Composition, method for producing artificial ordered and repetitions-containing antigen matrix, method for carrying out therapy and immunization

FIELD: medicine.

SUBSTANCE: composition is used for producing vaccines for preventing infectious diseases to occur, treating allergy and malignant neoplasm cases. Various invention embodiments comprise virus, virus-like particle, viral capsid particle, fag or their recombinant forms covered with any desired antigen highly ordered and having repetitions that is achieved owing to specific interactions taking place. Multi-functional process based on cassette-type system (alpha-vaccine techniques) allows one to produce antigen-coated viral particles like hepatitis B virus or measles virus.

EFFECT: causing marked immune response.

44 cl, 7 dwg

 

The technical field to which the invention relates.

This invention relates to the fields of molecular biology, Virology, immunology and medicine. The invention is a composition comprising an ordered and contains repetitions matrix antigen or antigenic determinants. The invention is also a method of producing antigen or antigenic determinants as ordered and contains repetitions of the matrix. Ordered and contains repetitions antigen or antigenic determinant is used for the manufacture of vaccines for the treatment of infectious diseases, treatment of allergies and as a pharmaceutical preparation for the prevention or treatment of cancer and to develop specific outspecifically antibodies.

The level of technology

The development of vaccines to prevent infectious diseases has had the greatest impact on human health than any invention in the field of medicine. It is established that through vaccination in the world to prevent three million deaths per year (see article Hillemann, Nature Medicine, 4:507 (1998)). The most common strategy vaccination using attenuated (i.e. less virulent) pathogens or closely related organisms was first demonstrated in 1796 by Edward Jenner, who conducted the vaccination against smallpox way is the introduction of a less dangerous cowpox virus. Although a number of live attenuated viruses (e.g., viruses, measles, mumps, rubella, varicella, adenovirus, poliovirus, influenza virus) and bacteria (such as Bacillus Calmette-guérin (Calmette-Guerin) (BCG) against tuberculosis) is successfully used for vaccination, there is a risk of serious complications associated with reversion (return) virulence and infection "vaccine" by the organism, in particular, immunocompromising entities.

The specific design of the weakened viruses currently achieved by recombinant DNA technology (i.e. genetic engineering) by creating a deletion or mutation variants. For example, it was shown that the introduction of engineering immunodeficiency virus monkeys (SIV) with a deletion in the nef gene protects macaques against subsequent infection by a pathogenic strain of SIV (see Daniel et al., Science, 258: 1938-1941 (1992)). However, the development of symptoms such as acquired immunodeficiency syndrome (AIDS), in animals that were injected attenuated SIV, a concern related to security issues (see Baba et al., Science, 267: 1820-1825 (1995)).

As an alternative approach you can use attenuated (weakened) viruses or bacteria as carriers of the antigen-coding genes of the pathogen that is considered too unsafe for introduction into the Atta is Pirovano form (for example, the human immunodeficiency virus (HIV)). After delivery of antigen-encoding gene of the host antigen is synthesized in situ. The cowpox viruses and similar poxvirus birds were used as such carriers for different genes in preclinical and clinical studies of a number of diseases (for example, see Shen et al., Science, 252:440 (1991)). One of the drawbacks of this strategy of vaccination is that it does not simulate the surface of the virion, because the recombinant protein is expressed on the surface of the host cell. In addition, immunocompromised subjects may develop complications, which is proved by the example of representing life-threatening disseminated infections cowpox (see Redfield N., Eng. J. Med., 316: 673 ((1998)).

The fourth approach to vaccination includes the use of selected components of the pathogen that are either cleared from the pathogen grown in vitro (e.g., hemagglutinin or neuraminidase of influenza), or resulting from the heterologous expression of a single viral protein (e.g., surface antigen hepatitis b). For example, recombinant mutant toxins (detoxificaton) used for vaccination against diphtheria, tetanus, cholera and pertussis toxins (see monograph Levine et al., Vaccines of new generation (New generation vaccines), 2nd ed., Marcel Dekker, nc., New York, 1997), and in the evaluation of recombinant HIV proteins (gp120 and gp160 full length) as a means for the induction of neutralizing antibodies against HIV were obtained unsatisfactory results (see Connor et al., J. Virol., 72: 1552 (1998)). Recently obtained promising results with soluble oligomeric gp160, which can induce a CTL response (cytotoxic T lymphocytes) and to cause the formation of antibodies with neutralizing activity against isolates of HIV-1 (see Van Cortt et al., J. Virol., 71:4319(1997)). Additionally, there may be used a peptide vaccine which is known for b - and T-cell epitopes of the antigen are associated with a molecule carrier, designed to improve the immunogenicity of the epitope by stimulating T cells-helper cells. However, one significant problem with this approach is that it provides a limited immune response to the protein as a whole. Moreover, the vaccine must be individually developed for different haplotypes of MHC (major histocompatibility complex). The most serious problem for this type of vaccine is that protective anti-virus antibodies recognize a complex three-dimensional structures that cannot be simulated peptides.

More new vaccination strategy is the use of DNA vaccines (see Dnnll et al., .Rev. Immunol., 15: 617(1997)), can any mod is the substance of the CTL responses with restriction of MHC class I (without the use of live vectors). This may provide greater protection against different strains of the virus through a focus on the epitopes of the conservative internal proteins common to many strains of the same virus. Because the antigen is formed with the use of post-translational modifications, conformation and oligomerization characteristic of mammals, it is more likely that he is close or identical to a protein of the wild type, which is formed during viral infection than recombinant or chemically modified proteins. However, this difference may be a disadvantage for the application of bacterial antigens, because it is not natural post-translational modification may result in reduced immunogenicity. In addition, viral surface proteins do not have a high level of organization in the absence of the protein matrix.

In addition to the prevention of infectious disease vaccine technology is used currently to solve the problems of immunity-related allergies. In subjects with allergic antibodies of IgE isotype formed by inadequate humoral immune response against specific antigens (allergens). Treatment of allergies by immunotherapy allergies requires weekly introduction of successively increasing doses of a specific antigen in the period up to 3-5 years. Likely is, formed a "blocking" antibody IgG that trap allergens in the secretions of the nose and respiratory tract, or shells before they react with IgE antibodies or mastocytoma. However, there is no permanent connection between the titers of IgG and relief of symptoms. Currently, this is an extremely lengthy and expensive process, which provides only for patients for whom each year show severe symptoms over a long period.

It has been established that introduction of only purified proteins is usually not sufficient to obtain a strong immune response; the selected antigen, as a rule, should be entered together with auxiliary substances called adjuvants. Put together adjuvants entered the antigen is protected from rapid degradation and adjuvant provides a prolonged release of antigen at a low level.

In contrast to isolated proteins viruses induce a rapid and effective immune responses in the absence of any adjuvants as with T cells, and without it (see Bachmann and Zinkernagel, Ann. Rev. Immunol., 15: 235-270 (1997)). Although viruses often consist of a small number of proteins, they are able to include stronger immune responses than the allocated of these components. For b-cell responses, it is known that one is m of the decisive factors immunogenicity of viruses is the frequency and order of surface epitopes. Many viruses have quasicrystalline surface, which presents an ordered sequence of epitopes that effectively cross-stitch epitope-specific immunoglobulins on b cells (see Bachmann and Zinkernagel, Immunol. Today, 17:553-555 (1996)). This cross-linking of surface immunoglobulin on b cells is a strong activation signal, which directly induces the development of the cell cycle and the production of IgM antibodies. In addition, stimulated B-cells are able to activate T cells-helper cells, which in turn induce switching in b cells the production of antibodies from IgM to IgG and the formation of long-term b-cell memory is the goal of any vaccination (see Bachmann and Zinkernagel, Ann. Rev. Immunol., 15: 235-270 (1997)). The structure of the virus is associated even with the generation of anti-antibodies in autoimmune diseases and as part of a natural response to pathogens (see T. Fehr et al., J. Exp. Med., 185: 1785-1792 (1997)). Thus the antigens on the viral particles, which are organized in an ordered and contains repetitions of the matrix, are highly immunogenic, as they can directly activate b cells.

In addition to a strong b-cell responses, viral particles are able to induce the formation of response of cytotoxic T-cells, another crucial component of the immune system. Yes the major cytotoxic T cells are particularly important to eliminate recitations viruses, such as HIV or hepatitis b virus, and to fight tumors. Cytotoxic T cells recognize native antigens, however, recognize the products of their degradation associated with MHC molecules of class 1 (see Townsend and Bodmer, Ann. Rev. Immunol., 7: 601-624 (1989)). Macrophages and dendritic cells are able to absorb and processional exogenous viral particles (but not soluble components) and provide educated degradation products of cytotoxic T-cells, causing their activation and proliferation (see article Kovacsovics-Bankowski et al., Proc. NatI. Acad. Sci. USA, 90: 4942-4946 (1993); Bachmann et al., Eur. J. Immunol., 25: 2595-2600 (1996)).

Viral particles as antigens have two advantages relative to the selected one of them components: (1) due to their surface structure with a high degree of repetition, they are able to directly activate b cells, resulting in high antibody titer and long-term memory b-cells, and (2) viral particles, but not of soluble proteins capable of inducing the response of cytotoxic T-cells even if the viruses are not infectious and adjuvants are missing.

The number of new vaccine strategies using the inherent virus immunogenicity. Some of these approaches focus on the granular nature of viral particles, for example, see Harding C.V. and R. Song (J. Immunology, 153:4925 (1994)), in which opisyvatsya, consisting of latex particles and antigen; Kovacsovics-Bankowski, M. and et al. (OEWG. NatI. Acad. Sci. USA, 90:4942-4946 (1993)), which describes a vaccine consisting of iron oxide particles and antigen; U.S. Patent No 5334394 received Kossovsky N., et al., which reveal the core particles coated with antigen; U.S. Patent No 5871747, which reveal the synthetic particles, bearing on the surface of one or more proteins covalently associated with them, as well as crustal particle with ecovalence related coating and at least one biologically active agent in contact with the specified covered particle bark (see, for example, application WO /94/15585).

However, the lack of data simulating viruses systems is that they are unable to recreate the orderly presentation of antigens found on the surface of viruses. Antigens associated with the surface in an arbitrary orientation, as found, induce a CTL response and does not induce or induce a weak response In cells. For an effective vaccine both components of the immune system must be strongly activated, as described above and in the article Bachmann and Zinkernagel, Ann. Rev. Immunol., 15: 235(1997).

In another example, recombinant viruses are used to deliver antigen. It was shown that the virus presented filamentous phage containing the antigen, fused with the capsid protein, has high immunogenicity (with whom. article R.N. Perham, et al., FEMS Environ. Rev., 17: 25-31 (1995); Willis et al., Gene, 128: 85-88 (1993); Minenkova et al., Gene, 128: 85-88 (1993)). However, this system is limited to very small peptides (5 or 6 amino acid residues) in the expression of the fused protein at a high level (see lannolo et al., J. Mol. Biol., 248: 835-844 (1995)) or limited by the low level of expression of larger proteins (see de la Cruz et al., J. Biol. Chem., 263: 4318-4322 (1988)). For small peptides observed only the CTL response and missing or there is only a weak response In cells.

In another system the proposed alpha viruses as vehicles for delivery of antigen (see U.S. Patent NoNo 5766602; 5792462; 5739026; 5789245 and 5814482). Problems with the use of recombinant viral systems described to date include the low density of expression of a heterologous protein on the surface of the virus and/or the complexity of a successful and reproducible obtaining new and different recombinant viruses for various applications.

In the further development of virus-like particles (the VLP) will be used in the field of production of vaccines due to their structural properties and their infectious nature. The VLP are supermolecular structure built in a symmetric manner of many protein molecules of one or more types. They lack the viral genome, SLE is a result of what they are noncommunicable. Often the VLP can be obtained in large quantities by heterologous expression and can be cleaned easily.

Examples include the VLP capsid proteins of hepatitis b virus (see Ulrich et al., Virus Res., 50: 141-182 (1998)), measles virus (see article Warnes et al., Gene, 160: 173-178(1995)); Sindbis virus, rotavirus (U.S. Patent NoNo 5071651 and 5374426), FMD virus (see article Twomey et al., Vaccine, 13: 1603-1610, (1995)), Norwalk virus (see article X. Jiang et al., Science 250: 1580-1583 (1990); MatsuiSM. et al., J. Clin. Invest. 87: 1456-1461 (1991)), the retroviral GAG protein (see Patent application PCT No. WO 96/30523), protein P1 of The retrotransposon, a surface protein of hepatitis b virus (see WO 92/11291) and human papilloma virus (see WO 98/15631). In some cases it is possible to use recombinant DNA technology to merge heterologous proteins the VLP (see Kratz R.A. et al., Proc. NatI. Acad. Sci. USA, 96:1915-1920 (1999)).

Thus in this technical field, there is a need to develop new and improved vaccines that stimulate a strong immune response CTL and b cells as effectively as natural pathogens.

The invention

The invention is a multifunctional new technology that allows to obtain particles coated with any desired antigen. Technology allows you to create highly effective vaccines against infectious diseases and develop vaccines for the treatment of the Oia allergies and malignant tumors.

In the first embodiment, the invention is a new composition comprising (A) is not natural molecular carrier and (B) an antigen or antigenic determinant.

Not natural molecular carrier includes crustal particle selected from the group consisting of cow particles are not of natural origin and cow particles of natural origin, as well as the organizer containing at least one first binding site and a specified organizer connected with this cow particle of at least one covalent bond.

The antigen or antigenic determinant has at least one second binding site, which is selected from the group consisting of a binding site not naturally occurring with the specified antigen or antigenic determinant, and binding that exists in nature with the specified antigen or antigenic determinant.

The invention is ordered and contains repetitions antigenic matrix obtained by the Association of the second binding with the first binding site through at least one ones connection. Thus, the antigen or antigenic determinant and not natural molecular carrier are connected together through the Association of the first and second binding centers with the formation of ordered and with the holding repetitions antigenic matrix.

In another embodiment, the crust particle of the above-mentioned composition contains a virus, virus-like particle, a phage, a viral capsid particle or a recombinant form. Alternative crustal particle can be represented by a synthetic polymer or a metal.

In a specific embodiment, the promoter may contain at least one first binding site. The first and second binding sites are particularly important elements of the composition, corresponding to the invention. In various embodiments of the invention the first and/or second binding site can be presented by the antigen and the antibody or fragment of antibody against antigen; Biotin and Avidya; streptavidin and Biotin; a receptor and its ligand; a ligand-binding protein and its ligand; polypeptides that interact by type latinboy lightning; the amino group and reacting it with a chemical group; a carboxyl group and reacting it with a chemical group; sulfhydryl group and reacting it with a chemical group; or a combination of both.

In a more preferred embodiment, the invention presents linking virtually any selected antigen with the surface of the virus, phage, viral particle or viral capsid particles. In izobreteny is developed strong antiviral immune response of the host by injection of antigen in quasicrystalline "virus-like" structure with obtaining a highly efficient immune response, i.e. vaccination against the presented antigen.

In one preferred embodiment, the crust particle can be selected from the group consisting of recombinant proteins of rotavirus, recombinant proteins of Norwalk virus, recombinant proteins of alphavirus, recombinant proteins of FMD virus, recombinant proteins of retrovirus, recombinant proteins of hepatitis b virus, recombinant proteins of tobacco mosaic virus, recombinant proteins of the virus of space for animals and recombinant proteins of human papilloma virus.

In another preferred embodiment, the antigen may be selected from the group consisting of: (1) a protein suited to induce an immune response against cancer cells; (2) a protein suited to induce an immune response against infectious diseases, (3) a protein suited to induce an immune response against allergens, and (4) a protein suited to induce an immune response in farm animals.

In a particularly preferred variant of the invention, the first binding site and/or the second binding site contain a polypeptide that interacts type latinboy lightning. In the most preferred embodiment, the first binding site and/or the second binding site is selected from the group consisting of (1) domain protein latinboy they say the AI JUN, and (2) domain protein latinboy lightning FOS.

In another preferred embodiment, the first binding site and/or the second binding site is selected from the group consisting of (1) genetically engineered lysine residue and (2) genetically engineered cysteine residue, i.e. two residue, which can be chemically communicate with each other.

Other embodiments of the invention include methods of obtaining compositions corresponding to the invention, and methods of therapeutic treatment using these compositions.

It should be understood that both the foregoing General description and the subsequent detailed description presented only as examples and explanations, and are intended to further explain the invention as it is claimed.

List of drawings

The Figure 1 shows the result of Western blotting showing the formation of viral particles containing protein E2-JUN, using the expression vector pCYTts::E2JUN.

The Figure 2 presents the result of Western blotting showing the formation of viral particles containing protein E2-JUN, expressed using the expression vector pTE5'2J::E2JUN.

The Figure 3 presents the result of a Western dot blot showing the expression of FOS antigen-ngh in bacteria and eukaryotes.

The Figure 4 presents the expression of HBcAg-JUN in cells of E. coli.

On Figo is e 5 presents the result of Western blotting, demonstrating that HBcAg-JUN is soluble in lysates of E. coli.

The Figure 6 presents the analysis using SDS-PAGE (polyacrylamide gel electrophoresis using sodium dodecyl sulfate) enrichment capsid particles HBcAg-JUN in the density gradient of sucrose.

The Figure 7 presents the analysis using SDS-PAGE in non conditions binding particles hGH-FOS and HBcAg-JUN.

Information confirming the possibility of carrying out the invention

1. Definition

The following definitions are presented to clarify the invention, which is believed by the inventors, is the invention.

Alphavirus: As used in this context, the term "alphavirus" refers to any of the RNA viruses included in the genus Alphavirus. Descriptions of this kind are contained in the work of Strauss and Strauss, Environ. Rev., 58: 491-562 (1994)). Examples of alpha viruses include viruses Aura, Bebaru, Cabassou, Chikungunya, Eastern encephalomyelitis of horses, Fortmorgan, Getah, Kyzylagach, Mayoaro, Middleburg, Mucambo, Ndumu, Pixuna, Tonate, Triniti, Una, Western encephalomyelitis of horses, Whataroa, Sindbis (SIN), Semliki forest (SFV), Venezuelan encephalomyelitis of horses (VEE) and Ross River.

Antigen: As used in this context, the term "antigen" is a molecule that can be bound by the antibody. The antigen is additionally capable of inducing a humoral immune response and/or cellular immune the initial response, leading to the formation of In - and/or T-lymphocytes. The antigen may have one or more epitopes (b - and T-epitopes). Specific reaction, related to the above, is intended to indicate that the antigen will react with high selectivity with its corresponding antibody and will not react with the multitude of other antibodies which may be stimulated by other antigens.

Antigenic determinants: As used in this context, the term "antigenic determinant" refers to that portion of the antigen that is specifically recognized - or T-lymphocytes. B-lymphocytes respond to foreign antigenic determinants through the production of antibodies, while T-lymphocytes are mediated cellular immunity. Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies or in the context of MHC T-cell receptor.

Association: As used in this context, the term "Association" in relation to the first and second binding sites used in relation to at least one ones connection. The nature of the Association may be covalent, ionic, hydrophobic, polar, or any combination thereof.

Center associate, first: As used in this context, the expression "first binding site" refers to the element the "organizer", which itself is associated with the cow particle in an orderly manner and with which may be associated (contact) the second binding site, located on the antigen or antigenic determinant. The first binding site may be a protein, polypeptide, peptide, sugar, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or their combination, or their chemically reactive group. The set of first binding sites present on the surface is not natural molecular media containing repetitions configuration.

The binding site, second: As used in this context, the expression "second binding site" refers to the element that is attached to the antigen or antigenic determinant, which may be associated the first binding site of the "organizer", which on the surface is not natural molecular media. The second binding site of the antigen or antigenic determinants may be a protein, polypeptide, peptide, sugar, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or a combination Ilyich chemically reactive group. On the antigen or antigenic determinant is present at least one second binding site.

The crust particle: As used in this context, the term "crust particle" refers to a rigid structure with its inherent organization with repetitions, which provides the basis for the accession of the "organizer". The crust particle, as used in this context, may be a product of the synthesis process or the product of a biological process.

CIS-acting: As used in this context, the term "CIS-acting" sequence refers to sequences of nucleic acids that binds replicase to catalyze RNA-dependent replication of RNA molecules. Data replication events lead to the replication of RNA molecules full length or partial RNA molecules, and thus, subgenomic the promoter of alpha viruses is a "CIS-acting sequence. CIS-acting sequences can be at the 5’end, 3’end, or both ends of the nucleic acid molecule or around them, as well as within the sequence.

Fusion: As used in this context, the term "fusion" refers to the combination of the amino acid sequence of different nature in the polypeptide chain through a combination of in reading frame encoding the x nucleotide sequences. The term "merger" unambiguously covers internal merge, i.e. the insertion of sequences of different nature in the polypeptide chain, in addition to merge with one of its ends.

Heterologous sequence: As used in this context, the term "heterologous sequence" refers to a nucleotide sequence that is present in the vector corresponding to the invention. The term "heterologous sequence" refers to any amino acid sequence or RNA encoded by the heterologous DNA sequence contained in the vector corresponding to the invention. Heterologous nucleotide sequence may encode protein molecules or RNA, which are normally expressed in the cell type in which they are present, or molecules that are normally not expressed in them (e.g., Sindbis structural proteins).

Selected: As used in this context, where the term "isolated" is used in relation to the molecule, this term means that the molecule has been removed from the natural to her environment. For example, polynucleotide or polypeptide that is present in vivo in the body of the living animal is not "isolated", but the same polynucleotide or polypeptide, separated from the materials, together with existing the x in its natural state, is "selected". In addition, recombinant DNA molecules contained in a vector is considered isolated with regard to the purposes of the present invention. The selected RNA molecules include presents RNA replication products of DNA and RNA in vivo or in vitro. Selected molecules of nucleic acids further include molecules, obtained by synthesis. In addition, the vector molecule contained in the recombinant cell host, are also highlighted. Thus, not all "isolated" molecules must be "cleansed."

Immunotherapy: As used in this context, the term "immunotherapy" is a composition for treating diseases or disorders. In particular, the term usually relates to a method of treating allergies or treatment of cancer.

Subject: As used in this context, the term "subject" refers to multicellular organisms and includes both plants and animals. Preferably, when multicellular organisms are animals, preferably vertebrates, even more preferably presents a mammal and most preferably a human.

Low or undetectable: As used in this context, the expression "low or undetectable" when used with respect to the level of gene expression refers to the expression level, to what that is either significantly lower than defined at the maximum induction of the gene (for example, at least five times lower), or is difficult to detect by methods used in the following examples section.

Lectin: As used in this context, proteins, obtained mainly from the seeds of leguminous plants, but also from many other plant and animal sources, which are binding sites for specific mono - or oligosaccharides. Examples include concanavalin a and agglutinin wheat germ, which is widely used as an analytical and preparative agents in studies of glycoproteins.

Nature (origin): As used in this context, the term "natural nature (origin)" means the whole or part are not synthetic and are or are formed in nature (i.e. in vivo).

Not natural: As used in this context, the term basically means not derived from nature, in particular, the term means "made by human hands".

Not of natural origin: As used in this context, the term "natural origin" means a synthetic or derived not from nature, in particular, the term refers to man-made.

Not natural molecular media: As used in this context, expressed the group of "not natural molecular carrier" refers to any product, made by human hands, which can serve for the production of rigid and contains repetitions of the sequence of the first binding. In the ideal case, the data of the first binding sites are located in geometrical order, but this is not necessary. Not natural molecular carrier may be organic or inorganic and may be synthesized by chemical means or by using biological process partially or completely. Not natural molecular carrier consists of (a) cow particles of natural or not natural origin and (b) of the promoter, which itself contains at least one first binding site and is connected to the particle bark at least one covalent bond. In a particular embodiment, not natural molecular carrier can be represented by a virus, virus-like particle, a viral capsid particle, a phage, recombinant or synthetic forms of the particle.

Ordered and contains repetitions matrix antigens or antigenic determinants: As used in this context, the term "ordered and contains repetitions matrix antigens or antigenic determinants" mainly repeats the structure of the antigen or antigenic determinants, which are characterized by the same spatial organization of antigens or EN is hennah determinants relative to the base. In one embodiment of the invention the pattern repeat may have a geometric shape. Perfect ordered and contains repetitions matrix antigens or antigenic determinants will be a distinct repetitions paracrystalline the structure of the antigen or antigenic determinant with distances of 5-15 nanometers.

Organizer: As used in this context, the term "promoter" is used to refer to the item associated with the particle bark involuntary manner that ensures the site of the formation of the nucleus for the establishment of an orderly and contains repetitions antigenic matrix. The organizer is any element that contains at least one first binding site, which is connected with the particle bark through at least one covalent bond. The promoter may be a protein, polypeptide, peptide, amino acid (i.e. the remainder of the protein, polypeptide or peptide), sugar, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or their combination, or their chemically reactive group.

Permissive temperature: As used in this context, the expression "permissive temperature" refers to temperatures at which the enzyme has a relatively high level of catalytic activity.

Purified: As used in this context, when the term "purified" is used in respect of the molecule, it means that the concentration of water molecules is increased relative to molecules associated with it in its natural environment. Associated naturally molecules include proteins, nucleic acids, lipids and sugars, but usually do not include water, buffers, and reagents added to maintain the integrity or facilitate purification of molecules that clear. For example, even if the mRNA dilute aqueous solvent during column chromatography using oligo dT, the mRNA molecules purified by the chromatography, if naturally associated nucleic acids and other biological molecules do not bind to the column and are separated from the data of RNA molecules.

Receptor: As used in this context, the term "receptor" refers to proteins or glycoproteins or fragments capable of interacting with another molecule, called a ligand. The ligand can belong to any class of biochemical or chemical compounds. The receptor does not necessarily have to be associated with a membrane protein. Soluble protein, such as, for example, binding protein maltose, or binding protein retinol, is also a receptor.

Balance: As used in the context the term "residue" is intended to denote a certain amino acid in the skeleton or the side chain of the polypeptide.

Temperaturesalinity: As used in this context, the expression "Temperaturesalinity" refers to the enzyme, which is easily catalyze a reaction at one temperature, but slowly catalyzes the same reaction or not catalyzes her at another temperature. Example temperaturecontrolled enzyme is a protein replicase encoded vector pCYTts, which has an easily identifiable activity replicate at temperatures not exceeding 34°and has a low or undetectable activity at 37°C.

Transcription: As used in this context, the term "transcription" refers to the formation of RNA molecules with DNA-matrices, which is catalyzed by RNA polymerase.

Recombinant cell-host: As used in this context, the term "recombinant cell host refers to the host cell into which has been introduced one or more nucleic acid molecules corresponding to the invention.

Recombinant virus: As used in this context, the expression "recombinant virus" refers to a virus that is genetically modified by human intervention. The expression covers any virus known in the art. In h is particularly the expression applies to alphavirus, genetically modified by man, and more specifically the term refers to the Sinbis virus, genetically modified by man.

Limiting temperature: As used in this context, the expression "limiting temperature" refers to temperatures at which the enzyme has a low or undetectable levels of catalytic activity. Known both as "heat"and "cold-sensitive mutants, and thus limiting the temperature may be above or below the permissive temperature.

The event RNA-dependent RNA replication: As used in this context, the term "event of RNA-dependent RNA replication" refers to the processes that lead to the formation of RNA molecules using RNA molecules as a matrix.

RNA-dependent RNA polymerase: As used in this context, the term " RNA-dependent RNA polymerase" refers to a polymerase that catalyzes the formation of RNA molecules using other RNA molecules. This term is used in this context as a synonym of the term "replicate".

Noncoding RNA: As used in this context, the term "noncoding RNA" refers to RNA sequence or molecule that does not encode an open reading frame or open frame encodes scity the project or its fragment, but the format in which is formed an amino acid sequence (e.g., there is no initiation codon). Examples of these molecules are molecules of tRNA, rRNA molecules and ribozymes.

Vector: As used in this context, the term "vector" refers to an agent (e.g., plasmid or virus)used to transfer genetic material into the cell is the master. The vector may consist of either DNA or of RNA.

One or any: When using the terms "one or any" in this description means "at least one" or "one or more"unless specified otherwise.

2. Songs sorted and contains repetitions matrices antigen or antigenic determinants and methods for their preparation

The described invention is a composition comprising an ordered and contains repetitions antigen or antigenic determinant. Moreover, the invention is a professional practitioner with the opportunity to construct an ordered and contains repetitions matrix antigen or antigenic determinants for various therapeutic purposes, which include prevention of infectious diseases, treatment of allergies and treatment of malignant tumors.

Compositions corresponding to the invention, generally contain two elements: (1) no natural molecular carrier and (2) ant the gene or antigenic determinant with at least one second binding site, able to form an Association through at least one ones relation with the said first center link.

Not natural molecular carrier includes (a) crustal particle selected from the group consisting of (1) cow particles are not of natural origin and (2) cow particles of natural origin, and (b) a promoter containing at least one first binding site, where the specified organizer associated with the specified cow particle of at least one covalent bond.

The antigen or antigenic determinant has at least one second binding site, which is selected from the group consisting of (a) binding site, which is not found in nature in the antigen or antigenic determinant; and (b) binding site, which is present in natural conditions in a given antigen or antigenic determinant.

The invention is ordered and contains repetitions antigenic matrix obtained at the expense of the Association of the second binding with the first binding with at least one ones connection. Thus, the antigen or antigenic determinant and not natural molecular carrier are connected together through the Association of the first and second binding centers with the formation of ordered and contains repetitions antigenic matrix is.

Practitioner can specially design the antigen or antigenic determinant and the second binding site so that the location (placement) of all antigens or antigenic determinants associated with non-natural molecular carrier (base), will be uniform. For example, you can put only the second binding site at the carboxyl or aminocore antigen or antigenic determinant, and this design will provide a uniform arrangement for all molecules of the antigen or antigenic determinants associated with non-natural molecular media. Thus, the invention is a convenient means to accommodate any antigen or antigenic determinants on non-natural molecular medium in a certain order and repetition.

For competent professionals in this field will understand that some embodiments of the invention include the use of technology, recombinant nucleic acids, such as cloning, polymerase chain reaction, purification of DNA and RNA, the expression of recombinant proteins in prokaryotic and eukaryotic cells, etc. these methodologies are well known to competent specialists in this field and they can easily be found in the published guidelines on laboratory methods (for example, see the monograph edited by J. Sambrook who et al., Laboratory guide to molecular cloning (Molecular Cloning, A Laboratory Manual), 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); the monograph edited by Ausubel F. et al. Modern methods of molecular biology (Current Protocols in Molecular Biology), John H. Wiley & Sons, Inc. (1997)). Fundamental laboratory techniques for working with tissue cultures and cell lines (see monograph cell Biology (Cell Biology) edited by Celis J., Academic Press, 2nd ed. (1998)) and technology-based antibodies (see monograph Harlow E. and Lane D., "Laboratory manual of antibodies" ("Antibodies: A Laboratory Manual,") Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); the work of M.P. Deutscher, "Guide to protein purification" ("Guide to Protein Purification,") Meth. Enzymol., 128, Academic Press, San Diego (1990); monograph Scopes R.K. "the Principles and practical aspects of protein purification" ("Protein Purification Principles and Practice") 3rd ed., Springer-Verlag, New York (1994)) also adequately described in the literature, which is included in this context by reference.

A. Constructing not natural molecular media

One of the elements of the composition, corresponding to the invention, is not natural molecular media containing crustal particle and organizer. As used in this context, the expression "not natural molecular carrier" refers to any product made by man, which can serve for the production of rigid and contains repetitions of the sequence PE is o binding. In particular, it is not natural molecular carrier includes (a) crustal particle selected from the group consisting of (1) cow particles are not of natural origin and (2) cow particles of natural origin, and (b) a promoter containing at least one first binding site and specified the organizer associated with the specified cow particle of at least one covalent bond.

As will be easily understood competent in this area specialists, crustal particle is not natural molecular carrier corresponding to the invention is not limited to any particular form. The crust particle may be organic or inorganic and may be synthesized by chemical means or by using a biological process.

In one of the embodiments of the invention are not natural crustal particle can be a synthetic polymer, a lipid micelle or metal. Data core particles known in the prior art and provide a basis for the creation of a new not natural molecular carrier (skeleton)of the invention. For example, core particles of synthetic resin or metal is described in U.S. Patent No 5770380, in which is disclosed the use of calixarenes organic substrate, which is connected to multiple peptide loops, to create a "simulator antibodies", which in U.S. Patent No 5334394 describe nanocrystalline particles, used as viral traps, which consist of a wide range of inorganic materials, including metals or ceramics. Preferred metals in this embodiment, include chromium, rubidium, iron, zinc, selenium, Nickel, gold, silver, platinum. Preferred ceramic materials in this embodiment include silicon dioxide, titanium dioxide, aluminum oxide, ruthenium oxide and tin oxide. Particles bark corresponding with this version of the implementation, can be made of organic materials, including carbon (diamond). Preferred polymers include polystyrene, nylon and nitrocellulose. For nanocrystalline particles of this type are especially preferred are particles made of tin oxide, titanium dioxide or carbon (diamond). Lipid micelles prepared by any means known in the art. For example, micelles can be obtained in accordance with the procedure described Baiselle and Millar (see Baiselle .J. and D.B. Millar, Biophys. Chem., 4: 355-361 (1975)) or Corti et al. (see Corti M, DegriorgioV., Sonnino S., Ghidoni R., Masserini M, and Tettamanti, G., Chem. Phys. Lipids, 38: 197-214 (1981)) or Lopez et al. (Lopez O., de la Maza, A., Coderch L, Lopez-lglesias C., Wehrii E. and Parra J.L, FEBS Lett., 426: 314-318 (1998)) or Topchieva and Karezin (Topchieva I. and Karaezin K., J. Colloid Interface Sci., 213:29-35 (1999)) or Morein et al. (Morein Century, Sundquist C., Hoglund, S., Dalsgaard, K., and Osterhaus, A., Nature 308: 457-60 (1984)), the cat who are introduced in this context by reference.

Particle bark can also be obtained using the biological process, which can be natural or not natural. For example, this type of option implementation may include crustal particle containing a virus, a virus-like particle, a phage, a viral capsid particle or a recombinant form. In a more specific embodiment, the crust particle may contain recombinant proteins of rotavirus, recombinant proteins of Norwalk virus, recombinant proteins of alphavirus, recombinant proteins of FMD virus, recombinant proteins of retrovirus, recombinant proteins of hepatitis b virus, recombinant proteins of tobacco mosaic virus, recombinant proteins of the virus of space for animals and recombinant proteins of human papilloma virus.

Being natural or not natural origin, crustal particle corresponding to the invention, characterized in that it contains a promoter that is joined to natural or not natural cow particle of at least one covalent bond. The organizer is an element associated with the cow particle is not random, which provides a plot of the kernel to create an ordered and contains repetitions antigenic matrix. In the ideal case, the organizer associated with cow particle in geometrical order, but this is not necessary is possible. A minimum requirement is that the organizer includes a first binding site.

As defined earlier, the promoter may be any element that contains at least one first binding site that is attached to the cow particle of at least one covalent bond. The organizer can be represented by a protein, polypeptide, peptide, amino acid (i.e. the remainder of the protein, polypeptide or peptide), sugar, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound ((Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or their combination, or their chemically reactive group. In a more specific embodiment, the promoter may include at least one first binding site containing the antigen, antibody or antibody fragment, Biotin, avidin, streptavidin, receptor, receptor ligand, a ligand, a ligand-binding protein, polypeptide, interacting by type latinboy lightning, amino group, a chemical group that reacts with the amino group, carboxyl group, a chemical group that reacts with a carboxyl group, a sulfhydryl group, a chemical group that reacts with a sulfhydryl group, or a combination of both.

In a preferred embodiment, the crust particle n is a natural molecular carrier contains a virus, a bacteriophage, a virus-like particle, a viral capsid particle or a recombinant form. Any virus known in the art as having ordered containing repetitions shell and/or the structure of the measles-containing protein, may be selected as a non-natural molecular carrier corresponding to the invention; examples of suitable viruses include sindbis and other alpha viruses; virus vesicular stomatitis, Rado (e.g., vesicular stomatitis virus), picorna-, toga-, orthomyxo, viruses, virus polyoma, parvovirus, rotavirus, a virus, a retrovirus, hepatitis b virus, tobacco mosaic virus, the virus of space for animals, the human papilloma virus (for example, see Table 1 article Bachman M.F. and Zinkernagel R.M., Immunol. Today, 77: 553-558 (1996)).

In one of the embodiments of the invention use genetic engineering of virus to create a fusion between the ordered and contains repeats of the viral envelope protein and a promoter containing the selected heterologous protein, peptide, antigenic determinant or a reactive amino acid residue. Other genetic manipulations known to specialists in this field can be used when constructing not natural molecular media, for example, it may be desirable to limit the replication capacity of recombinant virus by g the kinetic mutations. Viral protein selected for fusion with the protein of the organizer (i.e. the first binding site)must be ordered and contains repetitions structure, more preferably paracrystalline organization, in the optimal case, with distances of 5-15 nm on the surface of the virus. Creating a fused protein of this type will result in the formation of many sorted and contains repetitions of the organizers on the surface of the virus. Thus, the resulting ordered and contains repetitions organization of the first binding will reflect the normal organization of natural viral protein.

As will be discussed in more detail in this context, in the preferred embodiment of the invention the carrier is a recombinant alphavirus and, in a more particular case, the recombinant virus Sinbis. The alpha viruses are viruses with a positive strand of RNA that replicate their genomic RNA in the cytoplasm of infected cells and without the participation of the intermediate DNA (see Strauss and J. Strauss E., Environ. Rev., 58: 491-562 (1994)). Some representatives of the family of alpha viruses, in particular, Sindbis (see article C. Xiong et al., Science 243: 1188-1191 (1989); Schlesinger, S., Trends Biotechnol., 11: 18-22 (1993)), the virus Semliki Forest (SFV) (see the articleP. and Garoff, H., Bioechnology, 9: 1356-1361 (1991)and others (see ATU Davis N.L et al., Virology, 171: 189-204(1989)) have attracted great attention in terms of use as vectors for the expression of a number of different proteins on the viral basis (see article Lundstrom K., Curr. Opin. Biotechnol., 8: 578-582(1997); Liljestrm P., Curr. Opin. Biotechnol. 5: 495-500 (1994)) and as candidates for vaccine development. Was recently granted a number of Patents directed to the use of alpha viruses for the expression of heterologous proteins and vaccine development (see U.S. Patent NoNo 5766602; 5792462; 5739026; 5789245 and 5814482). Design alphaviruses media corresponding to the invention, may be made by means known in General in the field of recombinant DNA technology, as described in the aforementioned articles, which are included in this context by reference.

To obtain core particles on the basis of the virus to attach the antigen or antigenic determinants can be used many different recombinant host cells. For example, it is known that alpha viruses have a wide host range, Sindbis virus infects cultured cells of mammals, reptiles and amphibians, as well as some insect cells (see H. Clark, J. Natl. Cancer Inst. 51: 645 (1973); C. Leake, J. Gen. Virol., 35: 335 (1977); section written Stollar V. in the monograph "Togavirus The Togaviruses), Ed. by R. W. Schlesinger, Academic Press, (1980), str-621). Thus, for the practical implementation of the invention can be used for many is estvo recombinant host cells. Cells KSS, COS, Vero, HeLa and Cho are particularly suitable for obtaining heterologous proteins because they have activity glycosylation of a heterologous protein, similar activity in human cells (see E. Watson et al., Glycobiology 4: 227, (1994)), and can be tselection (see Zang, M. and et al., Bio/Technology, 13: 389 (1995)) or received genetic engineering (see article Renner W. et al., Biotech. Bioeng., 4: 476 (1995); K. Lee et al., Biotech. Bioeng. 50: 336 (1996)), with the aim of growing in serum-free medium, as well as in suspension.

The introduction of polynucleotide vectors into cells-owners may be effected by methods described in standard laboratory manuals (see, for example, the monograph edited by Sambrook J. et al., Laboratory guide to molecular cloning (Molecular Cloning, A Laboratory Manual), 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Chapter 9; the monograph edited by Ausubel F. et al. Modern methods of molecular biology (Current Protocols in Molecular Biology), John H. Wiley & Sons, Inc. (1997), Chapter 16), which includes such methods as electroporation, DEAE [(diethylamino)ethylcellulose)]-dextran-mediated transfection, transfection, microinjection, mediated by cationic lipid transfection, transduction, the load by scraping, ballistic introduction (bombing) and infection. Methods of introduction of exogenous DNA sequences in the year-the hosts discuss in Patent No 5580859, issued by Felgner P. et al.

Packed RNA sequences can also be used to infect host cells. Data are packaged RNA sequences can be introductionat in the cells of the host by introducing them into the environment for cultivation. For example, in a number of sources, including "Expression system Sindbis" ("Sindbis Expression System"), option (Invitrogen, catalog No. K750-1), described the preparation of noncommunicable alphavirus particles.

When using mammalian cells as recombinant host cells to obtain crustal particles on the basis of the virus in these cells, usually grown in tissue culture. The method of growing cells in culture are well known in the art (see the monograph edited by Celis J. cell Biology (Cell Biology), Academic Press, 2nd ed. (1998); the monograph edited by Sambrook J. et al., Laboratory guide to molecular cloning (Molecular Cloning, A Laboratory Manual), 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); the monograph edited by Ausubel F. et al. Modern methods of molecular biology (Current Protocols in Molecular Biology), John H. Wiley & Sons, Inc. (1977); monograph Freshney R. Culture of animal cells (Culture of Animal Cells), Alan R. Liss, Inc. (1983)).

Specialists in this field will understand that the first binding site may be any suitable protein, polypeptide, sugar, polynucleotide, peptide (aminoxy what LOTOS), natural or synthetic polymer, a secondary metabolite or be a fragment, or combination thereof, which may be for a specific accession to the selected antigen or antigenic determinate to the media. In one embodiment, the implementation of the centre's accession presents the protein or peptide can be selected from known in the art. For example, the first binding site can be selected from the following group: a ligand, receptor, lectin, avidin, streptavidin, Biotin, such epitope as, or T7 tag, ICC, Max, the immunoglobulin domains and any other amino acid sequence, which in the field of engineering known that it would be acceptable as a first binding site.

Next, the experts in this field should be understood that in another embodiment of the invention, the first binding site can be created is secondary to the organizer (i.e. protein or polypeptide), used in the design in frame fusion with the capsid protein. For example, protein can be used to merge with the protein shell with the amino acid sequence of which is known that it glycosylases in a specific way, and entered as a result, the sugar molecule is then the first binding site of viral media by linking Lek is in, which serves as a secondary binding site of the antigen. An alternative sequence of the promoter may be biotinylation in vivo and bitenova part can serve as the first binding site corresponding to the invention, or the sequence of the promoter may be subjected to chemical modification of various amino acid residues in vitro, with this modification serves as the first binding site.

In one of specific embodiments of the invention use the Sinbis virus. The genomic RNA of Sinbis virus packaged in a capsid protein, which is surrounded by a double lipid layer that contains three proteins, called E1, E2 and EZ. Data so-called membrane proteins are glycoproteins and glycosylated sites are located outside the double lipid layer, where the complexes of these proteins form "peaks", which can be seen on electron micrographs protruding from the surface of the virus. In a preferred embodiment of the invention, the first binding site is selected from a domain of the protein latinboy lightning JUN or FOS, which is fused in reading frame with the protein shell of E2. However, for all professionals in this field will be clear that you can use other membrane proteins in the design of the fused protein to the premises of the first binding site in the media corresponding to the invention.

p> In the most preferred embodiment of the invention, the first binding domain selected protein latinboy lightning JUN-FOS, which is fused in reading frame with the protein capsid (bark) of hepatitis C. However, for all professionals in this field will be clear that it is possible to use other proteins of the viral capsid in the design of the fused protein to the premises of the first binding site in the media corresponding to the invention.

In another preferred variant of the invention, the first binding site is selected lysine residue or cysteine, which is fused in reading frame with the protein capsid (bark) of hepatitis b virus. However, for all professionals in this field will be clear that you can use other viral capsid or virus-like particles in the construction of the fused protein to the premises of the first binding site in the media corresponding to the invention.

Example 1 is presented to demonstrate the design of protein, obtained by fusion in the frame of the read envelope protein E2 of Sinbis virus and protein domain latinboy lightning JUN using vector pTE5'2J described by Hahn et al. (see Proc. NatI. Acad. Sci. USA, 89: 2679-2683(1992)). The amino acid sequence of JUN, used for the first binding site, is as follows:

CGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVGC (SEQ ID NO: 59). In this case, the expected is that the binding site on the antigen would be domain protein latinboy lightning FOS and the amino acid sequence would be the following:

CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC (SEQ ID NO: 60).

Sequence data extracted from the transcription factors JUN and FOS, and each is flanked by a short sequence containing a cysteine residue on both sides. It is known that these sequences interact with each other. In the original hypothetical structure proposed for dimer JUN-FOS, it is assumed that the hydrophobic side chains of the monomer intertwined with the corresponding side chains of another monomer as lightning (see Landschuiz et al., Science 240:1759-1764 (1988)). However, as proven, this hypothesis is incorrect, and these proteins are known to form α-the spiral structure (see O'shea et al., Science 243: 538-542 (1989); O'shea et al., CelI, 68: 699-708 (1992); Cohen and Parry, Trends Biochem. Sci., 11: 245-248 (1986)). Thus, the term "lacinova lightning" is often used to refer to data of protein domains is more for historical reasons than from the point of view of structure. In this patent, the term "lacinova zip" used to refer to the sequences disclosed above, or sequences, mostly similar to the other sequences. Terms JUN and FOS are used to denote corresponding domains latinboy lightning, not for whole proteins JUN and FOS.

In one embodiment, the invention is to wear the El on the basis of E2 Sinbis virus E2JUN using expression pCYTts system (see Patent application U.S. No. 60/079562 filed March 27, 1998). Expression pCYTts system is a new expression vectors that provide a rigid regulation of gene expression in eukaryotic cells. DNA vectors of this system are transcribed to form RNA molecules, which are then replicated using temperaturescale replicate with the formation of additional RNA molecules. Obtained when replicating RNA molecules containing a nucleotide sequence that can be translated to form a protein of interest or which encodes one or more noncoding RNA molecules. Thus expression system allows for the production of recombinant virus particles Sinbis.

Example 2 presents the details of obtaining natural molecular media Sinbis E2-JUN, corresponding to the invention. Example 3 additionally presents another way to get media on the basis of recombinant Sinbis virus E2-JUN using vector pTE5'2JE2:JUN, obtained in Example 1. Thus, in the present invention two tools - expression pCYTts system (see Example 2) and the vector system pTE5'2J (see Example 3), using which you can get not natural molecular-based media of recombinant Sinbis virus E2-JUN. Analysis of viral particles, the scientists in each system, presented at the Figure 1 and Figure 2.

As previously shown, the invention comprises core particles viral nature that contain the virus, virus-like particle, a phage, a viral capsid particle or a recombinant form. Competent researchers know how to get the data core particles and attach them to the organizers. By providing other examples of the invention provides in this context, obtaining particles such as hepatitis b virus and capsid particles of measles virus as crustal particles (see Examples 17-22). In this embodiment, domain protein latinboy lightning JUN or domain protein latinboy lightning FOS can be used as a host and, therefore, as a first binding site for the non-natural molecular carrier corresponding to the invention.

In the Examples 23-29 shows the details of the receipt of core particles of hepatitis b virus carrying a peptide fused in reading frame with a reactive lysine residue, and antigens carrying genetically fused residue of cysteine, as the first and second binding sites, respectively.

Century Construction antigen or antigenic determinant with the second binding site

The second element of the composition, corresponding to the invention is an antigen or antigenic determinant with at less the th least one second binding site, able to be associated with at least one ones connection with the first binding site is not natural molecular media. The invention presents a composition that varies in accordance with the antigen or antigenic determinant selected from considerations of the desired therapeutical effect. Other compositions obtained by changing the molecules selected for the second binding site.

The antigens corresponding to the invention can be selected from the group consisting of the following components: (a) proteins suited to induce an immune response against cancer cells; (b) proteins suited to induce an immune response against infectious diseases; (C) proteins suited to induce an immune response against allergens, and (a) proteins suited to induce an immune response in farm animals.

In one specific embodiment of the invention, the antigen or antigenic determinant is a structure used for the prevention of infectious diseases. This method will be effective for the treatment of a wide range of infectious diseases that affect a wide range of hosts, such as human, cow, sheep, pig, dog, cat, other mammals, as well as for memleketim. Infectious diseases that are treated well and is known to the competent specialists in this field, their examples include infections of viral etiology such as HIV, influenza, herpes, viral hepatitis, a disease caused by viruses Epstein-Barr and polioviruses, viral encephalitis, measles, avian pox, etc. or infections of bacterial etiology such as pneumonia, tuberculosis, syphilis, etc., or infections of parasitic etiology such as malaria, trypanosomes, leishmaniasis, trihomoniaz, AMIBIOS etc. Thus, antigens or antigenic determinants selected for compositions corresponding to the invention, are well known in the field of medicine; examples of antigens or antigenic determinants include the following: antigens HIV, etc 140, 160 etc, antigens of influenza hemagglutinin and neuraminidase, a surface antigen of hepatitis b protein of circumsporozoite the causative agent of malaria.

In another specific embodiment, compositions corresponding to the invention, are immunotherapy, which can be used to treat allergies and cancer.

The selection of antigens or antigenic determinants for the composition and method of treating allergies should be known to the competent specialists in the field of medicine dealing with the treatment of these disorders; representative examples of this type of antigen or antigenic determinants include the following: bee venom phospholipase A2Bet v I allergen of birch pollen), 5 Dl m V (allergen poison wasps spotted), Der p I allergen house dust mites).

The selection of antigens or antigenic determinants for the composition and method of treatment of cancer must be known to the competent specialists in the field of medicine dealing with the treatment of these disorders; representative examples of this type of antigen or antigenic determinants include the following: Neg (breast cancer), GD2 (neuroblastoma), EGF-R (malignant glioblastoma), CEA(medullary thyroid cancer), CD52 (leukemia).

In a particular embodiment of the invention, the antigen or antigenic determinant is selected from the group consisting of: (a) a recombinant protein of HIV, (b) a recombinant protein of influenza virus, (C) a recombinant protein of hepatitis C virus, (d) a recombinant protein of Toxoplasma, (e) a recombinant protein of Plasmodium falciparum, (f) a recombinant protein of Plasmodium vivax, (g) a recombinant protein of Plasmodium ovale, (h) a recombinant protein of Plasmodium malariae, (i) a recombinant protein of breast cancer cells, (j) a recombinant protein of cancer cells kidney (k) a recombinant protein of prostate cancer cells, (1) recombinant protein cell skin cancer, (m) a recombinant protein of brain cancer cells of the brain, (n) a recombinant protein of leukemia cells, (o) a recombinant profiling (p) recombinant protein allergies bee sting (q) recombinant protein allergies to nuts, (r) recombinant belcoville food, recombinant proteins asthma and recombinant protein of Chlamydia.

After the antigen or antigenic determinant of a composition selected at least one second binding site may be introduced into the molecule to construct organized and contains repetitions of the sequence associated with non-natural molecular carrier corresponding to the invention. Representative examples of the second binding sites include, but are not limited to antigen, antibody, or antibody fragment, Biotin, avidin, streptavidin, receptor, receptor ligand, a ligand, a ligand-binding protein, polypeptide, interacting by type latinboy lightning, amino group, a chemical group that reacts with the amino group, carboxyl group, a chemical group that reacts with a carboxyl group, a sulfhydryl group, a chemical group that reacts with a sulfhydryl group, or a combination of both.

The Association between the first and second binding sites will be determined by the characteristics of the corresponding selected molecules, but will include at least one ones relationship. Depending on the combination of the first and second binding nature of the Association may be covalent, ionic, hydrophobic, polar, or their combination.

In one embodiment of the invention, the second binding site may be the represented domain protein latinboy lightning FOS or domain protein latinboy lightning JUN.

In the most specific embodiment of the invention selected for the second binding site is a domain protein latinboy lightning FOS, which is specifically associated with domain protein latinboy lightning JUN not natural molecular carrier corresponding to the invention. Association of protein domains latinboy lightning FOS and JUN provides the basis for the formation of organized and contains repetitions matrix antigen or antigenic determinants on the surface of the carrier. Domain protein latinboy lightning FOS can be fused in reading frame with the selected antigen or antigenic determinant on aminobenzo, carboxyl end or be localized within the protein, if desired.

Some merged design FOS are given as examples. The human growth hormone (see Example 4), phospholipase A2bee venom (PLA) (see Example 9), ovalbumin (see Example 10), etc 140 HIV (see Example 12).

To simplify the creation of the slit structures FOS describes the number of vectors that provide a choice of plan creation and design of antigens and antigenic determinants (see Example 6). Vectors pAV1-4 have been developed for the expression of the fused forms of FOS in .li; vectors pAV5 and pAV6 have been developed for the expression of the slit FOS proteins in eukaryotic cells. Brief description of the properties of these vectors is given is about the following:

1. pAVI: This vector was designed for secretion of the fused proteins containing FOS-end, periplasmatic space .li. Interest gene (g.o.i.) can be Legerova in sites StuI/NotI vector

2. V2: This vector was designed for secretion of the fused proteins containing FOS at N-end, periplasmatic space E. Li. Interest gene (g.o.i.) can be Legerova in sites NotI/EcoRV (or Notl/Hindlll) of the vector.

3. V3: This vector was designed for cytoplasmic production fused proteins containing FOS-end, .li. Interest gene (g.o.i.) can be Legerova in sites EcoRV/NotI vector.

4. V4: This vector was designed for cytoplasmic production fused proteins containing FOS at N-end, .li. Interest gene (g.o.i.) can be Legerova in sites NotI/EcoRV (or NotI/Hindlll) vector. The N-terminal methionine residue proteoliticeski removed during protein synthesis (see HireI et al., Rsos. NatI. Acad. Sci. USA, 86: 8247-8251 (1989)).

5. V5: This vector was designed for eukaryotic production fused proteins containing FOS-end. Interest gene (g.o.i.) can be inserted between the sequences encoding the signal sequence of hGH and domain FOS by ligating into the Eco47III sites/NotI vector. Alternative gene containing its own signalmousemoveevent, can be merged with the plot, coding FOS by ligating sites in StuI/NotI.

6. DAV6: This vector was designed for eukaryotic production fused proteins containing FOS at N-end. Interest gene (g.o.i.) NotI/StuI (or NotI/HindIII) of the vector.

Competent in this area specialists will be clear that the design of the fused protein FOS-antigen or antigenic determinant may include the addition in the form of certain genetic elements to facilitate the production of recombinant protein. Example 4 provides guidance on the establishment of a number of elements .li for regulation of translation and Example 7 provides guidance on the introduction of a eukaryotic signal sequence. Can be selected other genetic elements depending on the specific requirements of the practitioner.

The invention, which is also shown, including the products of fused protein FOS-antigen or FOS-antigenic determinant or bacterial (see Example 5)or eukaryotic cells (see Example 8). The choice of cell type for expression of fused protein is within the scope of knowledge of a competent specialist and depends on factors such as whether post-translational modification important aspect in creating a composition.

As previously noted, the invention discloses various methods konstruirovanie the fused protein FOS-antigen or FOS-antigenic determinants through the use of pAV vectors. In addition to ensuring expression in prokaryotes and eukaryotes, these vectors allow practitioners to choose between N - and C-terminal introduction to the antigen domain protein latinboy lightning FOS. Presents specific examples, in which FOS is drained by N - and C-ends with PLA (see Example 9) and ovalbumin (see Example 10). Example 11 demonstrates the purification of fused proteins PLA - and ovalbumin-FOS.

In the most specific embodiment, the invention relates to antigen or antigenic determinants encoded by the HIV genome. In particular, the HIV antigen is presented Dr. As shown in Examples 11-15, can be obtained Dr domain protein latinboy lightning FOS and synthesized and purified protein to attach to non-natural molecular carrier, corresponding to the invention. As is known to the competent person skilled in the art, other antigens and antigenic determinants of HIV can be used to create a composition corresponding to the invention.

In the most specific embodiment of the invention selected for the second binding site is a cysteine residue, which specifically binds to a lysine residue is not natural molecular carrier corresponding to the invention. Chemical bond lysine residue (Lys) residue of cysteine (Cys) provides the basis for education institutions is vannoy and contains repetitions of the sequence of the antigen or antigenic determinants on the surface of the carrier. The cysteine residue can be introduced by engineering in the reading frame of the selected antigen or antigenic determinants on aminobenzo, carboxyl end or be localized within the sequence, if this is desirable. In an example PLA, etc 140 HIV cysteine residue for binding to the lysine residue of the first binding site.

C. Obtaining particles of alpha-vaccine

The invention provides new compositions and methods of constructing ordered and contains repetitions antigenic matrices. As should know competent in this specialist area, conditions for the Assembly of ordered and contains repetitions antigenic matrix largely depend on the specific choice of the first binding site is not natural media and specific selection of the second binding site of the antigen or antigenic determinants. Thus, the choice of the practitioner researcher in creating the composition (i.e. the choice of the first and second binding, antigen and not natural media) will determine the specific terms of an Assembly of particles alfamacchine (combinations of ordered and contains repetitions antigenic matrix and not natural molecular media). Information regarding Assembly of particles alfamacchine, included in the professional expertise of the practitioner researcher, is there are many links to help the practitioner researcher (for example, see the monograph edited by Sambrook J. et al., Laboratory guide to molecular cloning (Molecular Cloning, A Laboratory Manual), 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); the monograph edited by Ausubel F. et al. Modern methods of molecular biology (Current Protocols in Molecular Biology), John H. Wiley & Sons, Inc. (1997); the monograph edited by Celis J. cell Biology (Cell Biology), Academic Press, 2nd ed. (1998); monograph Harlow E. and Lane D. Laboratory Handbook antibodies (Antibodies: A Laboratory Manual,") Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988), which are all included in this context by reference.

In a specific embodiment of the invention the protein domains latinboy zippers JUN and FOS are used respectively as the first and second binding sites, corresponding to the invention. When preparing particles alfamacchine antigen should be obtained and clear in conditions that promote the Assembly ordered and contains repetitions antigenic matrix on a non-natural media. In a special variant domain protein latinboy lightning JUN/FOS FOS-antigen or FOS-antigenic determinant should be treated regenerating agent (for example, dithiothreitol (DTT)to reduce or eliminate the possibility of formation of disulfide bonds (see Example 15).

For cooking is not natural media (i.e. recombinant Sinbis virus) in the embodiment, with domain protein latinboy lightning JUN/FOS R is combinatie viral particles E2-JUN should be concentrated, neutralized and treated regenerating agent (see Example 16).

The Assembly ordered and contains repetitions antigenic matrix version of the JUN/FOS carried out in the presence of a redox pair. Viral particles E2-JUN mixed with 240-fold molar excess of FOS-antigen or FOS-antigenic determinants for 10 hours at 4°C. Then the alpha particle vaccines concentrated and purified via chromatography (see Example 16).

In another embodiment of the invention the binding is non-natural molecular media antigen or antigenic determinant may be performed by chemical cross-stitching. In the most preferred embodiment, the chemical agent presents heterobifunctional cross-linking agent such as N-hydroxysuccinimidyl ester ∈-maleimidomethyl acid (see article Tanimori et al., J. Pharm. Dyn., 4: 812 (1981); Fujiwara et al., J. Immunol. Meth., 45: 195 (1981)), which contains the operations group that reacts with amino groups, and (2) maleimido group reactive SH-groups. Heterologous protein or polypeptide of the first binding site can be constructed so that it contains one or more lysine residues, which will serve as the reaction for the operations group heterobifunctional PE Cristo-linking agent. After completion of the chemical binding of the first binding is not natural molecular media maleimide group heterobifunctional cross-linking agent will be available to react with the SH group of the cysteine residue on the antigen or antigenic determinant. Obtaining antigen or antigenic determinants in this case may require the construction of a cysteine residue introduced into a protein or polypeptide selected as the second binding site so that it could react with free maleimide group on the cross-linking agent that is associated with the first binding is not natural molecular media.

3. Compositions, vaccines and their use, and methods of treatment

In one of the embodiments in the invention presents vaccines for prevention of infectious diseases in a wide range of species, particularly mammalian species such as human, monkey, cow, dog, cat, horse, pig, etc. Can be developed vaccine for the treatment of infections of viral etiology such as HIV, influenza, herpes, viral hepatitis, a disease caused by viruses Epstein-Barr and polioviruses, viral encephalitis, measles, avian pox, etc., or infections of bacterial etiology such as pneumonia, tuberculosis, syphilis, etc., or infections of the parasite is Noah etiology, such as malaria, trypanosomes, leishmaniasis, trihomoniaz, AMIBIOS etc.

In another embodiment, the invention presents vaccines for the prevention of cancer in a wide range of species, particularly mammalian species such as human, monkey, cow, dog, cat, horse, pig, etc. Can be developed vaccine for the treatment of all types of malignant tumors: lymphoma, carcinomas, sarcomas, melanomas, etc.

In another embodiment, the invention compositions corresponding to the invention, can be used to develop vaccines for the treatment of allergies. Antibodies of IgE isotype are important components of allergic reactions. Mastocyte bind IgE antibodies on their surface and release histamines and other mediators of allergic reactions in specific binding of antigen with IgE molecules that are associated with the surface of mastocytes. Inhibition of the formation of IgE antibodies as a consequence, is a promising target in terms of protection from allergies. It would be possible upon receipt of the response of T cells-helper cells. The responses of T cells-helper cells can be divided into the responses of T cells-helper type 1 (TH1) and type 2 (TH2) (see Romagnani, Immunol. Today, 18:263-266 (1997)). Cells TH1 secrete γ-interferon and other cytokines, which trigger the production of antibodies lgGI-3 in b-cell is.

On the contrary, a critical cytokine produced by cells of the TH2, presents IL-4 (interleukin-4), which directs the products lgG4 and IgE in b cells. In many experimental systems, the emergence of the answers TH1 and TH2 is mutually exclusive, because the cells of TH1 cupressinum the induction of T cellH2 and Vice versa. Thus the antigens that trigger a strong response of TH1, at the same time cupressinum development of responses TH2 and, therefore, production of IgE antibodies. Interestingly, in fact, all viruses induce the response of TH1 owner and do not cause the production of antibodies of IgE (see Coutelier et al., J. Exp. Med., 165: 64-69 (1987)). This sample isotype is not limited live viruses, but was marked to be inactivated or recombinant viral particles (see Lo-Man et al., Eur. J. Immunol., 28: 1401-1407 (1998)). Thus, using the process corresponding to the invention (for example, technology alpha vaccines), viral particles can be put to various allergens and used for immunization. These "viral structure of the allergen will receive an answer TH1, will form a "protective" antibody lgG1-3, which will prevent the production of IgE antibodies that cause allergic reactions. Because the allergen prezentuetsya viral particles, which resposne is raised by another set of T-cells, helper cells, than the allergen, it is likely that allergen-specific antibody lgG1-3 will be the induction even in patients with allergic subjects carrying pre-existing cells TH2, specific in relation to the allergen. The presence of high concentrations of IgG antibodies can inhibit the binding of allergen with IgE-related mastocytoma, thus inhibiting the release of histamine.

As a consequence, the presence of IgG antibodies can protect against lgE-mediated allergic reactions. Typical substances that cause allergies include pollen, grasses, ragweed, birch or juniper Mexican, house dust, mites, dander, mold, insect venom or medicinal substance (e.g., penicillin). Thus, immunization of subjects with viral particles, which are placed allergens, should be effective not only before but also after the onset of allergies.

Professionals in this field should be understood that, when the composition corresponding to the invention, is administered to a subject, they may be in preparation, which contains salts, buffers, adjuvants, or other substances which are desirable to improve the effectiveness of the composition. Examples of materials suitable for use in the manufacture of pharmaceutical compositions, described in numerous sources, including the Handbook of pharmaceutical Sciences, Remington (Remington''s Pharmaceutical Sciences (Ed. Osol, A., Mack Publishing Co., (1980)).

Consider that the composition corresponding to the invention, are "pharmacologically acceptable"if its administration can be tolerated by the principal recipient. In addition, the composition corresponding to the invention will be administered in therapeutically effective amounts (i.e. amounts that creates the desired physiological effect).

Composition, corresponding to this invention, can be entered in various ways known in the prior art, but the norm will be given by injection, infusion, inhalation, oral, or other suitable physical means. The composition may alternatively be administered intramuscularly, intravenously or subcutaneously. The components of the compositions for injection include sterile water (e.g., saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. Media or occlusive dressings can be used to increase the permeability of the skin and enhance absorption of the antigen.

In addition to vaccine technologies in other embodiments, the invention presents methods of medical treatment of cancer and allergies.

All patents and p is blikali, presented in this context, specifically incorporated by reference.

Examples

Enzymes and reagents used in the subsequent examples include DNA ligase T4 acquired company New England Biolabs; DNA polymerizatio, a set of plasmids Ql Spin Plasmid Kit, medium set of plasmids QIAGEN plasmid Midi Kit, kit for gel extraction QiaExll Gel Extraction Kit, the kit for purification of PCR (polymerase chain reaction) QIAquick PCR Purification Kit purchased from the company QIAGEN; set to clear MPHK QuickPrep Micro mRNA Purification Kit purchased in the firm Pharmacia; set for one-step RT-PCR (polymerase chain reaction using the reverse transcriptase) Superscript One-step RT PCR Kit, serum calf embryos (PCS), lactotropes and yeast extract, purchased the company Gibco BRL; oligonucleotides purchased from the company Microsynth (Switzerland); restrictase acquired firm Boehringer Mannheim, New England Biolabs or MBI Fermentas; Pwo polymerase and dNTP (dezoksinukleozidtrifosfaty)acquired firm Boehringer Mannheim. Environment HP-1 acquire the company CeII culture technologies (best Western, Switzerland). All standard chemicals acquire the company Fluka-Sigma-Aldrich, and all materials related to culture cells acquire the company RTR.

Manipulation of DNA is done using standard technology. DNA is prepared in accordance with the manufacturer's instructions, or from 2 ml of bacterial culture using the set of QIAprp Spin Plasmid Kit or from a 50 ml culture, using the kit QIAGEN Plasmid Midi Kit. For cutting by the restriction enzyme DNA incubated for at least 2 hours with the appropriate restriction enzyme at a concentration of 5-10 units (U) of enzyme/mg DNA in the manufacturer's recommended conditions (buffer, and temperature). Reaction of cutting more than one enzyme are at the same time, if the reaction conditions are suitable for all enzymes, in another case, the reaction is conducted sequentially. The DNA fragments selected for further manipulations, separated by electrophoresis in a 0.7 a 1.5% agarose gel, cut out from the gel and purified using the kit for the extraction of the gel QiaExll Gel Extraction Kit in accordance with instructions provided by the manufacturer. For ligation of DNA fragments of 100-200 PG of purified vector DNA incubated over night with a threefold molar excess of fragment insertions at 16°in the presence of 1 unit of DNA ligase T4 in the buffer supplied by the manufacturer (total volume of 10-20 μl). An aliquot (0.1 to 0.5 μl) of the ligation reaction is used to transform .li XL1-Blue (Stratagene). The transformation is performed by electroporation using a pulsating device for introduction of a gene Gene Pulser (BioRAD) and the cell for Gene Pulser 0.1 cm (BioRAD) at 200 ohms, 25 μf, 1,7 kV. After electroporation the cells incubated with shaking for 1 hour in 1 ml of medium S.O.B. (see Miller, 1972) before on the estate them on selective agar S.O.B.

Example 1:

The insertion of amphipatic helical domain JUN in E2

In vector pTE5'2J (see Hahn et al., Proc. NatI. Acad. Sci. USA, 89: 2679-2683, (1992)), being introduced sites restricts MluI and BstEII between codons 71 (Gln) and 74 (Thr) sequence that encodes a structural protein E2, which leads to the formation of vector pTE5'2JBM. The introduction of these sites restricts carried out by PCR using the following oligonucleotides:

Oligonucleotide 1:

E2insBstEII/BssHII:

5’-ggggACGCGTGCAGCAggtaaccaccgTTAAAgaaggcacc-3’ (SEQ ID NO:1) Oligonucleotide 2:

E2insMlulStul:

5’-cggtggttaccTGCTGCACGCGTTGCTTAAGCgacatgtagcgg-3’ (SEQ ID NO:2) Oligonucleotide 3:

E2insStuI:

5’-CCATGAGGCCTACGATACCC-3’ (SEQ ID NO:3)

Oligonucleotide 4:

E2insBssHII:

5’-GGCACTCACGGCGCGCTTTACAGGC-3’ (SEQ ID NO:4)

For PCR using 100 pmol each of the oligonucleotide with 5 ng DNA templates in a 100 μl reaction mixture containing 4 units of polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4. All the DNA concentration determined photometrically using the device GeneQuant (Pharmacia). Polymerase are added directly before PCR (origin - 95°). Temperature Cycling as follows and in the following order: 95°C for 2 minutes; 5 cycles consisting of stages: 95°C (45 sec), 53°C (60 sec), 72° (80 sec); and 25 cycles consisting of stages: 95°C (45 sec), 57°C (60 sec), 72° (80 sec).

Two of the fragment obtained by PCR analyses the comfort and purified by gel-electrophoresis using agarose. To obtain the final designs, conduct an Assembly using two PCR obtained PCR fragments using oligonucleotides 3 and 4 for amplification.

For Assembly by PCR using 100 pmol each of the oligonucleotide with 2 ng of purified fragments obtained by PCR, 200 μl reaction mixture containing 4 units of polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4. All the DNA concentration determined photometrically using the device GeneQuant (Pharmacia). Polymerase are added directly before PCR (origin - 95°). Temperature Cycling as follows and in the following order: 95°C for 2 minutes; 5 cycles consisting of the following stages: 95°C (45 sec), 57°C (60 sec), 72°C (90 sec); and 25 cycles consisting of the following stages: 95°C (45 seconds), 59°C (60 sec), 72° (90 seconds).

The final PCR product is purified using column for PCR Qia spin (Qiagen), and cut in a suitable buffer using 10 units of restricted BssHII and StuI, for 12 hours at 37°C. the DNA Fragments purified by gel-electrophoresis and are ligated into the cut BssHII/StuI and purified by gel-electrophoresis vector pTE5'2J (see Hahn et al., Proc. Natl. Acad. Sci. USA, 89:2679-2683 (1992)). The correct insertion of the PCR product is first analyzed by restriction analysis using BstEII and MIuI, and then by DNA sequencing fragment, extracting the aqueous PCR.

The DNA sequence encoding a domain amphipatic helix JUN, amplified by PCR from the vector pJuFo (see Crameri and Suter, Gene, 137:69 (1993))using the following oligonucleotides:

Oligonucleotide 5:

JUNBstEII:

5’-CCTTCTTTAAcggtggttaccTGCTGGCAACCaacgtggttcatgac-3’ (SEQ ID NO:5) Oligonucleotide 6:

MlulJUN:

5’-AAGCATGCTGCacgcgtgTGCGGTGGTCGGATcgcccggc-3’ (SEQ ID NO:6)

For PCR using 100 pmol each of the oligonucleotide with 5 ng DNA templates in a 100 μl reaction mixture containing 4 units of polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4. All the DNA concentration determined photometrically using the device GeneQuant (Pharmacia). Polymerase are added directly before PCR (origin - 95°). Temperature Cycling as follows and in the following order: 95°C for 2 minutes; 5 cycles consisting of the following stages: 95°C (45 sec), 60°C (30 sec), 72° (25 sec.); and 25 cycles consisting of the following stages: 95°C (45 sec), 68°C (30 sec), 72°C (20 sec).

The final PCR product purified by gel-electrophoresis and are ligated into the cut and purified by gel-electrophoresis pBluescript II(KS’). From the received vector emit a sequence of JUN by cutting MluI/BstEII, purified using QiaExII and are ligated into the vector pTE5'2JBM (pre-cut the same restrictases) to obtain a vector pTE5'2J:E2JUN.

Example 2:

Obtaining VI is usnih particles, containing E2-JUN using the pCYTts system

Structural proteins are amplified by PCR using pTE5'2J:E2JUN as template and oligonucleotides XbaIStruct (ctatcaTCTAGAATGAATAGAGGATTCTTTAAc) (SEQ ID NO: 12) and StructBsp-1201 (tcgaatGGGCCCTCATCTTCGTGTGCTAGTCAg) (SEQ ID NO: 87). For PCR using 100 pmol of each oligonucleotide and 5 ng DNA templates in a 100 μl reaction mixture containing 4 units of polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4. All the DNA concentration determined photometrically using the device GeneQuant (Pharmacia). Polymerase are added directly before PCR (origin - 95°). Temperature Cycling as follows: 95°C for 3 minutes with the following 5 cycles consisting of the following stages: 93°With (35 seconds), 54°With (35 sec), 72° (270 sec), and the subsequent 25 cycles consisting of the following stages: 92°With (30 seconds), 63°With (35 sec), 72° (270 sec). The PCR product purified by gel-electrophoresis and cut restrictase XbaI/Bsp1201 and are ligated into the vector pCYTts, pre-cut by the same enzymes (see Patent application U.S. No.60/079562 filed March 27, 1998).

Twenty micrograms pCYTtsE2:JUN incubated with 20 units of ScaI in a suitable buffer for at least 4 h at 37°C. the Reaction is stopped by extraction with phenol/chloroform followed by precipitation with isopropanol linearized DNA. The reaction restriction check gel-aletria Tom agarose. For transfection of 5.4 μg linearized pCYTtsE2:JUN mixed with 0.6 ág of the linearized pSV2Neo in 30 μl of H2O and add 30 μl of 1 M solution l2. After adding 60 μl of phosphate buffer (50 mm HEPES, 280 mm NaCl, 1.5 mm Na2HPO4pH 7,05) the solution is shaken for 5 seconds and then incubated at room temperature for 25 seconds. The solution is immediately added to 2 ml of medium HP-1 containing 2% FCS (2% FCS environment). Wednesday confluent at 80% cell culture VNC 6-hole Board, then replace the DNA-containing medium. After incubation for 5 hours at 37°C incubator with CO2The DNA-containing medium is removed and replaced by 2 ml of 15% glycerol in 2% FCS environment. The glycerol-containing medium is removed after a 30-second phase of the incubation and wash cells with 5 ml of medium HP-1, containing 10% FCS. Finally, add 2 ml of fresh medium HP-1, containing 10% FCS. Select stably transfetsirovannyh cells and grown on selective medium (HP-1 with the addition of G418) at 37°C incubator with CO2. When mixed population grows to confluently, culture is divided into two cups with the subsequent period of growth for 12 h at 37°C. One Cup with cells transferred in terms of cultivation at 30°for the induction of expression of viral particles, another Cup maintained at 37°C.

The expression of viral particles was determined by OSU Western blotting (see Figure 1). Culture medium (0.5 ml) precipitated with methanol/chloroform and resuspended sediment in sample buffer for SDS-PAGE. Samples heated for 5 minutes at 95°before making a 15% acrylamide gel. After SDS-PAGE the proteins transferred to nitrocellulose membrane Protan (Schleicher &Schuell, Germany), as proposed in section, written Bass and Yang, in the monograph edited by Creighton I.E. Practical approaches to the function of the protein (Protein Function: A Practical Approach, 2nd ed., IRL Press, Oxford (1997), p.29-55. The membrane blocked with 1% bovine albumin (Sigma) in TBS (10 × TBS/l: 87,7 g NaCl, 66,1 r Trizma hydrochloride (Sigma) and 9.7 r Trizma base (Sigma), pH 7.4) for 1 hour at room temperature followed by incubation with the antibody against EI/E2 (polyclonal serum) for 1 hour. The blot was washed 3 times for 10 minutes with TBS-T (TBS with the addition of 0.05% Tween 20) and incubated for 1 hour with alkaline phosphatase conjugate with IgG against rabbit (0.1 ág/ml, Amersham Life Science, England). After washing 2 times for 10 minutes in TBS-T and 2 times for 10 minutes in TBS conduct manifesting reaction using reagents for detection of alkaline phosphatase (10 ml AR buffer (100 mm Tris/HCl, 100 mm NaCl, pH 9,5) with 50 μl of NBT solution (7,7% nitro blue tetrazole (Sigma) in 70% dimethylformamide) and 37 μl of solution X-phosphate (5% of 5-bromo-4-chloro-3-indolylacetic in dimethylformamide).

Obtaining viral particles presented in Figure 1. The image of the Western blot shows that E2-JUN (lane 1) migrates in the direction of higher molecular weight in SDS-PAGE compared with E2 wild-type (lane 2) and the line of host cells VNC gives no background.

Example 3:

Obtaining viral particles containing E2-JUN, using vector pTE5'2JE2:JUN

Not containing RNase vector (1,0 µg) linearized cutting with Pvul. This is followed by transcription in vitro using the set for transcription in vitro SP6 (InvitroscripCAP made InvitroGen, Invitrogen BV, NVLeek, Netherlands). Received MMX’-cap analyze in pampering agarose gel.

Transcribed in vitro mRNA (5 μg) introduced by electroporation into cells VNC ADS: CCL10) in accordance with the instructions Invitrogen (Expression system Sindbis (Sindbis Expression system, Invitrogen BV, the Netherlands). After incubation for 10 minutes at 37°With medium containing FCS, replace environment HP-1, not containing FCS, followed by an additional incubation at 37°C for 10 hours. The supernatant is collected and analyzed by Western blotting for the formation of viral particles the same way as described in Example 2.

The result identical to the result obtained using pCYTtsE2:JUN, as shown in Figure 2.

Example 4:

The merging of human growth hormone (hGH) with domain latinboy lightning FOS (signal sequence is how it is!)

Gene hGH without human l the gender sequences amplified from the original plasmid (ATSS 31389) using PCR. Oligonucleotide 7 with an internal XbaI site create for annealing hGH gene on the 5’-end of the oligonucleotide 9 with an internal EcoRI site will primesouth at the 3’-end of the hGH gene. For PCR using 100 pmol of each oligonucleotide and 5 ng DNA templates in 75 μl of the reaction mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4).

PCR Cycling is as follows: 30 cycles at the annealing temperature 60°and elongation time of 1 min at 72°C.

Purified by gel-electrophoresis and selected PCR product used as template for a second PCR with the aim of introduction of the signal sequence is how it is! and consistency Shine-Dalgarno (Shine-Dalgarno). For PCR using 100 pmol of oligonucleotides 8 and 9, and 1 ng of the obtained PCR fragment-matrix in 75 μl of the reaction mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4). The temperature of annealing for the first five cycles 55°at the time of elongation for 60 s at 72°With other 25 cycles carried out at a temperature of annealing 65°With time and elongation for 60 s at 72°C.

Oligonucleotide 7:

gggtctagattcccaaccattcccttatccaggctttttgacaacgctatgctccgcgcccatcgtctgcaccag

ctggcctttgacacc (SEQ ID NO:7)

Oligonucleotide 8:

gggtctagaaggaggtaaaaaacgatgaaaaagacagctatcgcgattgcagtggcactggctggtttcgct accgtagcgcaggccttcccaaccattcccttatcc (SEQ ID NO:8)

Oligonucleotide 9:

cccgaattcctagaagccacagctgccctcc (SEQ ID NO:9)

The obtained recombinant hGH gene subcloning in pBluescript using XaI/EcoRI. The correct sequences of both chains is confirmed by DNA sequencing.

The DNA sequence encoding a domain amphipatic helix FOS, amplified by PCR from the vector pJuFo (see Crameri & Suter, Gene, 137:69 (1993)), using the oligonucleotides:

omp-FOS:

5’- ccTGCGGTGGTCTGACCGACACCC-3’ (SEQ ID NO: 10)

FOS-hgh:

5’- ccgcggaagagccaccGCAACCACCGTGTGCCgccaggatg-3’ (SEQ ID NO:11).

For PCR using 100 pmol of each oligonucleotide and 5 ng DNA templates in 75 μl of the reaction mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4). Perform the following temperature cycles: 95°C for 2 minutes followed by 5 cycles consisting of the following stages: 95°C (45 sec), 60°C (30 sec), 72° (25 sec), and the subsequent 25 cycles consisting of the following stages: 95°C (45 sec), 68°C (30 sec), 72°C (20 sec).

The PCR product was then purified, isolated and clone in the cut with StuI pBluescript-ompA-hGH. Hybrid gene then clone in plasmid pKK223-3 (Pharmacia).

Example 5:

Bacterial expression of FOS-hGH

ompA-FOS-hGH in pkk223-3 expressyour under the control of the inducible promoter, using JM101 as strain E. Li-owner. The expression is carried out in the flask shaking. Cells induce 1 mm IPTG (isopropylthioxanthone) (final concentration) at an OD (optical density) at 600 of 0.5. The expression continued for 10 hours at 37°C. the Cells with eraut by centrifugation at 3600× g at 10°C for 15 min to Sediment the cells frozen (-20°or liquid N2) and stored for 16 hours. Then the sediment was thawed at 4°and resuspending in 10 ml of 10 mm Tris - HCl, pH 7.4, containing 600 mm sucrose. After stirring for 15 min at 4°periplasmatic proteins released by osmotic shock procedure. Add chilled (4° (C) deionized water and mix the suspension for 30 min at 4°C. the Residue is diluted, resuspending and add lysozyme to degrade the cell walls of bacteria. Cells and spheroplast periplasmatic fraction is separated by centrifugation for 20 min at 11000×g at 4°C. Containing FOS-hGH supernatant analyzed by SDS-PAGE in reducing and non conditions and dot-blotting. Dot-blotting were carried out as described in Example 8, using antibody against hGH (Sigma) as the first antibody and the conjugate of alkaline phosphatase (AP)-antibody against mouse as the second antibody.

Correctly processionary FOS-hGH full length can be defined in regenerating and non conditions. Part of FOS-hGH is associated with other unidentified proteins using free cysteines present at amphipatic helix FOS. However, more than 50% downregulation of FOS-hGH is in his native monomial is nuclear biological chemical (NBC conformation (see Figure 3).

Purified FOS-hGH will be used to conduct the first experiments on the introduction JUN-containing viral particles.

Example 6:

The design of a series of pAV vectors for the expression of the fused proteins FOS

Design of a multifunctional vector system that provides either cytoplasmic production or secretion of proteins, merged with FOS at the N - or C-ends in .li or products of proteins, merged with FOS at the N - or C-ends in eukaryotic cells. Vectors pAV1 - pAV4 created for products merged with FOS proteins in .li cover the following cassette DNA that contain the following genetic elements in the following order: (a) a strong binding site of the ribosome and 5’-noncoding area, isolated from the gene .li is how it is! (aggaggtaaaaaacg) (SEQ ID NO: 13); (b) a sequence encoding a signal peptide of a protein of the outer membrane .li is how it is! (MKKTAIAIAVALAGFATVAQA) (SEQ ID NO: 14); (C) a sequence encoding a domain of FOS dimerization, flanked on both sides residues glycine and the residue cysteine (CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC) (SEQ ID NO: 15), and (d) segment encodes a short peptide linker (AAASGG (SEQ ID NO: 16) or GGSAAA (SEQ ID NO: 17)), the binding protein of interest with the domain of FOS dimerization. The corresponding encoding plots are given in capital letters. The organization of restriction enzymes cut sites provides ease of constructing the cast with FOS genes, or not containing a signal sequence. Cassette clone in the restriction site EcoRI/HindIII expression vector RK-3 (Pharmacia) for expression of fused genes under the control of a strong promoter tac.

pAVI

This vector design for secretion of the fused proteins containing FOS-end, periplasmatic space .li. Interest gene (g.o.i.) you can ligitamate in sites StuI/NotI vector.

pAV2

This vector design for secretion of the fused proteins containing FOS at N-end, periplasmatic space .li. Interest gene (g.o.i.) you can ligitamate in sites NotI/EcoRV (or Notl/Hindlll) of the vector.

V3

This vector design for cytoplasmic production fused proteins containing FOS-end, .li. Interest gene (g.o.i.) you can ligitamate in sites EcoRV/NotI vector.

V4

This vector design for cytoplasmic production fused proteins containing FOS at N-end, .coil. Interest gene (g.o.i.) you can ligitamate in sites NotI/EcoRV (or NotI/HindIII) of the vector. The N-terminal methionine residue is removed proteoliticeski when protein synthesis (see Hirel et al. Proc. NatI. Acad. Sci. USA 86: 8247-8251 (1989)).

In story pAV5 and pAV6, developing for eukaryotic products merged with FOS proteins, cover the following genetic elements placed in a different order: (a) segment encoding a leader peptide of human growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSA) (SEQ ID NO: 26); (b) a sequence encoding a domain of FOS dimerization, flanked on both sides by two glycine residues and a cysteine residue (CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC) (SEQ ID NO: 15); and (C) segment encodes a short peptide linker (AAASGG (SEQ ID NO: 16) or GGSAAA (SEQ ID NO: 17)), a binding protein of interest with the domain of FOS dimerization. The corresponding encoding plots are given in capital letters. The organization of restriction enzymes cut sites provide ease of design merged with FOS genes. Cassette clone in the restriction site EcoRI/HindIII expression vector pMPSVEH (see Artelt et al., Gene, 68:213-219 (1988)).

pAV5

This vector developed for eukaryotic production fused proteins containing FOS-end. Interest gene (g.o.i.) you can enter between the sequences encoding the signal sequence of hGH and domain FOS, by ligating into the Eco47III sites/NotI vector. Alternative gene containing its own signal sequence, can be drained from the site, coding FOS, by ligating sites in StuI/NotI.

V6

D. the config develop vector for eukaryotic production fused proteins contains FOS at N-end. Interest gene (g.o.i.) you can ligitamate in sites NotI/StuI (or NotI/HindIII) of the vector.

Construction of expression vectors pAVI-pAV6

To construct expression vectors pAVI-pAV6 synthesize the following oligonucleotides:

FOS-FOR1:

CCTGGGTGGGGGCGGCCGCTTCTGGTGGTTGCGGTGGTCTGACC(SEQ IDNO: 31);

FOS-FOR2:

GGTGGGAATTCAGGAGGTAAAAAGATATCGGGTGTGGGGCGGCC (SEQ ID NO: 32);

FOS-FOR3:

GGTGGGAATTCAGGAGGTAAAAAACGATGGCTTGCGGTGGTCTGACC (SEQ ID NO: 33);

FOS-FOR4:

GCTTGCGGTGGTCTGACC (SEQ ID NO: 34);

FOS-REV1:

CCACCAAGCTTAGCAACCACCGTGTGC (SEQ ID NO: 35);

FOS-REV2:

CCACCAAGCTTGATATCCCCACACCCAGCGGCCGCAGAACCACCGCAACCACCG (SEQ ID NO: 36);

FOS-REV3:

CCACCAAGCTTAGGCCTCCCACACCCAGCGGC (SEQ ID NO: 37);

OmpA-FOR-1:

GGTGGGAATTCAGGAGGTAAAAAACGATG (SEQ ID NO: 38);

hGH-FOR1:

GGTGGGAATTCAGGCCTATGGCTACAGGCTCC (SEQ ID NO: 39), and

hGH-FOR2:

GGTGGGAATTCATGGCTACAGGCTCCC (SEQ ID NO: 40).

To construct a vector V2 amplified plots encoding the signal sequence is how it is! and the domain of FOS, of the fused gene ompA-FOS-hGH in the vector RK-3 (see Example 5), using a pair of primers OmpA-FORI/FOS-REV2. The PCR product is cut with EcoRI/HindIII and are ligated into the same sites of vector RK-3 (Pharmacia).

To construct a vector pAV1 amplified plot, coding FOS, of the fused gene ompA-FOS-hGH in the vector RK-3 (see Example 5), using a pair of primers FOS-FORI/FOS-REV1. The PCR product is cut with HindIII and are ligated into the cut StuI/HindIII vector pAV2.

To construct a vector pAV3 amplificare the plot, encoding domain FOS, from the vector pAV1, using a pair of primers FOS-FOR2/FOS-REV1. The PCR product is cut with EcoRI/Hindlll and are ligated into the same sites of vector RK-3 (Pharmacia).

To construct a vector V4 amplified section that encodes a domain of FOS, of the fused gene ompA-FOS-hGR in the vector RK-3 (see Example 5), using a pair of primers FOS-FOR3/ FOS-REV2. The PCR product is cut with EcoRI/Hindlll and are ligated into the same sites of vector RK-3 (Pharmacia).

To construct a vector V5 amplified section that encodes the signal sequence of hGH fused gene hGH-FQS-hGH in the vector pSINrepS (see Example 7), using a pair of primers hGH-FOR1/hGHREV1. The PCR product is cut with EcoRI/NotI and are ligated into the same sites of vector pAV1. Then, the resulting cassette encoding a signal sequence hGH and domain FOS, isolate and clone in the vector pMPSVEH (see Artelt et al., Gene, 68: 213-219 (1988)), cut with the same enzymes.

To construct a vector V6 amplified plot, coding FOS, from the vector pAV2, using a pair of primers FOS-FOR4/FOS-REV3. The PCR product is cut with HindIII and clone in split Eco47III/HindIII vector pAV5. A cassette encoding a signal sequence hGH and domain FOS, then re-amplified from the received vector, using a pair of primers hGH-FOR2/FOS-REV3, digested with EcoRI/HindIII and are ligated into the vector pMPSVEH (see Artelt and co-is., Gene, 68:213-219 (1988)), cleaved with the same enzymes.

Example 7:

The design of FOS-hGH with human (hGH) signal sequence

For eukaryotic expression of fused protein FOS-hGH allocate fused gene OmpA-FOS-hGH from pBluescript::OmpA-FOS-hGH (see Example 4) by cutting with XbaI/Bsp1201 and clone in the vector pSINrepS (Invitrogen), cleaved with the same enzymes. The signal sequence of the hGH is synthesized using a PCR reaction mixture containing 50 pmol each primer, dATP, dGTP, dTTP, dCTP (no 200 μm each), and 2.5 units of DNA Taq polymerase (Qiagen), total volume of 50 μl in a buffer provided by the manufacturer, with the following amplification conditions: 92°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, 30 cycles) using overlapping oligonucleotides Sig-hGH-FOR

(GGGTCTAGAATGGCTACAGGCTCCCGGACGTCCCTGCTCCTGGCTTTTGGCCTG CTCTG) (SEQ ID NO: 41) and Sig-hGH-REV (CGCAGGCCTCGGCACTGCCCTCTTGAAGCCAGGGCAGGCAGAGCA GGCCAAAAGCCAG) (SEQ ID NO: 42). The PCR product is purified using the QiaExII kit, cut with StuI/XbaI and are ligated into the vector pSINrep5::OmpA-FOS-hGH cleaved by the same enzymes.

Example 8:

Eukaryotic expression of FOS-hGH

Not containing RNase vector (1,0 µg) (pSINrep5::OmpA-FOS-hGH) and 1.0 µg DHEB (see Bredenbeek et al., J. Virol, 67:6439-6446 (1993)) linearized, cutting with restriction enzyme Seal. This is followed by transcription in vitro using set for transcription in vitro SP6 (InvitroscripCAP made InvitroGen, Invitrogen BV, VLeek, The Netherlands). The obtained mRNA 5’-cap analyze in reducing conditions on an agarose gel.

Transcribed in vitro mRNA (5 μg) introduced by electroporation into cells VNC ADS: CCL10) in accordance with the instructions Invitrogen (Expression system Sindbis (Sindbis Expression system, Invitrogen BV, the Netherlands). After incubation for 10 minutes at 37°With medium containing FCS, replace environment HP-1, not containing FCS, followed by an additional incubation at 37°C for 10 hours. The supernatant is collected and analyzed by dot-blotting on education FOS-hGH.

Culture medium (2,5 μl) is applied in the form of spots on a nitrocellulose membrane and dried for 10 minutes at room temperature. The membrane blocked with 1% bovine albumin (Sigma) in TBS (10 × TBS/l: 87,7 g NaCl, 66,1 g Trizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH 7.4) for 1 hour at room temperature followed by incubation with 2 μg of rabbit antibodies against hGH person for 1 hour. The blot was washed 3 times for 10 minutes in TBS-T and incubated for 1 hour with alkaline phosphatase conjugated with IgG against rabbit (Jackson ImmunoResearch Laboratories, Inc.), diluted in the ratio 1:5000 in TBS-T. After washing 2 times for 10 minutes in TBS-T, the blot showing staining of AR, as described in Example 2. The results are presented in Figure 3.

Example 9:

The design of FOS-PLA (N - and C-terminal)/p>

The following gene construct by chemical synthesis of the gene encoding the catalytically inactive variant (see the articleet al., J. Allergy Clin. Immunol. 95: 1229-1235 (1995)) phospholipase A2bee venom (PLA).

To merge PLA with M-end domain of FOS dimerization plot amplified using the oligonucleotides PLA-FOR1 (CCATCATCTACCCAGGTAC) (SEQ ID NO: 45) and PLA-REV1 (CCCACACCCAGCGGCCGCGTATTTGCGCAGGTCG) (SEQ ID NO: 46). The PCR product cut with NotI and are ligated into the vector pAV1, pre-split restrictase StuI/NotI. To merge the PLA with the end of the dimerization domain FOS plot amplified using the oligonucleotides PLA-FOR2 (CGGTGGTTCTGCGGCCGCTATCATCTACCCAGGTAC) (SEQ ID N0:47) and PLA-REV2 (TTAGTATTTGCGCAGGTCG) (SEQ ID NO: 48). The product obtained by PCR, cut with NotI and are ligated into the vector pAV2, pre-split restrictase NotI/EcoRV.

Example 10:

Design fused gene FOS-ovalbumin (N - and C-terminal)

For cloning sequences encoding ovalbumin receive mRNA from the tissue of the chicken oviduct using the cleaning kit MPHKQuickPrep™ Micro mRNA Purification Kit (Pharmacia) according to the manufacturer's instructions. Using the set for one-step RT-PCR Superscript™ One-step RT-PCR Kit (Gibco BRL) synthesize cDNA encoding the Mature part of ovalbumin (corresponding to nucleotides 68-1222 mRNA (see McReynolds et al., Nature, 273: 723-728 (1978)) using the-W primers Ova-FORI (CCGGCTCCATCGGTGCAG) (SEQ ID NO: 49) and Ova-REVI (ACCACCAGAAGCGGCCGCAGGGGAAACACATCTGCC) (SEQ ID NO: 50). The PCR product is cut with NotI and clone in cut StuI/NotI vector pAVI with the purpose of the expression of the fused protein with a domain of FOS dimerization of the C-end. To obtain a fused protein with a domain of FOS dimerization at the N end of the site, encoding ovalbumin, amplified from the constructed vector (pAV1::Ova)using primers Ova-FOR2 (CGGTGGTTCTGCGGCCGCTGGCTCCATCGGTGCAG) (SEQ ID NO: 51) and Ova-REV2 (TTAAGGGGAAACACATCTGCC) (SEQ ID NO: 52). The PCR product is cut with NotI and clone in the cut with Notl/EcoRV vector pAV2. The cloned fragments is checked by analyzing the DNA sequence.

Example 11:

Production and purification of fused proteins FOS-PLA and FOS-ovalbumin

For cytoplasmic production fused proteins FOS suitable strain .li transform vectors pAV3::PLA, pAV4::PLA, pAV4::Ova, pAV3::Ova or pAV4::Ova. The culture is incubated in enriched medium in the presence of ampicillin with shaking at 37°C. Upon reaching an optical density (550 nm) add 1.1 mm IPTG and continue incubation for a further 5 hours. Cells are harvested by centrifugation, resuspended in a suitable buffer (such as Tris-HCl, pH to 7.2, 150 mm NaCl)containing Tnkase, RNase and lysozyme, and destroy through the chamber under pressure from the French). After centrifugation (Sorvall RC-5C, the rotor SS34, 15000 rpm, 10 min, 4° (C) sediment resuspended in the buffer for washing Taurus inclusions (20 mmtris-HCl, 23% sucrose, and 0.5% Triton X-100, 1 mm EDTA (etilendiamintetrauksusnoy acid), pH 8) at 4°and re-centrifuged as described above. This procedure is repeated until the supernatant, obtained after centrifugation, becomes almost transparent. Taurus inclusions resuspended in 20 ml of buffer to solubilize (5.5 M guanidine hydrochloride, 25 mm Tris-HCl, pH 7.5) at room temperature and removing insoluble material by centrifugation, followed by passing the supernatant through a sterile filter (0.45 µm). The protein solution stored at 4°C for at least 10 hours in the presence of 10 mm EDTA and 100 mm DTT, and then three times cialiswhat against 10 volumes of 5.5 M guanidine hydrochloride, 25 mm Tris-HCl, 10 mm EDTA, pH 6. The solution twice cialiswhat against 5 l of 2 M urea, 4 mm EDTA, 0.1 M NH4Cl, 20 mm sodium borate (pH 8.3) in the presence of a suitable redox couple (oxidized glutathione/restored glutathione; cystine/cysteine). Perelozheny protein is subjected to ion-exchange chromatography. Protein is stored in a suitable buffer with a pH above 7 in the presence of 2-10 mm DTT in order to save cysteine residues flanking domain FOS, in restored form. Before binding protein particles alphavirus DTT is removed, passing the protein solution through the column for gel filtration Sephadex G-25.

Example 12:

Design gp140-FOS/p>

Gene gp140 (Swiss-Prot:P03375), not containing internal website proteasome splitting, amplified by PCR from the original plasmid b4674 ADS 40829), containing the gene for others 160 full length, using the following oligonucleotides:

HIV-1:

5’-ACTAGTCTAGAatgagagtgaaggagaaatatc-3’ (SEQ ID NO: 53);

HIV-end:

5’-TAGCATGCTAGCACCGAAtttatctaattccaataattcttg-3’ (SEQ ID NO: 54);

HIV-Cleav:

5’-gtagcacccaccaaggcaaagCTGAAAGCTACccagctcgagaaactggca-3’ (SEQ ID NO: 55) and

HIV-Cleav2:

5'-caaagctcctattcccactgcCAGTTTCTCGAgctgggtagctttcag-3' (SEQ ID 10 N0:56).

For PCR I use 100 pmol of the oligonucleotide HIV-1 and HIV-Cleav2 and 5 ng DNA template in 75 μl of the reaction mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm gSO4). Cycling PCR is as follows: 30 cycles at the annealing temperature 60°With time and elongation for 2 min at 72°C.

For PCR II using 100 pmol of the oligonucleotide HIV-end and HIV-Cleav and 5 ng DNA template in 75 μl of the reaction mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4). Cycling PCR is as follows: 30 cycles at the annealing temperature 60°With time and elongation of 50 sec at 72°C.

Both obtained PCR fragments purified, isolated and used in the reaction Assembly by PCR. For Assembly by PCR using 100 pmol of oligonucleotides HIV-1 and HIV-end and 2 ng of each of the fragments obtained by PCR (PCR and PCR), 75 μl of the mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4). PCR Cycling OS is p as follows: 30 cycles at the annealing temperature 60° With time and elongation of 2.5 min at 72°C. the Product Assembly by PCR cut with XbaI and NheI. Amphipatic helix FOS merge in reading frame with the late Dr-140.

The DNA sequence encoding a domain amphipatic helix FOS, amplified by PCR from the vector pJuFo (see Crameri and Suter, Gene, 137:69 (1993)), using the oligonucleotides:

FOS-HIV:

5’-ttcggtgctagcggtggcTGCGGTGGTCTGACcgac-3’ (SEQ ID NO: 57) and

FOS-Apa:

5’-gatgctgggcccttaaccGCAACCACCGTGTGccgcc-3’ (SEQ ID NO: 58).

For PCR using 100 pmol of each oligonucleotide and 5 ng DNA template in 75 μl of the reaction mixture (4 units polymerase Taq or Pwo, 0.1 mm dNTP and 1.5 mm MgSO4). Temperature Cycling as follows: 95°C for 2 minutes followed by 5 cycles consisting of the following stages: 95°C (45 sec), 60°C (30 sec), 72° (25 sec), and the subsequent 25 cycles consisting of the following stages: 95°C (45 sec), 68°C (30 sec), 72° (20 sec). The resulting PCR product is cut with NheI and Bsp120L.

The final expression vector for GP140-FOS get when legirovanii both fragments obtained by PCR, the reaction ligating three fragments into pSinRep5. The resulting vector pSinRep5-GP140-FOS was determined by restriction analysis and DNA sequencing.

GP140-FOS clone in pCYTts using XbaI and Bsp120L to obtain a stable inducible cell lines that Express the dominant GP140-FOS.

Example 13:

Expression GP140FOS using pSinRep5-GP140-FOS

Not containing RNase vector (1,0 µg) (pSinRep5-GP140-FOS) and 1.0 µg DHEB (see Bredenbeek et al., J. Virol, 67: 6439-6446 (1993)) linearized, slitting restriction enzyme. This is followed by transcription in vitro using set for transcription in vitro SP6 (InvitroscripCAP made InvitroGen, Invitrogen BV, NVLeek, the Netherlands). The obtained mRNA 5’-cap analyze in reducing conditions on an agarose gel.

Transcribed in vitro mRNA (5 μg) introduced by electroporation into cells VNC ADS: CCL10) in accordance with the instructions Invitrogen (Expression system Sindbis (Sindbis Expression system, Invitrogen BV, the Netherlands). After incubation for 10 minutes at 37°With medium containing FCS, replace environment HP-1, not containing FCS, followed by an additional incubation at 37°C for 10 hours. The supernatant is collected and analyzed by Western blotting for the formation of soluble GP140-FOS exactly as described in Example 2.

Example 14:

Expression of GP140-FOS using pCYTts-GP140-FOS

Twenty micrograms pCYT-GP140-FOS linearized, slitting restriction enzyme. The reaction is stopped by extraction with phenol/chloroform followed by precipitation with isopropanol linearized DNA. The reaction restriction check gel-electrophoresis agarose. For transfection of 5.4 μg linearized pCYT-GP140-FOS mixed with 0.6 ág of the linearized pSV2Neo the 30 μl of N 2Oh and add 30 μl of 1 M solution l2. After adding 60 μl of phosphate buffer (50 mm HEPES, 280 mm NaCl, 1.5 mm Na2HPO4pH 7,05) the solution is shaken for 5 seconds and then incubated at room temperature for 25 seconds. The solution is immediately added to 2 ml of medium HP-1 containing 2% FCS (2% FCS environment). Wednesday confluent at 80% cell culture VNC (6-hole Board) then replace the DNA-containing medium. After incubation for 5 hours at 37°C incubator with CO2The DNA-containing medium is removed and replaced by 2 ml of 15% glycerol in 2% FCS environment.

The glycerol-containing medium is removed after a 30-second phase of the incubation and wash cells with 5 ml of medium HP-1, containing 10% FCS. Finally, add 2 ml of fresh medium HP-1, containing 10% FCS.

Select stably transfetsirovannyh cells and grown on selective medium (environment HP-1 with the addition of G418) at 37°C incubator with CO2. When mixed population grows to confluently, culture is divided into two cups with the subsequent period of growth for 12 h at 37°C. One Cup with cells transferred in terms of cultivation at 30°for the induction of expression of soluble GP140-FOS. Another Cup maintained at 37°C.

The expression of soluble GP140-FOS was determined by Western blotting. Culture medium (0.5 ml) precipitated with methanol/chlorof ROM and resuspending sediment in sample buffer for SDS-PAGE. Samples heated for 5 minutes at 95°before making a 15% acrylamide gel. After SDS-PAGE the proteins transferred to nitrocellulose membrane Protan (Schleicher &Schuell, Germany), as proposed in section, written Bass and Yang, in the monograph edited by Creighton I.E. Practical approaches to the function of the protein (Protein Function: A Practical Approach, 2nd ed., IRL Press, Oxford (1997), p.29-55. The membrane blocked with 1% bovine albumin (Sigma) in TBS (10 × TBS/l: 87,7 g NaCl, 66,1 rTrizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH of 7.4) for 1 hour at room temperature followed by incubation with the antibody against GP140 or GP160 for 1 hour. The blot was washed 3 times for 10 minutes with TBS-T (TBS with the addition of 0.05% Tween 20) and incubated for 1 hour with alkaline phosphatase conjugate with IgG against mouse/rabbit/monkey/human. After washing 2 times for 10 minutes in TBS-T and 2 times for 10 minutes in TBS conduct manifesting reaction using reagents for detection of alkaline phosphatase (10 ml AR buffer (100 mm Tris/HCl, 100 mm NaCl, pH 9,5) with 50 μl of NBT solution (7,7% nitro blue tetrazole (Sigma) in 70% dimethylformamide) and 37 μl of solution X-phosphate (5% of 5-bromo-4-chloro-3-indolylacetic in dimethylformamide).

Example 15:

Production and purification of GP140-FOS

Antibody against Dr covalently associated with NHS/EDC-activated dextran and Packed into a chromatographic column. The supernatant, containing GP140-FOS, placed the Ute on the column, and after sufficient washing elute GP140-FOS, using 0.1 M HCl. The eluate is neutralized at the time of collection, using 1 M Tris pH of 7.2, which is in test tubes for collection.

Since during the cleaning could be the formation of disulfide bonds, the collected sample is treated with 10 mm DTT in 10 mm Tris, pH 7.5 for 2 hours at 25°C.

DTT is removed during subsequent dialysis against 10 mm Mes, 80 mm NaCl, pH to 6.0. Finally, GP140-FOS mixed with alphavirus particles containing lacinova lightning JUN in E2, as described in Example 16.

Example 16:

Obtaining particles of alpha-vaccine

Viral particles (see Examples 2 and 3) concentrate using the device to filter Millipore Ultrafree Centrifugal Filter Devices that removes compounds of molecular weight of 100 KD, in accordance with instructions provided by the manufacturer. Alternative viral particles are concentrated by centrifugation in a sucrose gradient as described in the guide to the Expression system Sindbis (Sindbis Expression System (Invitrogen, San Diego, California). the pH of the suspension of the virus down to 7.5 and incubated viral particles in the presence of 2-10 mm DTT in a few hours. Viral particles purified from protein impurities on a column of Sephacryl S-300 (Pharmacia) (viral particles elute the free volume) in a suitable buffer.

Purified viral particles incubated with at least 240-fold molar excess of fused protein FOS-antigen in approaching the rd buffer (pH 7.5 to 8.5) in the presence of redox couples (oxidized glutathione/restored glutathione; cystine/cysteine) for at least 10 h at 4°C. After concentration of the particles using a device Millipore Ultrafree Centrifugal Filter Device that removes compounds with a molecular mass of 100 KD, the mixture is passed through a column for gel filtration Sephacryl S-300 (Pharmacia). Viral particles elute the free volume.

Example 17:

Merge amphipatic helix JUN with aminocom.com NSAD(1-144)

Spiral JUN merge with aminocom.com sequence of amino acids 1-144 HBcAg (design JUN-HBcAg). To construct the DNA sequence JUN-HBcAg sequence encoding the helix JUN and NSAD(1-144), separately amplified by PCR. The sequence JUN amplified from plasmid pJuFo using primers EcoRI-JUN(s) and JUN-SacII(as). Primer EcoRI-JUN(s) introducere the EcoRI site following the start codon ATG. Primer JUN-SacII(as) introducere linker encoding the amino acid sequence of GAAGS. The sequence NSAD(1-144) amplified from plasmid RESO (obtained in ATS No. 31518), using the primers JUN-HBcAg(s) and HBcAg(1-144)Hind(as). JUN-HBcAg(s) contains the sequence corresponding to the 3’-end sequence that encodes a helix JUN, which is a sequence encoding a linker GAAGS and the 5’-end sequence of HBcAg. HBcAg(1-144)Hind(as) introducere stop codon and a HindIII site after the codon 144 HBcAg gene. For PCR using 100 PM the l of each oligonucleotide and 50 ng DNA templates in 50 µl reaction mixtures, contains 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. For both reactions the temperature Cycling carried out as follows: 94°C for 2 min followed by 30 cycles consisting of the following stages: 94°C (1 min), 50°C (1 min), 72° (2 min).

Sequences of primers:

EcoRI-JUN(s):

(5’-CCGGAATTCATGTGCGGTGGTCGGATCGCCCGG-3’) (SEQ ID NO: 61);

JUN-SacII(as):

(5’-GTCGCTACCCGCGGCTCCGCAACCAACGTGGTTCATGAC-3’) (SEQ ID NO: 62);

JUN-HBcAg(s):

(5’-GTTGGTTGCGGAGCCGCOGGTAGCGACATTGACCCTTATAAAGAATTTGG-3’) (SEQ ID NO: 63);

HBcAg(1-144)Hind(as):

(5’-CGCGTCCCAAGCTTCTACGGAAGCGTTGATAGGATAGG-3’) (SEQ ID NO: 64).

The fusion of the two fragments obtained by PCR, carried out by PCR, using primers RI-JUN(s) and HBcAg(1-144)Hind(as). 100 pmol of each oligonucleotide used with 100 ng of purified obtained PCR fragments in a 50 µl reaction mixture containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. Use the following conditions PCR Cycling: 94°C for 2 min followed by 35 cycles consisting of the steps: 94°C (1 min), 50°C (1 min), 72° (2 min). The final PCR product analyzed by gel electrophoresis using agarose, cleaned and cut for 16 h in a suitable buffer restrictase EcoRI and HindIII. Fragment cut DNA are ligated into the cut with EcoRI/HindIII vector RCM to create expression vector pKK-JUN-HBcAg. The insertion of the PCR product identify restriction fragments length polymorphism analysis with p the power EcoRI/HindIII and by DNA sequencing of insertions.

Example 18:

Merge amphipatic helix JUN with carbonsilicon NSAD(1-144)

Spiral JUN merge with carbonsilicon sequence of amino acids 1-144 HBcAg (design HBcAg-JUN). To construct the DNA sequence of HBcAg-JUN sequence encoding the helix JUN and NSAD(1-144), separately amplified by PCR. The sequence JUN amplified from plasmid pJuFo using primers SacII-JUN(s) and JUN-HindIII(as). SacII-JUN(s) introducere linker encoding the amino acid sequence of LAAG. This sequence also contains SII. JUN-HindIII(as) introducere stop codon (TAA), followed by a HindIII site. The DNA sequence NSAD(1-144) amplified from plasmid RESO using primers EcoRI-HBcAg(s) and HBcAg(1-144)-JUN(as). EcoRI-HBcAg(s) introducere the EcoRI site before the start codon ATG sequence encoding HBcAg. HBcAg(1-144)-JUN(as) introducere sequence encoding a peptide linker (LAAG), which also contains a SacII site. For PCR using 100 pmol of each oligonucleotide and 50 ng DNA templates in a 50 μl reaction mixtures containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. Temperature Cycling carried out as follows: 94°C for 2 min followed by 30 cycles consisting of the following stages: 94°C (1 min), 50°C (1 min), 72° (2 min). Sequences of primers:

SacII-JUN(s):

(5’-TAGCCGCGGGTTGCGGTGGTCGGATCGCCCGG-3’) (SEQ ID NO: 65);

JUN-HindIII(as):

(5’-CGCGTCCCAAGCTTTTAGCAACCAACGTGGTTCATGAC-3’) (SEQ ID NO: 66);

EcoRI-HBcAg(s):

(5’-CCGGAATTCATGGACATTGACCCTTATAAAG-3’) (SEQ ID NO: 67) and HBcAg-JUN(as):

(5’-CCGACCACCGCAACCCGCGGCTAGCGGAAGCGTTGATAGGATAGG-3’)(SEQID NO: 68).

The fusion of the two fragments obtained by PCR, carried out by PCR, using primers EcoRI-HBcAg(s) and JUN-HindIII(as). To merge using a PCR of 100 pmol each of the oligonucleotide used with 100 ng of purified obtained PCR fragments in a 50 µl reaction mixture containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. Use the following conditions PCR Cycling: 94°C for 2 min followed by 35 cycles consisting of the steps: 94°C (1 min), 50°C (1 min), 72°C (2 min). The final PCR product analyzed by gel electrophoresis using agarose and cut for 16 h in a suitable buffer restrictase EcoRI and HindIII. Fragment cut DNA purified by gel-chromatography and are ligated into the cut with EcoRI/HindIII vector RCM to create expression vector pKK-HBcAg-JUN. The insertion of the PCR product to determine DNA restriction analysis with EcoRI/HindIII and by DNA sequencing of insertions.

Example 19:

The insertion of amphipatic helix JUN in the epitope with/E1 NSAD(1-144)

It is known that the epitope with/E1 (residues 72-88) NSAD is located at the end on the surface of the capsid of hepatitis C. a Fragment of this site (residues 76-82) protein genetic and replace spiral JUN to obtain binding to antigens (design HBcAg-JUNIns). The DNA sequence of HBcAg-JUNIns get by PCR, and the sequence of the helix JUN and two sequences encoding fragments NSAD (amino acid residues 1-75 and 83-144), amplified separately by PCR. The sequence JUN amplified from plasmid pJuFo using primers BamHI-JUN(s) and JUN-SacII(as). BamHI-JUN(s) introducere linker sequence encoding a linker sequence of the peptide GSGGG, which also contains a BamHI site. JUN-SacII(as) introducere sequence encoding a peptide linker GMGS, followed by a sequence complementary to the 3’-end sequence that encodes a JUN. The DNA sequence NSAD(1-75) amplified from plasmid RESO using primers EcoRIHBcAg(s) and HBcAg75-JUN(as). EcoRIHBcAg(s) introducere the EcoRI site followed by a sequence corresponding to the 5’-end sequence of HbcAg. HBcAg75-JUN(as) introducere linker encoding the peptide GSGGG, after 75 amino acids HbcAg, followed by a sequence complementary to the 5’-end sequence that encodes a helix JUN. Fragment NSAD (83-144) amplified using primers JUN-HBcAg83(s) and HBcAg(1-144)Hind(as). JUN-HBcAg83(s) contains the sequence corresponding to the 3’-end sequence that encodes JUN, followed by a linker encoding the peptide GAAGS, and the sequence corresponding to the 5’-to the CC sequence, coding NSAD(83-144). HBcAg(1-144)Hind(as) introducere stop codon and a HindIII site after the codon 144 gene NSAD. For PCR using 100 pmol of each oligonucleotide and 50 ng DNA templates in 50 µl reaction mixtures (2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4). Temperature Cycling carried out as follows: 94°C for 2 min followed by 35 cycles consisting of the steps: 94°C (1 min), 50°C (1 min), 72° (2 min).

Sequences of primers:

BamHI-JUN(s):

(5’-CTAATGGATCCGGTGGGGGCTGCGGTGGTCGGATCGCCCGGCTCGAG-3’) (SEQ ID NO:69);

JUN-SacII(as):

(5’-GTCGCTACCCGCGGCTCCGCAACCAACGTGGTTCATGAC-3’) (SEQ ID NO: 70);

EcoRIHBcAg(s):

(5’-CCGGAATTCATGGACATTGACCCTTATAAAG-3’) (SEQ ID NO: 71);

HBcAg75-JUN (as):

(5’-CCGACCACCGCAGCCCCCACCGGATCCATTAGTACCCACCCAGGTAGC-3’) (SEQ ID NO: 72);

JUN-HBcAg83(s):

(5’-GTTGGTTGCGGAGCCGCGGGTAGCGACCTAGTAGTCAGTTATGTC-3’) (SEQ ID NO: 73), and

HBcAg(1-144)Hind(as):

(5’-CGCGTCCCAAGCTTCTACGGAAGCGTTGATAGGATAGG-3’) (SEQ ID NO: 74).

The merging of the three obtained PCR fragments as follows. First, the fragment encoding NSAD (1-75), merge sequence that encodes JUN, by PCR using the primers EcoRIHBcAg(s) and JUN-SacII(as). Secondly, the resulting product is poured with a fragment of a HbcAg (83-144) by PCR using primers EcoRI HBcAg(s) and HBcAg HindIII(as). For mergers by PCR 100 pmol each of the oligonucleotide used with 100 ng of purified obtained PCR fragments in a 50 µl reaction mixture containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. The same PCR cycles used to produce specific fragments. The final PCR product is cut for 16 h in a suitable buffer using restricted EcoRI and HindIII. The DNA fragment are ligated in cut EcoRI/HindIII vector RCM, receiving vector pKK-HBcAg-JUNIns. The insertion of PCR product was determined by restriction analysis using EcoRI/HindIII and by DNA sequencing of insertions.

Example 20:

Merge amphipatic helix JUN with carbonsilicon nucleocapsid protein (N) of measles virus

Spiral JUN merge with carbonsilicon a truncated fragment of the protein N measles virus containing residues of amino acids 1-473 (design N473-JUN). To construct a DNA sequence that encodes a N473-JUN, sequence, encoding a helix JUN, and a sequence encoding a N473-JUN, amplified separately by PCR. The sequence JUN amplified from plasmid pJuFo using primers SacII-JUN(s) and JUN-HindIII(as). SacII-JUN(s) introducere sequence encoding a peptide linker LAAG. This sequence also contains the website Sacli. Antisense primer JUN-HindIII(as) introducere stop codon (TAA), followed by a HindIII site. The sequence N (1-473) amplified from plasmid pSC-N, containing the sequence encoding the complete protein N measles virus (provided by M. Billeter, Zurich), using primers EcoRI-Nmea(s) and Nmea-JUN(a). EcoRI-N(mea)(s) introducere website EcoRJ before the start codon ATG sequence that encodes n N(mea)-JUN(as) is complementary to the 3’-end sequence that encodes N(1-473), followed by a sequence complementary to the sequence that encodes a peptide linker (LAAG). For PCR using 100 pmol of each oligonucleotide and 50 ng matrix Dncv 50 μl reaction mixtures containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. Temperature Cycling carried out as follows: 94°C for 2 min followed by 35 cycles consisting of the steps: 94°C (1 min), 50°C (1 min), 72° (2 min).

Sequences of primers:

SacII-JUN(s):

(5’-CTAGCCGCGGGTTGCGGTGGTCGGATCGCCCGG-3’) (SEQ ID NO: 75);

JUN-HindIII(as):

(5’-CGCGTCCCAAGCTTTTAGCAACCAACGTGGTTCATGAC-3’) (SEQ IDNO:76);

EcoRI-Nmea(s):

(5’-CCGGAATTCATGGCCACACTTTTAAGGAGC-3’) (SEQ ID NO: 77) and

Nmea-JUN(as):

(5’-CGCGTCCCAAGCTTTTAGCAACCAACGTGGTTCATGAC-3’) (SEQ ID NO: 78).

The merger of the two obtained PCR fragments is carried out in the following PCR reaction using primers EcoRI-Nmea(s) and Nmea-JUN(as). To merge using a PCR of 100 pmol each of the oligonucleotide used with 100 ng of purified obtained PCR fragments in a 50 µl reaction mixture containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. Temperature Cycling carried out as follows: 94°C for 2 min followed by 35 cycles consisting of the steps: 94°C (1 min), 50°C(1 min), 72° (2 min). The PCR product is cut for 16 h in a suitable buffer using restricted EcoRI and HindIII. The DNA fragment purified and are ligated into the cut EcoRI/HindIII vector RCM, receiving plasmid pKK-N473-JUN. The insertion of PCR product was determined by restriction analysis using EcoRI/HindIII and by DNA sequencing of insertions.

Example 21:

Expression and partial purification of HBcAg-JUN

The E. coli strain xl-1 blue transformed pKK-HBcAg-JUN. 1 ml of the daily culture of bacteria used for inoculation of 100 ml of LB medium containing 100 μg/ml ampicillin. This culture is grown for 4 hours. At 37°until OD at 600 nm reaches a value of approximately 0.8. The induction of the synthesis of HBcAg-JUN carried out by adding IPTG to a final concentration of 1 mm. After induction, the bacteria continue to shake at 37°C for 16 hours. Bacteria are harvested by centrifugation at 5000×g for 15 minutes the Precipitate frozen at -20°C. the Sediment was thawed resuspended in the buffer for lysis of bacteria (10 mm Na2PO4, pH 7.0, 30 mm NaCl, and 0.25% Tween-20, 10 mm EDTA, 10 mm DTT) supplemented with 200 μg/ml lysozyme and 10 ál benzonase (Merck). Cells incubated for 30 min at room temperature and destroy the camera under pressure from the French). Triton X-100 is added to the lysate to a final concentration of 0.2% and the lysate incubated for 30 min on ice with periodic is shaking. In Figure 4 represent the expression of a protein HBcAg-JUN in .li after induction with IPTG. Cells .li carrying the expression plasmid pKK-HBcAg-JUN or control plasmid, used for induction of expression of HBcAg-JUN using IPTG. Before adding IPTG take a sample of the bacterial culture, the carrier plasmid pKK-HBcAg-JUN (lane 3), and culture carrying the control plasmid (lane 1). After 16 h after addition of IPTG again take samples of the culture containing pKK-HBcAg-JUN (lane 4), and the control culture (lane 2). The expression of protein monitorium using SDS-PAGE and subsequent staining of Kumasi.

Then the lysate centrifuged for 30 min at 12,000×g to remove insoluble cellular debris. The supernatant and the residue analyzed by Western blotting using monoclonal antibodies against NSAD (YVS1841 acquired firm Accurate Chemical and Scientific Corp., Westbury, NY, USA), showing that significant amounts of protein NSAD-JUN is soluble (see Figure 5). Briefly, lysates obtained from cells .li expressing NSAD-JUN, and from control cells centrifuged at 14,000×g for 30 minutes the Supernatant (=soluble fraction) and sediment (=insoluble fraction) share and diluted with SDS sample buffer to equal volumes. Samples are analyzed using SDS-PAGE with subsequent Western blotting using mono is analnogo antibodies against NSAD YVS 1841. On track 1 presents the soluble fraction, control cells; lane 2 presents insoluble fraction, control cells; lane 3 presents the soluble fraction of cells expressing NSAD-JUN; track 4 presents the insoluble fraction of cells expressing HBcAg-JUN.

The clarified lysate of cells used for centrifugation in a stepwise gradient using a step gradient of sucrose, consisting of 4 ml of 65% sucrose solution, which impose 3 ml of 15% sucrose solution, which cause 4 ml lysate of bacteria. The sample is centrifuged for 3 h at 100,000×g at 4°C. After centrifugation, 1 ml fractions from the top of the gradient is collected and analyzed by SDS-PAGE and subsequent staining of Kumasi (see Figure 6). On track 1 presents the total E. coli lysate before centrifugation. On tracks 1 and 2 show the fractions 1 and 2 from the top of the gradient. On tracks 4-7 presents fractions 5-8 (15% sucrose). Protein HBcAg-JUN determined by staining Kumasi.

Protein HBcAg-JUN accumulates at the interface between 15 and 65% sucrose, which indicates that it forms the capsid particle. A large part of the bacterial proteins remains do not contain sucrose upper layer of the gradient, resulting centrifugation of particles HBcAg-JUN in a stepwise gradient of PR which leads to enrichment, and partial purification of particles.

Example 22:

Covalent binding of hGH-FOS with HBcAg-JUN

In order to demonstrate binding of the protein to particles HBcAg-JUN, choose human growth hormone (hGH), merged on carboxylic spiral FOS, as a model protein (hGH-FOS). Particles HBcAg-JUN mixed with partially purified hGH-FOS and incubated for 4 h at 4°to bind proteins. The mixture is then cialiswhat during the night against a 3000-fold volume of buffer for dialysis (150 mm NaCl, 10 mm solution of Tris-HCl, pH 8.0) to remove DTT present in the solution HBcAg-JUN, and the solution hGH-FOS, thus enabling covalent binding of proteins through the formation of disulfide bonds. As controls solutions HBcAg-JUN and hGH-FOS also cialiswhat against the buffer used for dialysis. Samples of all three dialetheic solutions of proteins analyzed by SDS-PAGE in non conditions.

Linking hGH-FOS c HBcAg-JUN determined by Western blot turns with the antibody against hGH (see Figure 7). hGH-FOS associated with HBcAg-JUN, must migrate from the estimated molecular weight of approximately 53 KD, whereas unbound hGH-FOS migrates with the estimated molecular weight of 31 KD. Product dialysis analyzed by SDS-PAGE in the absence of reducing agent (lane 3) and in the presence of a reducing agent (lane 2) and determine okresu is of Kumasi. As a control hGH-FOS, which is not mixed with the core particles also contribute to the gel in the presence of a reducing agent (lane 1).

Shift hGH-FOS to molecular weight of approximately 53 KD mark in the presence of capsid protein HBcAg-JUN, suggesting that efficient binding of the hGH-FOS with HBcAg-JUN.

Example 23:

The insertion of a peptide containing a lysine residue in the epitope with/E1 NSAD(1-149)

Epitope with/E1 (residues 72-88) NSAD is located at the end on the surface of the capsid of hepatitis b virus (NSAD). A fragment of this site (Proline 79 and Alanine 80) genetic replaced by the peptide Gly-Gly-Lys-Gly-Gly (design HBcAg-Lys). The introduced lysine residue contains in the side chain reaction the amino group, which can be used for intermolecular chemical cross-linking of the particles NSAD with any antigen that contains a free group of cysteine.

The DNA sequence of HBcAg-Lys receive by PCR: Two fragments that encode fragments NSAD (amino acid residues 1-78 and 81-149), separately amplified by PCR. The primers used for PCR data, also being introduced DNA sequence encoding the peptide Gly-Gly-Lys-Gly-Gly. Fragment NSAD (1-78) amplified from RESO using primers EcoRIHBcAg(s) and Lys-HBcAg(as). Fragment NSAD (81-149) amplified from RESO using Prime the market Lys-HBcAg(s) and HBcAg(1-149)Hind(as). Primers Lys-HBcAg(as) and Lys-HBcAg(s) being introduced complementary DNA sequences at the ends of the two PCR products, providing a fusion of the two PCR products in the subsequent PCR Assembly. The collected fragments amplified by PCR, using primers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).

For PCR using 100 pmol of each oligonucleotide and 50 ng DNA templates in a 50 μl reaction mixtures containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. For both reactions the temperature Cycling carried out as follows: 94°C for 2 min followed by 30 cycles consisting of the steps: 94°C (1 min), 50°C (1 min), 72° (2 min).

Sequences of primers:

EcoRIHBcAg(s):

(5’-CCGGAATTCATGGACATTGACCCTTATAAAG-3’) (SEQ ID NO: 79);

Lys-HBcAg(as):

(5’-CCTAGAGCCACCnTGCCACCATCTTCTAAATTagtacccacccaggtagc-3’) (SEQ ID NO: 80);

Lys-HBcAg(s):

(5’-GAAGATGGTGGCAAAGGTGGCTCTAGGGACCTAGTAGTCAGTTATGTC-3’)

(SEQ ID NO: 81);

HBcAg(1-149)Hind(as):

(5’-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3’) (SEQ ID NO: 82).

To merge using two PCR obtained using PCR fragments 100 pmol of primers EcoRIHBcAg(s) and HBcAg(1-149)Hind(as) is used with 100 ng of the two obtained when purified PCR fragments in a 50 µl reaction mixture containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm gSO4. Use the following conditions PCR Cycling: 94°C for 2 min followed by 30 cycles consisting of the steps: 94°C (1 min), 50°C (1 min), 72° (2 min). Assembled p is oduct PCR analyzed by gel electrophoresis using agarose, clean and cut for 19 h in a suitable buffer restrictase EcoRI and HindIII. Fragment cut DNA are ligated into the cut with EcoRI/HindIII vector RCM to create expression vector pKK-HBcAg-Lys. The insertion of the PCR product to determine DNA restriction analysis with EcoRI/HindIII and by DNA sequencing of insertions.

Example 24:

Expression and partial purification of HBcAg-Lys

The E. coli strain XL-1 blue transformed PKK-HBcAg-Lys. 1 ml of the daily culture of bacteria used for inoculation of 100 ml of LB medium containing 100 μg/ml ampicillin. This culture is grown for 4 h at 37°until OD at 600 nm reaches a value of approximately 0.8. The induction of the synthesis of the HBcAg-Lys carried out by adding IPTG to a final concentration of 1 mm. After induction, the bacteria continue to shake at 37°C for 16 h, the Bacteria are harvested by centrifugation at 5000×g for 15 minutes the Precipitate frozen at -20°C. the Sediment was thawed resuspended in the buffer for lysis of bacteria (10 mm Na2HPO4, pH 7.0, 30 mm NaCl, and 0.25% Tween 20, 10 mm EDTA, 10 mm DTT) supplemented with 200 μg/ml lysozyme and 10 ál benzonase (Merck). Cells incubated for 30 min at room temperature and destroy the camera under pressure from the French). Triton X-100 is added to the lysate to a final concentration of 0.2% and the lysate incubated for 30 min on ice with periodic internal is jivani. Cells .li carrying the expression plasmid pKK-HBcAg-Lys or a control plasmid, used for induction of expression of HBcAg-Lys with IPTG. Before adding IPTG take a sample of the bacterial culture, the carrier plasmid PKK-HBcAg-Lys, and culture carrying the control plasmid. After 16 h after addition of IPTG again take samples of the culture containing PKK-HBcAg-Lys, and the control culture. The expression of protein monitorium using SDS-PAGE and subsequent staining of Kumasi.

Then the lysate centrifuged for 30 min at 12,000×g to remove insoluble cellular debris. The supernatant and the residue analyzed by Western blotting using monoclonal antibodies against HBcAg (YVS1841 acquired firm Accurate Chemical and Scientific Corp., Westbury, NY, USA), showing that significant amounts of protein HBcAg-Lys is soluble. Briefly, lysates obtained from cells of E. Li, expressing HBcAg-Lys, and from control cells, centrifuged at 14,000×g for 30 minutes the Supernatant (=soluble fraction) and sediment (=insoluble fraction) share and diluted with SDS sample buffer to equal volumes. Samples are analyzed using SDS-PAGE with subsequent Western blotting using monoclonal antibodies against NSAD YVS1841.

The clarified lysate of cells used for centrifugation in a stepwise gradient used is of a stepped gradient of sucrose, consisting of 4 ml of 65% sucrose solution, which impose 3 ml of 15% sucrose solution, which cause 4 ml lysate of bacteria. The sample is centrifuged for 3 h at 100000×g at 4°C. After centrifugation, 1 ml fractions from the top of the gradient is collected and analyzed by SDS-PAGE and subsequent staining of Kumasi. Protein HBcAg-Lys accumulates at the interface between 15 and 65% sucrose, which indicates that it forms the capsid particle. A large part of the bacterial proteins remains do not contain sucrose upper layer of the gradient, resulting centrifugation of particles HBcAg-Lys in a stepwise gradient leads to enrichment and partial purification of particles.

Example 25:

Chemical binding of FLAG peptide to HBcAg-Lys using heterobifunctional cross-linking linker SPDP

Synthetic FLAG peptide with a cysteine residue on aminocore (the amino acid sequence of CGGDYKDDDDK) chemically bind with purified particles HBcAg-Lys with the aim of obtaining immune response against the FLAG peptide. 600 ál of 95% purified solution of particles HBcAg-Lys (2 mg/ml) incubated for 30 min at room temperature with heterobifunctional cross-linking linker M-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (0.5 mm). After completion of the reaction the mixture cialiswhat overnight against 1 l of 50 mm is ostatniego buffer (pH 7,2) with 150 mm NaCl to remove free SPDP. Then 500 µl derivatizing capsid HBcAg-Lys (2 mg/ml) is mixed with 0.1 mm FLAG peptide (containing aminobenzoic cysteine) in the presence of 10 mm EDTA to prevent catalyzed by metal oxidation of sulfhydryl. The reaction monitorium to increase the optical density of the solution at 343 nm, which is caused by the release of pyridine-2-thione of SPDP in the reaction with the free cysteine of the peptide. The reaction derivatizing Lys residues from the peptide ends in approximately 30 minutes

Particles that carry FLAG, is injected mice.

Example 26:

Design pMPSV-gp140Cys

Gene Dr amplified by PCR from pCytTSgp140FOS using oligonucleotides gp140 CysEcoRI and SaIIgp140. For PCR using 100 pmol of each oligonucleotide and 50 ng DNA templates in a 50 μl reaction mixtures containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. For both reactions the temperature Cycling carried out as follows: 94°C for 2 min followed by 30 cycles consisting of the steps: 94°C (0.5 min), 55°C (0.5 min), 72° (2 min).

The PCR product is purified using the QiaEXII kit, cut with SaII/EcoRI, and are ligated into the vector pMPSVHE, cut with the same enzymes.

Sequences of oligonucleotides:

Gp140CysEcoRI:

5’-GCCGAATTCCTAGCAGCTAGCACCGAATTTATCTAA-3’ (SEQ ID NO: 83);

SaIIgp140

5’-GGTTAAGTCGACATGAGAGTGAAGGAGAAATAT-3’ (SEQ ID NO: 84).

Example 27:

Expression pMPSVgp14Cys

pMPSVgp140Cys (20 µg) linearized, slitting restriction enzyme. The reaction is stopped by extraction with phenol/chloroform followed by precipitation with isopropanol linearized DNA. The reaction restriction is checked by gel-electrophoresis in agarose. For transfection of 5.4 μg linearized pMPSVgp140-Cys mixed with 0.6 ág of the linearized pSV2Neo in 30 μl of N2Oh and add 30 μl of 1 M solution l2. After adding 60 μl of phosphate buffer (50 mm HEPES, 280 mm NaCl, 1.5 mm PA2RHO4pH 7,05) the solution is shaken for 5 seconds and then incubated at room temperature for 25 seconds. The solution is immediately added to 2 ml of medium HP-1 containing 2% FCS (2% FCS environment). Wednesday confluent at 80% cell culture VNC (6-hole Board) then replace the DNA-containing medium. After incubation for 5 hours at 37°C incubator with CO2The DNA-containing medium is removed and replaced by 2 ml of 15% glycerol in 2% FCS environment. The glycerol-containing medium is removed after a 30-second phase of the incubation and wash cells with 5 ml of medium HP-1, containing 10% FCS. Finally, add 2 ml of fresh medium HP-1, containing 10% FCS.

Select stably transfetsirovannyh cells and grown on selective medium (environment HP-1 with the addition of G418) at 37°C incubator with CO2. When mixed population grows to confluently, culture is divided into two cups with the consequences of the current period of growth for 12 h at 37° C. One Cup with cells transferred in terms of cultivation at 30°for the induction of expression of soluble GP140-Cys. Another Cup maintained at 37°C.

The expression of soluble GP140-Cys was determined by Western blotting. Culture medium (0.5 ml) precipitated with methanol/chloroform and resuspended sediment in sample buffer for SDS-PAGE. Samples heated for 5 minutes at 95°C before making a 15% acrylamide gel. After SDS-PAGE the proteins transferred to nitrocellulose membrane Protan (Schleicher &Schuell, Germany), as proposed in section, written Bass and Yang, in the monograph edited by Creighton I.E. Practical approaches to the function of the protein (Protein Function: A Practical Approach, 2nd ed., IRL Press, Oxford (1997), p.29-55. The membrane blocked with 1% bovine albumin (Sigma) in TBS (10 × TBS/l: 87,7 g NaCl, 66,1 g Trizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH of 7.4) for 1 hour at room temperature followed by incubation with the antibody against GP-140 or GP-160 for 1 hour. The blot was washed 3 times for 10 minutes with TBS-T (TBS with the addition of 0.05% Tween 20) and incubated for 1 hour with alkaline phosphatase conjugate with IgG against mouse/rabbit/monkey/human. After washing 2 times for 10 minutes in TBS-T and 2 times for 10 minutes in TBS conduct manifesting reaction using reagents for detection of alkaline phosphatase (10 ml AR buffer (100 mm Tris/HCl, 100 mm NaCl, pH 9,5) with 50 μl of a solution of the NBT (7,7% nitro blue tetrazole (Sigma) in 70% dimethylformamide) and 37 μl of solution X-phosphate (5% of 5-bromo-4-chloro-3-indolylacetic in dimethylformamide).

Example 28

Cleaning GP140Cys

Antibody against Dr covalently associated with NHS/EDC-aktvirovannom dextran and Packed into a chromatographic column. The supernatant, containing GP140Cys, placed on the column, and after sufficient washing elute GP140Cys using 0.1 M HCl. The eluate is neutralized at the time of collection, using 1 M Tris pH of 7.2, which is in test tubes for collection.

Since during the cleaning could be the formation of disulfide bonds, the collected sample is treated with 10 mm DTT in 10 mm Tris, pH 7.5 for 2 hours at 25°C.

DTT is removed during subsequent dialysis against 10 mm Mes, 80 mm NaCI, pH 6.0. Finally, GP140Cys mixed with alphavirus particles containing the remainder JUN in E2, as described in Example 16.

Example 29

The design of PLA2-Cys

Gene PLA2 amplified by PCR from pAVSPLAfos using oligonucleotides EcoRIPLA and PLA-Cys-hind. For PCR using 100 pmol of each oligonucleotide and 50 ng DNA templates in a 50 μl reaction mixtures containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm gSO4. For both reactions the temperature Cycling carried out as follows: 94°C for 2 min followed by 30 cycles consisting of the steps: 94°C (0.5 min), 55°C (0.5 min), 72° (2 min).

The PCR product is purified using the QiaEXII kit, cut with EcoRI/HindIII, and are ligated into the vector pAV3, cut those who e enzymes.

Sequences of oligonucleotides:

EcoRIPLA:

5’-TAACCGAATTCAGGAGGTAAAMGATATGG-3’ (SEQ ID NO: 85)

PLACys-hind:

5’-GAAGTAAAGCTTTTAACCACCGCAACCACCAGAAG-3’ (SEQ ID NO: 86).

Example 30

Expression and purification PLA-cys

For cytoplasmic products labeled Cys .li XL-1 Blue transformed with vectors pAV3::PLA and pPLA-Cys. The culture is incubated in enriched medium in the presence of ampicillin at 37°C, shaking. Upon reaching an optical density (550 nm) was added 1 mm IPTG and continue incubation for a further 5 hours. Cells are harvested by centrifugation, resuspended in a suitable buffer (such as Tris-HCl, pH to 7.2, 150 mm NaCl)containing Tnkase, RNase and lysozyme, and destroy through the chamber under pressure from the French). After centrifugation (SorvaII RC-5C, the rotor SS34, 15000 rpm, 10 min, 4° (C) sediment resuspended in the buffer for washing Taurus inclusions (20 mm Tris-HCl, 23% sucrose, and 0.5% Triton X-100, 1 mm EDTA, pH 8) at 4°and re-centrifuged as described above. This procedure is repeated until the supernatant, obtained after centrifugation, becomes almost transparent. Taurus inclusions resuspended in 20 ml of buffer to solubilize (5.5 M guanidine hydrochloride, 25 mm Tris-HCl, pH 7.5) at room temperature and removing insoluble material by centrifugation, followed by passing the supernatant through a sterile filter,45 μm). The protein solution stored at 4°C for at least 10 hours in the presence of 10 mm EDTA and 100 mm DTT, and then three times cialiswhat against 10 volumes of 5.5 M guanidine hydrochloride, 25 mm Tris-HCl, 10 mm EDTA, pH 6. The solution twice cialiswhat against 5 l of 2 M urea, 4 mm EDTA, 0.1 M NH4Cl, 20 mm sodium borate (pH 8.3) in the presence of a suitable redox couple (oxidized glutathione/restored glutathione; cystine/cysteine). Perelozheny protein is subjected to ion-exchange chromatography. Protein is stored in a suitable buffer with a pH above 7 in the presence of 2-10 mm DTT in order to save cysteine residues in the reduced form. Before binding protein particles alphavirus DTT is removed, passing the protein solution through the column for gel filtration Sephadex G-25.

1. A composition comprising (A) is not natural molecular media, including crustal particle selected from the group consisting of a virus, virus-like particle of a bacteriophage, viral capsid particles and their recombinant forms, and organizer containing at least one first binding site and a specified organizer connected with this cow particle of at least one covalent bond, and (B) an antigen or antigenic determinant with at least one second binding site, where the specified second binding site selected from the group consisting of a binding site, not vstrechaiuschih is camping in nature with the specified antigen or antigenic determinant, and binding that exists in nature with the specified antigen or antigenic determinant, and the specified second binding site is capable of Association through at least one ones relation with the said first binding site and the specified antigen or antigenic determinant and the specified media interact in specified Association with the formation of ordered and contains repetitions antigenic matrix.

2. The composition according to claim 1, characterized in that the holder is a polypeptide or residue and the specified second binding site is a polypeptide or residue.

3. The composition according to claim 1, characterized in that the said first and/or the second binding sites represent the antigen and the antibody or fragment of antibody against him, Biotin and avidin, streptavidin and Biotin, d) receptor and its ligand, landspitali protein and its ligand, interacting polypeptides latinboy lightning, amino group and a chemical group reactive with her, a carboxyl group and a chemical group reactive with her, sulfhydryl group and a chemical group reactive with her, or their combination.

4. The composition according to claim 3, characterized in that the second binding does not occur in nature with the specified antigen or anti the military determinant.

5. The composition according to claim 1, characterized in that the crustal particle presents recombinant alpha-virus.

6. The composition according to claim 5, characterized in that the recombinant alpha-virus presents Sindbis virus and the first binding site and said second binding site both contain the interacting polypeptide latinboy lightning.

7. The composition according to claim 6, wherein the first binding site and said second binding site are polypeptides latinboy zippers JUN and/or FOS.

8. The composition according to claim 1, characterized in that the crustal particle is a virus-like particle.

9. The composition of claim 8, wherein the first binding site is an amino group and the specified second binding site is a sulfhydryl group.

10. The composition according to claim 8, characterized in that the said virus-like particle is a capsid protein of hepatitis C.

11. The composition of claim 10, wherein the first binding site and said second binding site both contain the interacting polypeptide latinboy lightning.

12. The composition according to claim 11, wherein the first binding site is a polypeptide JUN and the specified second centre of binding the Oia is a polypeptide FOS.

13. The composition of claim 10, wherein the first binding site is a lysine residue and said second binding site is a cysteine residue.

14. The composition according to claim 8, characterized in that the said virus-like particle is a capsid protein of measles virus.

15. The composition according to 14, wherein the first binding site and said second binding site both contain the interacting polypeptide latinboy lightning.

16. The composition according to item 15, wherein the first binding site and said second binding site are polypeptides latinboy zippers JUN and/or FOS.

17. The composition according to claim 1, characterized in that the crustal particle selected from the group consisting of recombinant proteins of rotavirus, recombinant proteins of Norwalk virus, recombinant proteins, alpha virus, recombinant proteins of FMD virus, recombinant proteins of retrovirus, recombinant proteins of hepatitis b virus, recombinant proteins of tobacco mosaic virus, recombinant proteins of the virus of space for animals, recombinant proteins of human papilloma virus.

18. The composition according to 17, wherein the first binding and the second binding site both contain the interacting polypeptide latinboy lightning.

19. The composition according to 17, wherein the first binding site is an amino group and the specified second binding site is a sulfhydryl group.

20. The composition according to 17, wherein the first binding site and said second binding site are polypeptides latinboy zippers JUN and/or FOS.

21. The composition according to claim 1, characterized in that the antigen is selected from the group consisting of proteins, suitable for the induction of immune responses against cancer cells, proteins, suitable for the induction of immune responses against infectious diseases, proteins, suitable for the induction of immune responses against allergens and proteins suitable for the induction of an immune response in farm animals.

22. The composition according to item 21, characterized in that the antigen is a recombinant protein of HIV, a recombinant protein of influenza virus, a recombinant protein of hepatitis C virus, a recombinant protein of Toxoplasma, a recombinant protein of Plasmodium falciparum, the recombinant protein of Plasmodium vivax, a recombinant protein of Plasmodium ovale, a recombinant protein of Plasmodium malariae, a recombinant protein of breast cancer cells, recombinant protein cells of kidney cancer, a recombinant protein of prostate cancer cells, recombinant protein cell skin cancer, recomb nanny protein cells, brain cancer, recombinant protein of leukemia cells, recombinant profiling, recombinant protein allergies bee sting, recombinant protein allergies to nuts, recombinant protein allergies to foods or recombinant protein asthma or recombinant protein of Chlamydia.

23. The composition according to item 21, wherein the first binding and the second binding site both contain interacting polypeptides latinboy lightning.

24. The method of obtaining natural ordered and contains repetitions antigenic matrix, characterized in that the gain is not natural molecular media, including crustal particle selected from the group consisting of a virus, virus-like particle of a bacteriophage, viral capsid particles and their recombinant forms, and organizer containing at least one first binding site and a specified organizer connected with this cow particle of at least one covalent bond, get an antigen or antigenic determinant with at least one second binding site, where the specified second binding site selected from the group consisting of binding, newstrategies in nature with the specified antigen or antigenic determinant, and binding that exists in nature with the specified antigen or antigenic determinant, moreover, the specified second binding site is capable of Association through at least one ones relation with the said first center link, and combine specified not natural molecular media and the specified antigen or antigenic determinant, with the specified antigen or antigenic determinant and the specified media interact in specified Association with the formation of ordered and contains repetitions antigenic matrix.

25. The method according to paragraph 24, wherein as indicated organizer using the polypeptide or its residue and as specified second binding site using a polypeptide or residue.

26. The method according to paragraph 24, wherein said first and/or the second binding sites represent the antigen and the antibody or fragment of antibody against him, Biotin and avidin, streptavidin and Biotin, a receptor and its ligand, landspitali protein and its ligand, interacting polypeptides latinboy lightning, amino group and a chemical group reactive with her, a carboxyl group and a chemical group reactive with her, sulfhydryl group and a chemical group reactive with her, or their combination.

27. The method according to p, wherein the first binding does not occur in nature with the specified antigen or antigenic determinant.

28. The method of therapeutic treatment, wherein the subject is administered a composition according to claim 1.

29. Pharmaceutical compositions the Oia, containing the composition according to claim 1 and an acceptable pharmaceutical carrier.

30. The method of immunization, wherein the subject is administered a composition comprising (A) is not natural molecular media, including crustal particle selected from the group consisting of a virus, virus-like particle of a bacteriophage, viral capsid particles and their recombinant forms, and organizer containing at least one first binding site and a specified organizer connected with this cow particle of at least one covalent bond, and (B) an antigen or antigenic determinant with at least one second binding site, where the specified second binding site selected from the group consisting of the binding site not naturally occurring with the specified antigen or antigenic determinant, and binding that exists in nature with the specified antigen or antigenic determinant, and the specified second binding site is capable of Association through at least one ones relation with the said first binding site and the specified antigen or antigenic determinant and the specified media interact in specified Association with the formation of ordered and contains repetitions antigenic matrix.

31. The method according to item 30, wherein the specified immunodeficiency who isace leads to the formation of an immune response.

32. The method according to item 30, wherein the specified immunization leads to the formation of humoral immune response.

33. The method according to item 30, wherein the specified immunization leads to the formation of the cellular immune response.

34. The method according to item 30, wherein the specified immunization leads to the formation of humoral immune response and cellular immune response.

35. The method according to item 30, wherein the specified immunization leads to the formation of a protective immune response.

36. Vaccine composition comprising (A) is not natural molecular media, including crustal particle selected from the group consisting of a virus, virus-like particle of a bacteriophage, viral capsid particles and their recombinant forms, and organizer containing at least one first binding site and a specified organizer connected with this cow particle of at least one covalent bond, and (B) an antigen or antigenic determinant with at least one second binding site, where the specified second binding site selected from the group consisting of a binding site not naturally occurring with the specified antigen or antigenic determinant, and binding that exists in nature with the specified antigen or antigenic determinant, and the WTO is the second binding site is capable of Association through at least one ones relation with the said first binding site and said antigen or antigenic determinant and the specified media interact in specified Association with the formation of ordered and contains repetitions antigenic matrix.

37. Vaccine composition according p, characterized in that it further contains an adjuvant.

38. Vaccine composition according p, characterized in that the holder is a polypeptide or residue and the specified second binding site is a polypeptide or residue.

39. Vaccine composition according p, characterized in that the said first and/or the second binding sites represent the antigen and the antibody or fragment of antibody against him, Biotin and avidin, streptavidin and Biotin, a receptor and its ligand, landspitali protein and its ligand, interacting polypeptides latinboy lightning, amino group and a chemical group reactive with her, a carboxyl group and a chemical group reactive with her, sulfhydryl group and a chemical group reactive with her, or their combination.

40. Vaccine composition according p, characterized in that the crustal particle contains virus-like particle.

41. Vaccine composition according p, characterized in that the crustal particle includes a particle similar to a virus hepatitis C.

42. Vaccine composition according p, characterized in that the crustal particle includes a particle similar to the measles virus.

4. Vaccine composition according p, characterized in that the crustal particle contains a virus.

44. Vaccine composition according to item 43, wherein the specified crustal particle contains a Sindbis virus.

Priority items:

30.11.1998 and 08.07.1999 according to claims 1, 2, 24, 25, 28-30, 36 and 38;

30.11.1998 on PP-9, 11-13, 15, 16, 18-20, 23, 26, 27, 31-35, 37, 39, 40, 43 and 44;

08.07.1999 on PP, 14, 17, 21, 22, 41 and 42.



 

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The invention relates to medicine and relates to chimeric live attenuated infectious virus, which is used to create chimeric vaccines flavivirus

The invention relates to biotechnology, Virology and immunology, and can be used to create a vaccine against the virus of classical swine fever (CSFV)

The invention relates to biotechnology, in particular immunobiotechnology, and for the creation of recombinant modified vaccinia virus Ankara (MVA), is able to replicate in human cells

The invention relates to medicine, namely to methods of specific prophylaxis of viral infections

FIELD: antibiotics, pharmacy.

SUBSTANCE: invention relates to stabilization of rapamycin or rapamycin derivative with immunosuppressive properties and sensitive to oxidation. Method for stabilization involves addition of antioxidant to the purified rapamycin in small concentrations - below 1% as recalculated for rapamycin mass. Also, invention relates to a solid mixture containing rapamycin and antioxidant taken in the catalytic amount. Antioxidant represent preferably 2,6-di-tert.-butyl-4-methylphenol. The stabilized rapamycin shows high stability against oxidation, it can be stored as formulation without package before it's the following treatment and can be used in an unmodified form for preparing the required galenic composition.

EFFECT: improved stabilizing method.

14 cl, 3 dwg, 2 ex

The invention relates to a linear cyclodextrin copolymers and oxidized cyclodextrin, which can be used as a carrier for the delivery of various therapeutic agents
The invention relates to medicine, namely to ophthalmology, and is intended for the treatment of inflammatory processes in the eyeball

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes

The invention relates to experimental medicine, cardiology

The invention relates to pharmaceutical compositions comprising a stable water-insoluble complex, consisting of stable amorphous form of the therapeutically active compound dispersed on a molecular level in the water-insoluble ionic polymer

The invention relates to pharmaceutical compositions comprising a stable water-insoluble complex, consisting of stable amorphous form of the therapeutically active compound dispersed on a molecular level in the water-insoluble ionic polymer

The invention relates to pharmaceutical compositions comprising a stable water-insoluble complex, consisting of stable amorphous form of the therapeutically active compound dispersed on a molecular level in the water-insoluble ionic polymer

The invention relates to the pharmaceutical industry, in particular the production of medicines used for colds, relieving headaches and neuralgia

FIELD: antibiotics, pharmacy.

SUBSTANCE: invention relates to stabilization of rapamycin or rapamycin derivative with immunosuppressive properties and sensitive to oxidation. Method for stabilization involves addition of antioxidant to the purified rapamycin in small concentrations - below 1% as recalculated for rapamycin mass. Also, invention relates to a solid mixture containing rapamycin and antioxidant taken in the catalytic amount. Antioxidant represent preferably 2,6-di-tert.-butyl-4-methylphenol. The stabilized rapamycin shows high stability against oxidation, it can be stored as formulation without package before it's the following treatment and can be used in an unmodified form for preparing the required galenic composition.

EFFECT: improved stabilizing method.

14 cl, 3 dwg, 2 ex

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