
  • the competitive quantification of RNA directly from the sample material without having to accept the disadvantages mentioned above
  • the present invention therefore relates to protein-coated polyribonucleic acids which are characterized in that they contain a virus RNA or bacteriophage RNA, in which the natural nucleic acid sequence is varied by singular or multiple insertions and / or substitutions
  • the protein-coated polyribonucleic acids consist of an RNA bacteriophage whose nucleic acid sequence is varied as described above.
  • bacteriophages are the phages Q-beta, VK, PP7, F2, FR, GA, SP, MS, MS2, fcan, Ml 2 or R17
  • the phage Q-beta is preferred.
  • bacteriophages can be handled and propagated without a greater risk
  • sequence variations preferably leads to the genome sequence being changed to such an extent that the underlying bacteriophage or virus can no longer multiply independently without the addition of further elements
  • sequence variations consist in the insertion or substitution of freely determinable sequences with a length of 20 to 900 bases or with one to three sequence sections simultaneously, each comprising a length of 17 to 50 bases.
  • Sequences are preferably substituted, ie the total length of the sequence is not or only slightly varied.
  • the newly introduced freely definable sequence sections have approximately the same length as the sequences deleted from the virus or phage genome.
  • the newly introduced freely definable sequence sections consist of synthetic sequences or, if necessary, varied genome sequences of other organisms.
  • the related genome sequences are preferably those of diagnostic relevance, that is to say that precisely these sequence segments are used in diagnostic methods. These sequence sections are, for example, the binding sites for PCR primers or binding sites for oligonucleotide probes.
  • genome sequences or sections which may be varied are those from human-pathogenic RNA viruses, for example retro and flaviviruses.
  • sequences from the HIV, HCV, HGV, influenza, Picorna, yellow fever, Hanta or Dengue viruses are of particular diagnostic relevance.
  • genome sequences or sections which may be varied are coding sequences from mammals, for example sections from the messenger RNA of growth factors (cytokines), the cellular amount of which is determined in diagnostic methods.
  • cytokines growth factors
  • the synthetic sequences can be functional ribonucleic acids, for example ribozymes or aptamers.
  • genomic regions of the phage are selected which either do not concern a functional region for the phage or only the reading frame for a single protein.
  • the sequence variation is preferably limited to a genome region of the phage which encodes only a protein which is necessary for the multiplication of the phage.
  • phages that carry the desired sequence variation cannot reproduce on their own if the missing factor is not supplied by the host cells becomes.
  • Bacterial cells which provide this missing factor are thus used to produce the protein-coated polyribonucleic acid according to the invention by means of phages.
  • This factor consists of the unchanged coding gene for the protein, the reading frame of which is affected by the sequence variation.
  • the gene can be integrated in the host genome or can lie on an independent genetic element that is introduced into the host organism.
  • Bacterial cells which cannot be infected by a phage are preferably used to prepare such a phage (genetic marker F " ). As a result, the phages cannot multiply independently and mutations due to multiple passages of the phage are excluded. The genome of one RNA phages are under a high selection pressure.
  • the protein-coated polyribonucleic acids consist of the bacteriophage Q-beta, in the nucleic acid sequence of which the 3 'region of the gene for the' read-through protein 'is completely or partially replaced by other, freely definable sequences.
  • sequence sections of approximately the same length are preferably exchanged. Insertions are preferably inserted at sites that have little effect on the folding structure of the RNA phage genome. Such sites are, for example, the ends of stem loops.
  • the reading frame for the 'Coat' Protein is preferably closed with a constitutive Stop Codon [TGA] and otherwise not varied
  • the present invention also relates to a process for the preparation of the protein-coated polyribonucleic acids described above, which is characterized in that:
  • RNA phage genome or RNA virus genome which is cloned as a DNA sequence, by insertion or substitution and the genome thus varies
  • RNA virus genome which is cloned as a DNA sequence, by insertion or substitution and the genome thus varies
  • introducing the varied genome sequence into a suitable host cell by known methods, in which viruses or phages are produced which contain the desired varied polyribonucleic acid packaged in a protein envelope
  • RNA phage genome or from an RNA virus genome as a vector, which is cloned as a DNA sequence
  • a freely definable nucleic acid sequence is inserted into the cloned genome by insertion or substitution.
  • Such a variation almost inevitably leads to a loss of essential functions for the virus or the phage.
  • the insertion therefore takes place at a position in which no functional region of the phage is affected or preferably at a location that destroys the coding region for only one protein.
  • the loss of function is complemented by the provision of this gene in the host cell
  • the varied genome sequence is brought into host cells, in which it serves as a template for producing the varied ribonucleic acid.
  • the host cells produce viruses or phages which contain the desired polyribonucleic acid
  • RNA virus or an RNA phage is used as a vector for the production of the protein-coated polyribonucleic acid according to the invention.
  • vectors can be, for example, bacteriophages, retroviruses, influenza viruses or plant viruses which contain single-stranded or double-stranded RNA as the genome.
  • the preparation of the protein-coated ones Ribonucleic acid takes place in the respective natural or in a compatible host cell of the virus or phage used as a vector
  • the freely definable sequence that is to be brought into the vector is either produced synthetically or isolated from another organism and, if appropriate, varies. It is present as a DNA sequence. This can be a DNA fragment or a cloned DNA
  • the freely definable sequence In order to insert the freely definable sequence into the vector, one uses, for example, targeted clomeration via residual interfaces.
  • the selected interfaces are preferably singular, that is to say they do not exist anywhere else in the vector or in the freely definable sequence
  • the freely definable sequence does not contain any suitable residual interfaces, these are added to the sequence. They can be artificially added or inserted using methods of directed mutagenesis or by means of the polymerase chain reaction by amplifying the freely definable sequence with primers that are primed with their 3 ' Areas are complementary to this sequence and have the interfaces attached to their 5 'ends
  • RNA virus or an RNA bacteriophage The ribonucleic acid genome of an RNA virus or an RNA bacteriophage is isolated and transcribed into 'Copy DNA' (cDNA) (reverse transcribed)
  • Plasmids are used for the clomerization, which allow a transfer of the cloned genome sequences into their original or a compatible host organism
  • the cloned genomic sequence is placed behind regulatory elements which allow a transcription of this sequence in the host organism.
  • regulatory elements which allow a transcription of this sequence in the host organism.
  • the genomic sequence is transcribed in the host organism, the ribonucleic acid genome is formed and the RNA virus or RNA bacteriophage used is expanded.
  • Regulatory factors are preferred Used elements that can be induced, that is, elements that allow experimental regulation of expression.
  • areas are preferably substituted instead of inserted so that the overall length of the genomic sequence is influenced as little as possible.
  • the failure of certain functions can be complemented. Areas in a phage or in a virus genome which code for only one protein can be destroyed, replaced or deleted, so that the varied phage or virus genome is no longer a template for the error-free expression of this protein can serve. If this protein can be produced within the host cell independently of the varied phage or virus genome, the loss of function which otherwise occurs with the loss of this protein is complemented.
  • a variation in the genome of the phage or of the virus preferably takes place in a region coding only for one protein, without destroying further, for example, regulatory elements.
  • the variations are preferably made at locations where the folding pattern of the genomic RNA is influenced as little as possible.
  • No functional virus or phage can arise from a cloned genome that carries a variation that leads to the loss of an essential function for the virus or for the phage.
  • the loss of a specific function prevents the propagation of the affected phage or virus in host cells that do not produce an additional compensating factor.
  • Cloned genomes with such a variation are safe against an uncontrolled spread of a virus or a phage.
  • a functional virus or phage cannot be replicated from the protein-enveloped ribonucleic acid, the natural sequence of which is varied
  • locations or regions are preferably selected which do not cause any loss of function for the phage or where only the function of a protein-coding gene is affected by insertion or substitution. If no loss of function is caused, no system for complementation is preferred however, a location or area is selected for variation that is only one
  • Reading frame for a protein is destroyed, but is not otherwise functionally important.
  • the failure of this reading frame is complemented by a copy on another genetic element, for example a plasmid.
  • the functional failure has the advantage that the phage or virus varied in this way does not exist without this element can reproduce and spread
  • restriction sites into a cloned virus or phage genome
  • the insertion of freely definable sequences is preferably carried out by cloning via restriction sites.
  • interfaces for restriction enzymes are inserted at suitable sites in the cloned genomic sequence using genetic engineering methods that match the restriction ends of the freely definable sequence to be inserted
  • These interfaces can be inserted, for example, via directed mutagenesis or via the polymerase chain reaction. When using the latter, the entire cloned genome sequence is amplified with primers which carry the desired restriction sites at their 5 'ends.
  • the primers bind in the genomic sequence, and part of the genomic sequence can be deleted by the selection of the binding site Copy of the entire sequence, which may contain deletions in the target area of the genomic sequence.
  • the PCR amplification product can be used directly after the restriction digestion to insert a freely definable sequence
  • the amplification product can be circularized and cloned to be used as a template for the insertion of new sequence segments.
  • the freely definable sequence is inserted into the cloned genomic sequence. It is inserted or it substitutes an area within the genomic sequence. This area is determined by the positioning of the restriction interfaces used for this.
  • the freely definable sequence provided with restriction sites and the genomic sequence modified with restriction sites are digested with the respective restriction enzymes and ligated together.
  • the ligation products are cloned. The clones are checked to ensure that they correspond to the desired sequence.
  • the host cells for the phages or for the viruses are equipped with the affected gene, which has become functionless in the manipulation of the genomic sequence in order to be able to produce the protein and to complement the associated functional failure.
  • the coding sequence can be provided for the host cell either by inserting this gene sequence into its genome or by inserting a vector. This complementing sequence can also be contained on the same vector that carries the varied genomic sequence.
  • the gene for this protein product is isolated from the phage genome or from the virus genome and is cloned in bacteria.
  • the complementing gene is placed under the control of a suitable regulatory element and brought into the host cell by means of a suitable plasmid or vector.
  • the regulatory element controls the permanent or inducible expression of the gene.
  • the host cell has the corresponding protein to complement the functional loss generated by the variation in the cloned genomic sequence.
  • a suitable plasmid or vector is with the simultaneous Presence of the vector compatible in the same host cell.
  • the origin of replication (ori) of the plasmids used must be compatible with one another.
  • the vectors must carry different selectable resistance markers
  • the host cells associated or compatible with the respective system i.e. the RNA viruses or RNA phages, are used.
  • the host cells express the gene which complements the loss of function caused by the variation of the cloned genome sequence (F3)
  • the cloned gene sequence which bears the desired variations (E3) is brought into the host cells.
  • the host cells which contain the varied gene sequence and the complementing sequence, express constitutively or after induction phage or virus particles which, instead of a phage or virus genome, carry a varied genome which contains the freely definable sequence
  • the phages or virus particles produced are isolated from the medium in the same way as the naturally occurring phages or viruses or after lysis of the cells from them by centrifugation, precipitation or extraction.
  • the phages or virus particles can also be used with the surrounding host cells, which may be inactivated
  • Another object of the invention is the use of the protein-coated polyribonucleic acids according to the invention, in particular in diagnostic methods for Example for the qualitative and quantitative detection of ribonucleic acids, for the positive control of the detection of virus RNA, for the control of the efficiency of methods for the purification and isolation of ribonucleic acids or for the control of the efficiency of the reverse transcription of ribonucleic acids
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard to document or test the suitability of the method used or to determine the detection limit of the method used.
  • the protein-enveloped polyribonucleic acid used as standard can correspond in its freely determinable proportion to the sequence of the ribonucleic acid sequence to be detected in whole or in part.
  • Methods for testing for the presence of a specific ribonucleic acid in a sample consist of sample preparation and isolation of the ribonucleic acid and its detection.
  • the processing is carried out by extraction, for example by means of phenol / chloroform extraction, by enzymatic digestion, for example by means of proteinase, by treatment with detergents or with amphiphilic substances, by treatment with denaturing salts, by heating, by frequent freezing and thawing or by mechanical digestion or a combination of these methods.
  • the further enrichment of the nucleic acid can be achieved by binding to a solid phase, for example to anion exchangers, silica gel or Membranes or on glass or by extraction.
  • Detection is carried out by direct hybridization of a suitably labeled oligonucleotide probe to the target nucleic acid or after transcription in DNA, amplification of a specific DNA fragment and detection of this Fr agmentes on hybridization, determination of length, restriction digestion or DNA sequencing
  • the protein-coated polyribonucleic acids according to the invention are added to the sample material and processed with the sample or analyzed in a separate batch
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard to document or test the suitability of the method used or to determine the detection limit of the method used.
  • the protein-coated polyribonucleic acid used as standard has a sequence similar to that to be detected or is a variation of this sequence and accompanies all steps in the diagnostic method, in order to use it instead of the sequence to be detected, or in a subset of the sample to be determined, or subsequently, if the sequence to be detected was not ascertainable
  • Virus nucleic acid and messenger RNA mRNA
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard to serve as a quantity reference and to generate calibration curves for methods with which the amounts of the unknown samples can be determined.
  • the protein-encased polyribonucleic acid used as standard can correspond in its freely determinable proportion to the sequence of the ribonucleic acid sequence to be detected in whole or in part
  • the protein-coated polyribonucleic acids according to the invention can be used as standard to work as a competitor sequence.
  • the protein-coated polyribonucleic acid used as standard corresponds freely in its Determinable part of the sequence of the ribonucleic acid sequence to be detected.
  • the standard is processed and analyzed in different quantities with the unknown sample to be determined. The quantity is determined by competitive methods
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard in order to document the suitability of the method used as an accompanying sample.
  • the nucleic acid sequence used is not identical to the sequence of the nucleic acid to be detected
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard to check the detection capability of a laboratory, an institution or a person or a method
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard to document the suitability and efficiency of the method used.
  • the protein-coated polyribonucleic acid used as standard can be used in whole or in part in its freely definable part of the sequence of the ribonucleic acid sequence to be detected correspond
  • the protein-coated polyribonucleic acids according to the invention can be used as a standard in order to document the suitability and efficiency of the process used
  • the protein-coated polyribonucleic acids according to the invention can be used as standard and function control for reagents and laboratory kits for isolating ribonucleic acids and their reverse transcription in DNA
  • the protein-coated polyribonucleic acids according to the invention can be used as a sample in order to check the efficiency of cleaning and disinfection measures, in particular for the inactivation of RNA viruses
  • the protein-coated polyribonucleic acids according to the invention can be used to mark samples of any kind.
  • the freely definable portion in the sequence can be used to encode origin, type or other information and can later be analyzed and 'read' by means of a suitable diagnostic method
  • the protein-coated polyribonucleic acids according to the invention can be produced in order to use the RNA isolated therefrom as hybridization samples.
  • the RNA is preserved until its preparation by its protective protein coat.
  • the freely definable sequence contains the areas which are used for hybridization with a complementary sequence.
  • the polyribonucleic acids according to the invention can contain, for example, labeled nucleotides which are incorporated in the host organism and which can be used for their detection.
  • Hybridized polyribonucleic acids which represent variations of a phage genome can also be replicated in vitro by means of the RNA-dependent RNA polymerase specific for this RNA Replicase can be detected
  • the protein-coated polyribonucleic acids of the present invention can be used to make RNA for binding purposes.
  • the RNA is preserved through its protective protein envelope until it is isolated.
  • the freely definable sequence contains areas that can bind certain structures (aptamers)
  • the polyribonucleic acids according to the invention can contain, for example, labeled nucleotides which are incorporated in the host organism and which can be used for their detection.
  • Polyribonucleic acids which bind a structure and which represent the variations of a phage genome can also be replicated in vitro by means of of the RNA-specific RNA polymerase replicase for this RNA-specific or can be detected by RT-PCR amplification
  • the protein-coated polyribonucleic acids according to the invention can be used to produce functional ribonucleic acids, for example ribozymes.
  • the sequences are preserved by their protective protein envelope until they are used.
  • the freely definable sequence contains regions which contain functional sequences
  • the plasmid pBRT7Qß was provided by Prof Dr Hans Weber, Institute of Molecular Biology, University of Zurich [published in Barrera I, et al, J Mol Biol 232 (1993) 512-521].
  • the plasmid carries the genome of the phage Q- beta behind a T7 promoter, a ß-lactamase gene (ampicillin resistance) and the ColEl origin of replication
  • This plasmid leads to the production of Q-beta phages in transformed Escherichia coli (E coli) which can produce the T7 RNA polymerase.
  • E coli Escherichia coli
  • the plasmid pBRT7Qß is produced with the aid of two oligonucleotide primers, a PCR product which comprises the entire plasmid and whose ends are in the gene for the read-through protein a1.
  • the primers carry restriction sites at their 5 'ends. This product is religated a plasmid with the Q-beta phage sequence and a variation in the al gene.
  • 100 ng plasmid pBRT7Qß are mixed with 60 pmol each of the oligonucleotide primers [82593], 5'-ATT CTA GAG CCG TGG CAA TGG TTA TAT TGA CCT TGA TGC G (sequence 2) and [82594], 5'-TAT CTA GAA AGC TTA ATA CGCTGG GTT CAG CTG ATC ⁇ AT AGC ATC G (page sequence 3), with 1 ⁇ l 10 mM deoxyribonucleotides (dNTPs), 10 mM Tris-HC1, pH 8.3, 2 mM magnesium chloride, with 2 u Taq DNA polymerase (Hoffmann La -Roche) and 0.2 u Pwo DNA polymerase (Boehringer Mannheim) amplified in a volume of 100 ⁇ l.
  • dNTPs deoxyribonucleotides
  • the reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 55 ° C and 2 min at 72 ° C and finally 20 min at 72 ° C (Perkin Elmer GeneAmp 2400).
  • the amplification product is extracted with 50 ⁇ l phenol (Roth) and with chloroform / isoamy alcohol (25: 1). 10 ⁇ l of 5 M potassium acetate and 350 ⁇ l of ethanol are added to the aqueous phase.
  • the precipitated DNA is pelleted by centrifugation. The pellet is washed with 70% ethanol and dried in vacuo.
  • the DNA is dissolved in 20 ⁇ l of water and cut with the restriction enzyme Hind III (Boehringer Mannheim) according to the manufacturer's instructions (2 hours at 37 ° C.).
  • the reaction mixture is made up to 100 ⁇ l with water and extracted with phenol as above and precipitated with ethanol.
  • the DNA pellet is in 20 ul
  • DH5alpha (DH5alpha) are transformed with 0.5 ⁇ l of the ligation mixture. The colonies obtained are picked and dressed. Plasmid DNA is isolated from overnight cultures
  • the plasmid DNA is controlled by restriction digestion and DNA sequencing (Replicon
  • sequence of the plasmid pBRT7Qßd is disclosed as sequence 4.
  • the plasmid contains a varied Q-beta phage genome, the reading frame for the 'coat' protein is closed with a TAA stop codon.
  • the gene for the read-through protein carries a deletion of 264 base pairs and two different unique restriction sites Hind III (AAGCTT) and Xba I (TCTAGA) at this point.
  • Immunodeficiency virus (HIV) genome The plasmid pBRT7Qßd is linearized with the residual enzyme Hind III (Boehringer Mannheim, according to the manufacturer) and dephosphorylated with alkaline phosphatase (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol.
  • Plasmid DNA is isolated overnight (Nucleobond Kit, Machery-Nagel, according to the manufacturer's instructions). The plasmid DNA is checked by means of a residual digestion and DNA sequencing
  • sequence 7 The sequence of the plasmid pBRT7QßSK145 is disclosed as sequence 7
  • the Q-beta phage genome on the plasmid pBRT7Qß is used as the template.
  • the reaction solution contains 100 ⁇ M deoxyribonucleotides (dNTPs), 50 ⁇ M of both oligonucleotide primers in 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and with 2 u Taq DNA polymerase (Hoffmann La-Roche) in a volume of 50 ⁇ l.
  • the reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400)
  • sequence 10 The product is extracted with phenol and precipitated
  • the product is with the restriction enzymes Xba I and Hind III digested (Boehringer Mannheim, according to the manufacturer), with phenol extracted and fallen
  • the plasmid pBRT7Qßd is cut with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol.
  • the cut plasmid and the cut amplification product are ligated together, E. coli are transformed and plasmid DNA is prepared and verified by DNA sequencing.
  • the sequence of the plasmid pBRT7QßSK145-431 is disclosed as Sequence 11.
  • the plasmid contains the sequences SK145 5'-AgT ggg ggg ACA TCA AgC AgC CAT gCA AAT (sequence 12) and SK431 5'-TgC TAT gTC AgT TCC CCT Tgg TTC TCT (sequence 13) at a distance of 214 base pairs; including the sequences SK145 and SK431, the fragment has a length of 271 base pairs.
  • a fragment with restriction sites is amplified from the HIV I genome using the polymerase chain reaction.
  • the PCR is carried out with the oligonucleotides [84673] 5'-ATT AAA gCT TAg Tgg ggg gAC ATC AAg C (sequence 14) and [84675] 5'-TA TCT AgA TgC TAT gTC AgT TCC CTT Tg (sequence 15) and a cDNA Template of HIV I performed.
  • the reaction solution contains 100 ⁇ M deoxyribonucleotides (dNTPs), 50 ⁇ M of both oligonucleotide primers, 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and 2 u Taq DNA polymerase (Hoffmann La-Roche) in a volume of 50 ⁇ l.
  • the reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400).
  • the reaction product is cut with the restriction enzymes Hind III and Xba I (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol.
  • the plasmid pBRT7Qßd is cut with the restriction enzymes Xba 1 and Hind III (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol. The cut plasmid and the cut amplification product are ligated together, E. coli are transformed. Plasmid DNA is prepared and verified by DNA sequencing.
  • sequence 16 The sequence of the plasmid pBRT7QßSK 145-102-431 is disclosed as sequence 16. It contains a 142 base pair (sequence 17) long sequence section from HIV 1.
  • a fragment with restriction sites is amplified from the HCV genome using the polymerase chain reaction.
  • the PCR is carried out with the oligonucleotides [84672] 5'-ATT AAA gCT TgC AgA AAg CgT CTA gCC (sequence 18) and [84671] 5 'TAC TCTAgA CTC gCA AgC ACC CTA TC (sequence 19) and a cDNA template from HCV .
  • the reaction solution contains 100 ⁇ M deoxyribonucleotides (dNTPs), 50 ⁇ M of both oligonucleotide primers in 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and with 2 u Taq DNA polymerase (Hoffmann La-Roche) in a volume of 50 ⁇ l.
  • the reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400).
  • the reaction product is cut with the restriction enzymes Hind III and Xba I (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol.
  • the plasmid pBRT7Qßd is cut with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol.
  • Plasmid DNA is prepared and verified by DNA sequencing.
  • sequence 20 contains a 216 base pair (sequence 21) long sequence section from the HCV genome
  • the plasmid pP + QßRT was provided by Dr Donald R Mills, Dept Microbiology, State University of New York, U.S.A. It is 8J kb in size and carries the pSMl
  • Origin of replication which is compatible with the ColEl ori from the pBRT7Qß plasmid.
  • the plasmid carries a gene for resistance to trimethoprim and the gene for the readthrough protein al under the control of the P + promoter. This plasmid leads to a constitutive expression of al Protein in transformed E. coli [Arora et al., J. Mol. Biol. (1996) 258 433-446],
  • Competent E. coli from strain BL21 (DE3) (Stratagene) or BL21 (DE3) are transformed with pP + QßRT and a pBRT7Qß plasmid (e.g. pBRT7QßSK145).
  • the clones are selected for ampicillin and trimethoprim resistance (LB agar with 75 mg / 1 ampicillin, Sigma, 50 mg / 1 trimethoprim, Sigma).
  • the cells are grown overnight in a preculture (LB medium with ampicillin and trimethoprim, 37 ° C) and in one
  • phage supernatant 100 ul phage supernatant are extracted twice with 50 ul phenol (Roth) and with chloroform / isoamyl alcohol (25 J).
  • the aqueous supernatant is treated with DNase I (Boehringer Mannheim) (1 g / 1, 30 min at room temperature).
  • the solution is extracted with phenol as above and precipitated with ethanol (0.5 M LiCl, 3 volumes of ethanol) and pelleted by centrifugation.
  • the pellet is washed with 70% ethanol and extracted.
  • the ribonucleic acid obtained is dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 7.5; ImM EDTA) and determined photometrically.
  • TE buffer 10 mM Tris-HCl, pH 7.5; ImM EDTA
  • Ribonucleic acid according to Example 7 is used as a template.
  • the oligonucleotide primers SK145 (sequence 12) and SK43 1 (sequence 13) or for pBRT7QßSK145-102- 431 the combination GAG3, 5'-ATCAATgAggAAgCTgCAgAATggg (sequence 22) and SK43 1 were used.
  • RNA from the plasmid pBRT7QßHCV (sequence 20)
  • the PCR amplification is carried out in a volume of 50 ⁇ l with 100 ⁇ M deoxyribonucleotides (dNTPs), 50 ⁇ M of both oligonucleotide primers in 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and with 2 u Taq DNA polymerase (Hoffmann La-Roche) carried out. 5 ⁇ l of RNA solution from Example 7 are used as templates. The reaction conditions are 35 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400).
  • RNA from the plasmid pBRT7QßSK 145-431 and the primers SKI 45 and SK431 results in a 271 bp fragment
  • RNA from the plasmid pBRT7QßSK145-102-431 and the primers SK145 and SK431 results in a 142 bp fragment
  • a 99 bp fragment is produced
  • RNA from the plasmid pBRT7QßHCV and the primers HC3 and HC4 a 216 bp fragment of double-stranded DNA is produced. which are detected in the agarose gel.
  • ribonucleic acid is obtained from 100 ⁇ l of phage supernatant in accordance with Example 7.
  • the product is diluted in steps of 110 in TE buffer from 10E-1 to 10E-9 Reverse transcriptase and PCR amplification treated with the oligonucleotide binders SKI 45 and SK 431.
  • the PCR amplification is carried out in five batches each.
  • RNAeasy isolation kit Qiagen, according to the manufacturer
  • RT-PCR RNAeasy isolation kit
  • 1 ml of whole blood from a non-HIV infected blood donor is mixed with 12.5 ⁇ l of a 10E-6 diluted phage supernatant from pBRT7QßSK145-102-431, corresponding to approximately 25,000 genome copies of HIV.
  • the mixed blood sample is separated into five aliquots A, B, C, D and E, each containing 5000 genome equivalents, and each with 10 ⁇ l phage supernatant from pBRT7QßSKl 45-431 with dilutions of 10E-8, 10E-7, 2xl0E-7, 10E-6 and 2xlOE-5 offset, corresponding to 200, 2000, 4000, 20000 and 40,000 genome equivalents.
  • the samples are treated according to example 10.
  • Approach A shows a product of 142 bases long
  • approach B the same product and little product with a length of 271 bases long
  • batch C shows the same product quantities and batches D and E only show the product with 271 bases long.
  • the amount of genomic equivalents from the sequence pBRT7QßSK145-102-431 is approximately 4,000 or the equivalent of 16,000 per ml
  • the polymerase chain reaction is carried out with the oligonucleotide binders FAS5, 5'-gCgC AgcTT TTC TgC CAT AAg CCC TgT CC AgC ATg CCA CgT AAg CgA AA (sequence 26) and FAS3, 5'-ACCg TCTAGA Ag AAg ACA AAg CCA CCC CAA TgAgCCCC Sequence 27) on the template Lambda DNA (2 ⁇ g, Boehringer Mannheim) generates a fragment of approximately 380 base pairs (40 pmol primer FAS5 and FAS3, 100 ⁇ M dNTPs, 10 mM Tris-HCl, pH 8.3, 2 mM magnesium chloride, 2 u Taq DNA polymerase (Hoffmann La-Roche) amplified in a volume of 50 ⁇ l
  • the reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 63 ° C and 2 min at 72 ° C and finally 20 min
  • the cut plasmid and the cut amplification product are ligated together, E. coli are transformed. Plasmid DNA is prepared and verified by DNA sequencing.
  • the plasmid pBRT7QßFas contains the Q-beta phage genome with an insertion of 369 base pairs in the Hind III interface sequence 28). Phage particles are produced in accordance with Example 6.
  • Fas is a protein from the tumor necrosis factor (TNF) family.
  • the protein is involved in apoptosis.
  • the amount of mRNA for fas can be interesting for medical reasons.
  • the sequence of the mRNA for fas is stored in the gene bank under the number M67454.
  • Human cells from the cell culture are mixed with different amounts of protein-coated RNA from the vector pBRT7QßFas. They contain an RNA that can compete with the mRNA for fas in RT-PCR.
  • the cells are disrupted and the RNA is isolated (RNAeasy, QIAGEN, according to the manufacturer).
  • the RNA is reverse transcribed (M-MuLV, peqlab, according to the manufacturer's instructions) and amplified by PCR.
  • the oligonucleotide primers used are 82689, 5'-TTC TgC CAT AAg CCC TgT CC (sequence 29) and 82690, 5'-AgA AgA CAA AgC CAC CCC AA (sequence 30).
  • the PCR product from the natural mRNA has a length of 368 base pairs
  • the product from the competitive RNA from the phage has a length of 369 base pairs.
  • the products can be distinguished by restriction digestion with the enzyme Sph I or by differential hybridization. With equal amounts of amplification product from the natural sequence and from the competitor sequence, there are equal amounts of RNA in the cells and in the competitive sample.
  • the plasmid pBRT7Qßd is cut with the restriction enzyme Hind III (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol.
  • coli are transformed and plasmid DNA is prepared and verified by DNA sequencing GGTCGGATCC T in its Hind III interface, phage particles are produced in accordance with example 6.
  • the RNA with the same sequence 5'-GGAGC UCAGCCUUCA CUGCAUGAUA AACCGAUGCU GGGCGAUUCU CCUGAAGUAG GGGAAGAGUU GUCAUGUAUG GGGUCGGACGAC gin UGGGGGGACGinG (UGGGGGGACGinG) ., Nucleic Acids. Res., (1996) 6, 1029-103 6].
  • the plasmid pBRT7Qßd is cut with the restriction enzyme Hind III (Boehringer Mannheim, according to the manufacturer), extracted with phenol and precipitated with ethanol
  • RNA is isolated from the phage by means of phenol extraction. From the RNA, which contains degenerate areas, those with special
  • Phage particles obtained with the plasmid pBRT7QßAPT are extracted with phenol and precipitated with ethanol.
  • the RNA contains an aptamer sequence that binds the amino acid arginine.
  • a solution with 0.5 ⁇ g RNA in 1 ml buffer 250 mM NaCl, 50 mM Tris-HCl, pH 7.6, 5 mM magnesium chloride) is added to D-arginine agarose or L-arginine agarose (OJ ml), 10 incubated for min and washed with 300 ml buffer.
  • the agarose is eluted with 1 ml buffer at 95 ° C.
  • RNA M-MuLV, peqlab, according to the manufacturer.
  • the oligonucleotide primers 82808, 5'-gAT CCA gAA CCC gAC CgC (sequence 36) and 82809, 5'-CCA TCg gCg TgA TAg gCC (sequence 37) are used. Only when starting with the eluate from L-arginine agarose does a 764 bp DNA fragment arise, which demonstrates the binding of the RNA to L-arginine

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Abstract

L'invention concerne des acides polyribonucléiques enveloppés de protéines et contenant un virus ARN ou un bactériophage à ARN inoffensif, d'apparition naturelle, dont la séquence d'acide nucléique naturelle varie. L'invention concerne également un procédé de production et son utilisation de préférence aux fins de diagnostic. Pour le diagnostic, des séquences d'acides nucléiques sont insérées dans le génome d'un virus ARN ou d'un phage à ARN ou bien des sections sont substituées. Comme support, on utilise de préférence le phage Q-béta auquel cas, de préférence, le gène de la protéine Poursuite de la lecture a1 est modifié. La perte fonctionnelle du phage ou du virus liée à la manipulation génétique est compensée par la préparation d'une copie du gène concerné dans l'organisme hôte du virus ou du phage et ce, de préférence, sur un élément génétique séparé. Cet élément complémentaire permet de produire dans l'hôte naturel des dérivés du virus ou du phage, qui portent des sections d'acides nucléiques pertinentes en termes de diagnostic, et qui sont stables car protégés contre la dégradation par l'enveloppe de protéines du virus ou du phage. Pendant le retraitement des échantillons et pendant la transcription inverse de l'ARN lors du diagnostic, ils se comportent de façon similaire aux échantillons à identifier. Ces dérivés permettent de valider les diagnostics et peuvent servir de contrôles ou de norme compétitive pour la quantification d'ARN ou de virus ARN. Les dérivés de virus ou de phages utilisés comme normes sont inoffensifs pour les hommes et ne sont pas capables de se reproduire de façon autonome.
PCT/EP1998/005258 1997-08-22 1998-08-19 Acides polyribonucleiques enveloppes de proteines, procede de production correspondant et leur utilisation WO1999012948A2 (fr)

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FR2606030B1 (fr) * 1986-10-31 1989-10-20 Pasteur Institut Retrovirus recombinant defectif, son procede de fabrication et ses applications, notamment pour l'etude de l'interaction des retrovirus sauvages avec des lignees cellulaires infectables par celui-ci
CN1038306A (zh) * 1988-03-21 1989-12-27 维吉恩公司 重组反转录病毒
WO1993023573A1 (fr) * 1992-05-15 1993-11-25 New England Deaconess Hospital Quantification de l'arn viral par amplification competitive de chaine par polymerase
ES2126620T5 (es) * 1992-10-06 2010-05-21 Siemens Healthcare Diagnostics Products Gmbh Retrovirus del grupo de los vih y su utilizacion.
DE4339922C1 (de) * 1993-09-03 1994-10-06 Max Planck Gesellschaft Vektor für Leber-Gentherapie
CA2192324C (fr) * 1994-06-10 2006-10-10 Venkatachala Natarajan Virus de recombinaison et methode pour le quantifier
CA2227339A1 (fr) * 1995-07-19 1997-02-06 Merck & Co., Inc. Particules synthetiques de type virus hpv11
EP0854929A1 (fr) * 1995-09-27 1998-07-29 Medical Research Council Virus recombines comprenant une proteine pouvant etre clivee par une protease
JP2000501285A (ja) * 1995-11-15 2000-02-08 メルク エンド カンパニー インコーポレーテッド 合成ｈｐｖ１１ウイルス様粒子
AUPO023496A0 (en) * 1996-05-31 1996-06-27 Commonwealth Scientific And Industrial Research Organisation Modified small RNA viruses
US5939262A (en) * 1996-07-03 1999-08-17 Ambion, Inc. Ribonuclease resistant RNA preparation and utilization
DE19737442C2 (de) * 1997-08-22 1999-06-24 Olfert Landt Tib Molbiol Synth Proteinumhüllte Polyribonukleinsäuren, ein Verfahren zu ihrer Herstellung und ihre Verwendung

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