Interleukin-1 inhibitor, method for production thereof, dna molecule encoding interleukin-1 inhibitor, and precursor thereof

FIELD: biotechnology, gene engineering, medicine.

SUBSTANCE: polypeptide (IL-li) is obtained by recombinant DNA technology having activity of interleukin-1-alpha and interleukin-1-beta inhibitor. Disclosed is DNA sequences encoding inhibitor precursor and maturated form thereof. Described is method for production of vector constructs containing said DNA sequences, as well as transformed cells producing of recombinant IL-li.

EFFECT: method for large-scale production of interleukin-1-beta inhibitor, useful in medicine.

21 cl, 26 dwg, 11 ex

 

This application is isolated from application No. 4894343/13, filed November 26, 1990, with priority dated may 27, 1988

A. IL-1

Interleukins-1 represent a class of proteins produced by many cell types, including monocytes and some macrophages. This class includes at least two proteins with molecular weight of 17-18 kilodaltons, known as interleukin-1 alpha and interleukin-1 beta. These proteins have important physiological effects on a number of different target cells involved in inflammatory and immune reactions. Proteins are co-Metheny (phytohemaglutinin) for T-lymphocytes, inducing fibroblasts and chondrocytes to secrete latent collagenase and improving the surface adhesion force of endothelial cells to neutrophils. In addition, they affect the hypothalamus as pyrogens, they stimulate the catabolism of muscle protein, and they make the hepatocytes to synthesize a class of proteins known as acute-phase reactive substances". Thus, interleukin-1 (IL-1), obviously, represent an important part of the body's response to infection and injury.

C. Pathological Role of IL-1

However, despite the generally favorable effects of IL-1, there are occasions when their effects become negative. For example, IL-1 can increase the level of sod is Rania collagens in gouty joint and involved as a mediator of acute and chronic stages of immunopathology in rheumatoid arthritis. IL-1 may be responsible for modifying the function of endothelial cells by chemotaxis and migration of leukocytes and lymphocytes in the synovial tissue inducyruya capillary proliferation and stimulating the accumulation of macrophages in the synovial lining during the acute phase of this disease. In the phase discontinuity of tissue IL-1 is involved as a mediator in the induction of tissue rupture, stimulating the release of enzymes from fibroblasts and chondrocytes.

In addition, excessive production of IL-1 was demonstrated in the skin of patients with psoriasis, and high levels of IL-1 can be found in synovial fluid of patients with psoriatic arthritis. IL-1 released by cells in inflammatory synovial membrane with psoriatic arthritis, can cause rupture of the tissues, stimulating the release of enzymes from other cells. Joint pathology of Reiter syndrome similar to the observed in psoriatica arthritis and rheumatoid arthritis. In addition, IL-1 can be detected in synovial fluid of patients suffering from osteoarthritis. The release of IL-1 in chondrocytes involved in the rupture of the articular cartilage in this disease.

IL-1 can also increase the severity of autoimmune diseases. For example, reduced production of IL-1 are described from the peripheral blood cells of patients suffering from common is lupus erythematosus. In addition, some changes in the function of b-lymphocytes may be associated with impaired production of IL-1 or suitability of IL-1.

Excessive production of IL-1 was demonstrated in peripheral monocytes of patients with scleroderma, and IL-1 is involved as a potential agent of fibrosis by stimulating the production of collagen by fibroblasts. The mechanism of tissue damage when the ringworm may also include cell-mediated immunity, and therefore IL-1 may be involved as a mediator in this pathophysiological process.

Acute and chronic collagen pulmonary disease is the excessive production of collagen lung fibroblasts, which can be stimulated by IL-1. Recent studies on animal models regarding pulmonary hypertension show that IL-1 may be responsible for the induction of changes in endothelial cells that lead to a narrowing of the pulmonary arteries. It is this narrowing leads to pulmonary hypertension and further secondary violation. Thus, inhibitors of IL-1 could be a suitable means for the treatment of these lung diseases.

Recent studies show that IL-1 can directly damage the beta cells in the islets of Langerhans, which are responsible for the production of insulin, the Destruction of cells through IL-1 is now regarded as the primary event in the acute phase of juvenile diabetes.

Infiltration of monocytes and macrophages in the kidney dominates in many forms of acute and chronic glomerulonephritis. The release of IL-1 in these cells can lead to local accumulation of other inflammatory cells, leading eventually to inflammatory destruction and fibrosis of reccie in the kidneys.

It is shown that the crystals found in the tissues or fluids gout or pseudopodia can directly simulate the macrophages to release IL-1. Thus, IL-1 may be an important mediator in the inflammatory cycle in such cases.

IL-1 is able to induce calcium loss from the bones and may be responsible for osteoporosis, which is observed in inflammatory diseases of the joints.

Keratinocytes from patients suffering from psoriasis, release large amounts of IL-1. This mediator can be responsible for secondary cell proliferation that occurs in the skin of patients suffering from this disease.

IL-1 is one of the important endogenous pyrogens, and may be responsible for inducing an appreciable degree of fever observed in some infectious diseases, such as acute febrile illness caused by bacteria or viruses.

Sarcoidosis is characterized by granulomatous lesions in many organs telephoneno, that IL-1 is able to induce the formation of granulomas in vitro and may be involved in this process in patients suffering from sarcoidosis.

Excessive production of IL-1 was demonstrated in peripheral monocytes from Crohn's disease and ulcerative colitis. Local release of IL-1 in the intestine may be an important mediator in the inflammatory stimulation cycle in such cases.

Some lymphomas differ fever, osteoporosis and even secondary arthritis. Excessive production of IL-1 demonstrated some lymphoma cells in vitro and may be responsible for some of the clinical manifestations of these zlokacestvennosti. In addition, IL-1, produced some malignant lymphocytes may be responsible for certain violations, such as fever, acute phase response and the total depletion that occur with leukemia.

The release of IL-1 by astrocytes in the brain are believed to be responsible for the induction of fibrosis, which can occur as a result of brain damage from occlusion of vessels.

C. the Use of an inhibitor of IL-1

Under these and other circumstances, when IL-1 has negative effects, there is a need for clinical use of the inhibitor effects of IL-1. Because IL-1 is komitehs T-lymphocytes, it is crucial for the development of autoimmune and other immune diseases. Therefore, systemic administration of inhibitors of IL-1 might be useful for immuno-suppressive agents. When the local introduction of such inhibitors of IL-1 can serve in preventing the breakdown of tissues in inflammatory joint and other sites of inflammation. Actually, to prevent the destruction of tissue, some inhibitors of IL-1 could be even more effective than when introduced in combination with inhibitors of collagenase.

Therapeutic effect against the influence of IL-1 may be possible at the level of synthesis, secretion, or binding of the target cells with the protein. IL-1 is synthesized monocyte/macrophages and other cells in response to lipopolysaccharide, fragments of complement and viruses. Any molecule that blocks the binding of these inducing agents with producer cells or preventing their effect on the physiology of these cells could serve as a regulator of the activity of IL-1. IL-1 is not secreted traditional system secretion because the selected mRNA, encoding at least two precursor proteins With a molecular mass of 50 KD, but does not contain a hydrophobic signal sequence. The release of the active from the inactive protein precursor, apparently, requires the implementation of p is atualiza this predecessor. The inhibitor of the release of one of the IL-1 or more IL-1 from their predecessors theoretically could regulate the action of IL-1. Apparently, the IL-1 acts on target cells via the classical receptor-mediated pathways, although this receptor is not already highlighted. Therefore, it is likely that the molecule, preventing the binding of IL-1 to its receptors or regulating these receptors can regulate the action of IL-1. In addition, although still quite clear intracellular processes after receptor binding of IL-1, it is possible that there are agents that can interfere with cellular responses to other receptor-mediated events, and therefore may block the action of IL-1. For the reasons mentioned above, sourced proteins and small molecules able to inhibit IL-1 one or more of the above methods. The applicant has unexpectedly found that there are at least two protein inhibitors of IL-1 with the properties of ingibirovaniya IL-1. These molecules obtained in purified form, which allows the specialist in this field of knowledge to determine their amino acid sequence. Moreover, characterized by obtaining cells that produce these proteins and mRNA, which leads to their synthesis. Finally, the identified antisera, which facilitate the task of screening Bible is flowed expression of cDNA for genes coding these inhibitors. These reagents together allow encoding cDNA encoding inhibitors of IL-1. In turn, these genes create the possibility of large-scale production of inhibitors of IL-1, suitable for use in the technology of preparation of drugs useful in the treatment of pathophysiological conditions mediated by IL-1.

The present invention relates to inhibitors of IL-1 ("IL-1"), more specifically, the inhibitor of IL-1 with monocyte origin. In addition, the present invention relates to biologically active analogues of these inhibitors.

The aim of the present invention to provide a purified forms of inhibitors of IL-1, which are active against IL-1 alpha or IL-1 beta or combinations thereof. Another objective of the present invention is the obtaining of these inhibitors in purified forms in order to facilitate determination of their amino acid sequences. An additional objective of the present invention to provide amino acid sequences of specific inhibitors of IL-1. In addition, identification of biologically active analogs of such inhibitors of IL-1 with advanced or equivalent properties is also one of the purposes of the present invention.

In addition, the purpose of the present invention is to obtain the system is s recombinant DNA for the production of inhibitors of IL-1 in accordance with the invention. Another objective of the present invention is the creation of purified forms of inhibitors of IL-1 which would be valuable pharmaceutical active against IL-1.

Additional objectives and advantages of the present invention will be presented in part in the description and in part will be understood from this specification or from practice of the invention. Objectives and advantages may be realized and obtained by means and methods specified in the attached claims.

To achieve the objectives in accordance with the present invention are disclosed inhibitors of IL-1, which exhibit inhibitory activity against IL-1. Preferred inhibitors isolated in purified form from monocyte-conditioned medium from monocytes grown on plates coated with immunoglobulin G (IHH).

Preferred inhibitors in accordance with the present invention are inhibitors 1, 2 and 3. Inhibitors 1 and 2 represent proteins manipulated in positions characteristic of proteins with a molecular mass 22-23 KD, polyacrylamide gel electrophoresis with dodecyl-sodium sulfate, and which elute at 52 mm and 60 mm NaCl, respectively, of the column of liquid Express chromatography of proteins Mono Q when specific conditions. In addition, to achieve the objectives in accordance with the present which m invention the applicant discloses pharmaceutical compositions containing at least one of the active components, the inhibitor of IL-1 of the present invention or its biologically active analogues.

Moreover, to achieve the objectives of the present invention also discloses a system of recombinant DNA to create these inhibitors of IL-1 and their analogues. A preferred variant of this system includes at least one cDNA clone, or its synthetic equivalent, encoding at least one inhibitor of IL-1, together with vectors and cells that make up the expression system that can Express the disclosed here inhibitors of IL-1. Also available antisera for use in the identification of these cDNA clones. In addition, the available expression systems to obtain these inhibitors of IL-1 using the indicated cDNA clones, their analogues or other DNA sequences encoding these inhibitors.

Brief description of drawings

Figa and 1b depict protein profile chromatography Mono Q two metabolically labeled monocytic of supernatant. Cells were cultured on plates coated with IHH (1A) or fetal calf serum (1b).

Figa shows painted silver gel fractions of the areas indicated on Figa and 1b.

Fig.2b is autoradiogram gels shown in Figa.

Figa, b and C show data regarding the nutrient purified inhibitor of IL-1 of Example 1. Figa data leads chromatography with a picture of radioactivity. Fig.3b is painted silver gels relative to samples of the fractions shown in Figa, Figs shows autoradiogram gels on Fig.3b.

Figa and b show the results of the gel filtration chromatograms of an inhibitor of IL-1, purified on a column of Mono q

Figa and b show the results of Western analysis of mouse antisera.

6 depicts the construction of plasmids pSVXVPL 2-11-1i.

7 depicts the construction of plasmids RMK - SGE:IL-1i.

Figa-d depict data on IL-1i-α (inhibitor of IL-1 alpha), Figa and 8b show the chromatography data. Figs depicts painted silver gel on samples of the fractions indicated on Fig.8b. Fig.8d depicts autoradiogram.

Figa and 9 present data on inhibitor of IL-1-beta (IL-1i-β). Figa displays chromatography. Fig.9 displays the data polyacrylamide gel electrophoresis with sodium dodecyl sulfate.

Figure 10 shows the data on the fractionation of peptizyde IL-1i-α.

11 reflects the data on the fractionation of peptides of IL-1i-α.

Figa is a photograph of a gel with GT10-IL 1i-2A, digested using EcoRI after electrophoresis in accordance with Example 6.

Fig.12b reflects these autoradiogram Sa is used for blotting of the gel, shown in Figa.

Fig depicts a portion of a DNA sequence proteincoding region lambda GT10-IL 1i-2A and predicted amino acid sequence in accordance with Example 6.

Fig depicts the nucleotide sequence GT-10-IL 11i-2A.

Fig depicts the peptide, including, inter alia, the sequence of the IL-1i and secretory leader sequence of the Monomeric residues.

Below are more preferred variants of the invention, which, together with the following examples help to explain the principles of the present invention.

A. the Inhibitor from Human Monocytes

As noted above, the present invention relates to inhibitors of IL-1, which is selected in purified form. Preferably, the inhibitors of IL-1 of the present invention is obtained from medium conditioned by human monocytes, in which monocytes grown in containers covered with IHH. In addition, the present invention covers mainly the purified inhibitors of IL-1 any origin, which are biologically equivalent to the inhibitor derived from human monocytopenia environment.

In the description of the invention and its formula, the term "biologically equivalent" applicant means the composition of the present invention capable of preventing the action of IL-1 similarly, is however not necessarily in the same degree, as a native inhibitor of IL-1, isolated from monocytes. The term "mainly homologous" in the description and the claims is meant a degree of homology to the native inhibitor of IL-1, isolated from monocyte-conditioned medium, exceeding the degree of homology, which is any of the previously described inhibitors of IL-1. Preferably, if the degree of homology is greater than 70%, more preferably greater than 80% and most preferably greater than 90%. Especially preferred group of inhibitors has more than 95% homology to the native inhibitor. The percent homology is calculated as the percentage of amino acid residues in the smaller of the two sequences, which are located on the same level with identical amino acid residues in the sequence being compared, when you can enter four span length of 100 amino acids in order to facilitate this arrangement, as indicated by Dayhoff M in Atlas of Protein Sequence and Structure, Volume 5, str (1972), national Organization for Biochemical Research, Washington, D.C., and this work is mentioned here as a reference.

Preferred inhibitors of IL-1 of the present invention derived from the monocyte-conditioned medium and they first isolated in purified Faure is E. For the purposes of this application the "pure form" or "purified form" regarding the disclosed inhibitors of IL-1 means a drug that is substantially free of other proteins, which are not proteins, inhibitors of IL-1. Preferably, the inhibitors of IL-1 of the present invention have at least 90% purity, more preferably 95% purity.

At least three purified inhibitor of IL-1 selected methods of Example. They include inhibitor 1 inhibitor 2 inhibitor 5. Inhibitor 1 manifests itself as a molecule with a molecular mass 22-23 KD when polyacrylamide gel electrophoresis with sodium dodecyl sulfate with an approximate isoelectric point of 4.8, and elute from the column liquid Express chromatography of proteins Mono Q at 52 mm NaCl in Tris buffer, pH of 7.6. Inhibitor-2 is a protein with a molecular mass 22-23 KD, isoelectric point of 4.8, but his elute from the column Mono Q at 60 mm NaCl. Inhibitor 3 is a protein with a molecular mass of 20 KD and aluinum from the column Mono Q at 48 mm NaCl. Inhibitors 1, 2 and 3 are related immunologically and functionally. These inhibitors in purified form allows the applicant to create their amino acid sequences. With the use of purified inhibitors described for the first time in this application, and methods, such as those described here and the technical guidelines for Protein sequencing machine (ABI you can display a significant part of the amino acid sequences of these inhibitors. Example 3 shows the amino-acid sequence obtained from the three types of inhibitors of IL-1, namely IL-1i-X, IL-1i-α and IL-1i-β.

The applicant has found at least one antibody against the inhibitor of IL-1. Other polyclonal and monoclonal antibodies against this and other inhibitors of IL-1 can be obtained well-known in the art methods. One specific polyclonal antibody described in Example 4.

C. Recombinant Inhibitor

1. Total

Currently disclosed method of recombinant DNA to obtain an inhibitor of IL-1. In one embodiment of the invention the active site is biologically equivalent with respect to the natural inhibitor of IL-1, isolated from man. The sequence of natural or synthetic DNA can be used directly for inhibitors of IL-1. This method involves:

(a) obtaining a DNA sequence capable of directing cell-owner to obtain a protein having the activity of inhibiting IL-1,

(b) cloning the DNA sequence into a vector capable of transferrates and replicate in the cell-the master and the specified vector contains the operating elements required for expression sequence,

(C) transferring the vector containing the synthetic DNA sequence and operational elements into the cell host, able to Express the DNA encoding the inhibitor of IL-1,

(d) culturing host cells under conditions suitable for amplification of the vector and expression of the inhibitor,

(e) collecting inhibitor, and

(f) the granting of an inhibitor of active tertiary structure, whereby it has the activity of inhibiting IL-1.

2. DNA sequence

The DNA sequence provided for use in this method, discussed in part in Example 5, and partly in Example 6. It is assumed that these sequences include synthetic and natural DNA sequences. Natural sequence, in addition, include segments of cDNA or genomic DNA.

Example 6 provides a molecular clone DNA that encodes a protein that is identical highlighted in Examples 1-3. Example 6 a plaque GT10-IL 1i-2A separated from the GT10 library. Multiply the phage within the plaque and the DNA is isolated and digested with EcoRI. The EcoRI fragment size 1850 base pairs carries the coding sequence for an inhibitor of IL 1. Fig shows a partial DNA sequence of the EcoRI fragment.

With regard to these doctrines and practices specialist in this field of knowledge is able to get other synth is political polynucleotide sequence. As an example of this prior art relating to polynucleotide synthesis, we can cite the work of Matt euccl, PPM and Carutners, M.N., J. Am. Chem. Soc. 103: 3185 (1981) and Beaucage, S.L. and Caruthers, M.N., Tetrahedron lett. 22: 1859 (1981), as well as the instructions with oligonucleotide synthesizer ABI.

These synthetic sequences can be identical to the natural sequence, described in more detail below, or they can contain other nucleotides. In one embodiment, if the synthetic sequence containing the nucleotides that differ from those found in natural DNA sequences of the present invention, it is assumed that these different sequences all encode the same polypeptide, which has the same primary structure as the IL-1i, isolated from monocytes. Alternatively, a synthetic sequence containing different nucleotides that encodes a polypeptide that has the same biological activity as IL-1, and described in this application.

In addition, the DNA sequence may be a fragment of a natural sequence that is a fragment of polynucleotide, which occurs in nature and which is isolated and purified for the first time by the present applicant. In one embodiment, the DNA sequence is a restriction fragment from the i.i.d. from a cDNA library.

Alternatively, a DNA sequence isolated from a human genomic library. An example of such library, usable in this embodiment shown Lawn et al. Cell, 75: 1157-1174 (1978), and this work is given here as a reference.

In a preferred version, this option assumes that natural DNA sequence of the get method, which involves:

(a) obtaining a human cDNA library from cells, preferably monocytes, capable of generating an inhibitor of IL-1 in the vector and the cell is able to amplify and Express all or part of this cDNA,

(b) sensing library of human genomic DNA, at least one probe capable of contact with the gene or its protein product inhibitor of IL-1,

(c) identifying at least one clone containing the gene encoding the inhibitor, due to the ability of the clone to associate at least one probe for the gene or its protein product,

(d) isolation of the gene or part of the gene encoding the inhibitor of the clone or clones,

(e) linking gene or suitable fragments with operating elements necessary for the maintenance and expression of the gene in the cell host.

Natural DNA sequences that are suitable in this way, can also be identified and allocated in porosimetry, which provides:

(a) obtaining a library of human genomic DNA, preferably propagated in the host E. coli recA recBC,

(b) sensing library of human genomic DNA, at least one probe capable of contact with the gene or its protein product inhibitor of IL-1,

(C) identifying at least one clone containing the gene encoding the inhibitor, due to the ability of the clone to associate at least one of the zones for the gene or its protein product,

(d) isolation of the gene encoding the inhibitor of clone (clones), which identified, and

(e) linking gene or suitable fragments with operating elements necessary for the maintenance and expression of the gene in the cell host.

When selecting natural DNA sequences suitable for use in the above method, it is preferable to identify two restriction site located within and in close proximity to the end parts of the corresponding gene or sections of the gene. The DNA segment containing the corresponding gene, then remove remaining genomic material using the appropriate restriction endonucleases. After excision of the 3' and 5' ends of the DNA sequence and any connection exon subjected to the reconstruction of the corresponding DNA-PEFC is guatelmala able to encode the N - and C-ends of the protein inhibitor of IL-1 and is able to synthesize DNA sequence with its operating elements.

3. Vectors

(a) Microorganisms, in particular, E. coli

Vectors are provided for use in the present invention include any vectors, in which you can type discussed here DNA sequence, together with any preferred or necessary operating elements, and which you can then migrate into the cell host and replicate in this cell. Preferred vectors are those whose restriction sites are well documented and which contain operating elements, which are preferred or required for transcription of the DNA sequence. However, provided some variants of the present invention that use of undisclosed to the present time vectors, which could contain one or more cDNA sequences discussed in this application. In particular, it is preferable that all of these vectors had some or all of the following characteristics: (1) possession of a minimum number of sequences of the host body, (2) the sustainable maintenance and reproduction in the desired host, (3) the ability to be in a desirable host with a high number of copies, (4) possession of a regulated promoter, located so as to promote transcription of the desired gene, (5) the presence of at least the ne marker DNA sequence, the coding of breeding trait that is present on a portion of the plasmid separate from the part where incarcerous DNA sequence, (6) a DNA sequence that is able to terminate transcription.

In various preferred embodiments of the invention these cloning vectors that contain and are able to Express the DNA sequences of the present invention contain various operating elements. These "operating elements", as they are discussed in this application include at least one promoter, at least one sequence of the Shine - Dalgarno and the initiation codon, and at least one termination codon. Preferably, if these "operating elements" also include at least one operator, one of the leader sequence for proteins to be exported from the intracellular space, at least one gene for a regulatory protein, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation vector DNA.

Some of these operating elements can be present in each of the preferred vectors of the present invention. It is assumed that any additional operating elements can be included in the data vectors using methods known in the village is authorized technical field in particular, in the light of these provisions.

In practice, it is possible to construct each of these vectors in a way that allows unimpeded selection, Assembly and interchangeability. This facilitates the Assembly of numerous functional genes from combinations of these elements and coding region DNA sequences. In addition, many of these items can be used in more than one owner. It is also anticipated that in certain embodiments of the implementation of the present invention, the vectors may contain a DNA sequence capable of functioning as regulators ("operators"), and other DNA sequences that can encode regulatory proteins.

(I) Regulators

In one embodiment of the present invention, these sliders to prevent expression of the DNA sequence in the presence of certain environmental conditions that allow transcription and subsequent expression of the protein encoded by the DNA sequence. In particular, preferably, if the regulatory segments inserzioni in the vector so that it doesn't happen the expression of a DNA sequence, or is substantially limited in the absence of, for example, isopropylthio-beta-D-galactoside. In this situation transformed microorg the mechanisms, containing a DNA sequence, can be grown to the desired density before the expression of IL-1i. In this embodiment the expression of a desired protein induce by adding substances in the microbial environment, which can cause the expression of a DNA sequence after reaching the desired density.

(II) Promoters

The expression vectors must contain promoters that can be used by the body host for the expression of its own proteins. Although usually use lactose promoters, isolated and characterized other microbial promoters, allowing individuals to use them for the expression of recombinant IL-1i.

(III) a transcription Terminator

Putative transcription terminators provide stabilization of the vector. In particular, the sequence described in the work of Rosenberg, M. and Court, D., Ann. Rev. Genet. 13: 319-353 (1979)will be used in the present invention.

(IV) Netransliruemye sequence

It should be noted that in the preferred embodiment of the invention it is possible to reconstruct the 3' or 5' end of the coding region in order to carry out the introduction of a 3' or 5' netrenirovannykh sequences in gene transcript. Among these netrenirovannykh sequences can be mentioned those which are identified Schmeissner, ., McKenney, K., Rosenberg, M. and Court, D., J. Molec. Biol., 176: 39-53 (1984), and the work put into the description by reference.

(V) Sites of ribosomal binding

Microbial expression of foreign proteins requires the presence of certain operating elements, which are not limited to, include the sites of ribosomal binding. The website ribosomal binding is a sequence that the ribosome recognizes and binds to it in the beginning of protein synthesis, as indicated in the work of Gold, L., et al., Ann. Rev. Microbio. 35: 557-580, or work Marguis, D.M., et al., Genc 42: 175-183 (1986), which are introduced in this description as a reference. The preferred site of ribosomal binding is GAGGGCGAAAAA (ATC).

(VI) a Leader sequence and translational linker

In addition, preferably, the DNA encoding the appropriate secretory leader (signal) sequence present at the 5' end of the DNA sequence, as outlined in the work of Watson, M.E., Nucl. Acids. Res. 12: 5145-5163, which is introduced in this description by reference, if the protein you need to secrete from the cytoplasm, DNA for a leader sequence may be in the position that provides the production of a hybrid protein in which the leader sequence directly adjacent to the inhibitor and covalently linked, that is, between the two On The To-coding sequences should not be signals of transcription or translation termination. The presence of the leader sequence is preferably partially on one or more of the following reasons. First, the presence of the leader sequence may facilitate the processing of an inhibitor of IL-1 by the host. In particular, the leader sequence can direct the cleavage product of the initial broadcast by the leader peptidases, removing the leader sequence and leaving the polypeptide with the amino acid sequence, which has the potential protein activity. Secondly, the presence of the leader sequence can facilitate the purification of an inhibitor of IL-1 by removing the protein from the cell cytoplasm. In some types of microorganisms host the presence of a suitable leader sequence facilitates the transportation of a native protein in periplasmatic space, as in the case of some E. coli. In the case of certain E. coli, and Saccharomyces strains of Bacillus and Pseudomonas appropriate leader sequence facilitates the transport of the protein through the cell membrane into the extracellular environment. In this case, the protein can be purified from the extracellular protein. Thirdly, in the case of some proteins, obtained in accordance with the present invention, the presence of the leader sequence may be required for the location of a native protein in which, in which he may be ordered to take his active structure, which has the corresponding protein activity.

In one preferred embodiment of the present invention additional DNA sequence feature directly in front of the DNA sequence which encodes an inhibitor of IL-1. Additional DNA sequence capable of functioning as a translational linker (crosslinking agent), that is, it is a DNA sequence that encodes RNA that serves to position the ribosome in the immediate vicinity of the site of ribosomal RNA binding inhibitor with which it comes into contact. In one embodiment of the present invention translational linker can be obtained using DNA sequences TAACGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGATCTTGGAGGATGATTAAATG and methods currently known to experts in the field related to PA linkers.

(VII) the translational terminator

The estimated translational terminators serve the purpose of the increment of the mRNA. They can be either natural, as described in the work of Kohli, J. Mol. Gen. Genet. 182: 450-439, or synthetic, as described by Pettersson, R.F. Gene 24: 15-2 (1983), both of which were put in this description as a reference.

(VIII) Breeding marker

Preferably, the o-cloning vector contained a breeding marker, which causes the expression of the breeding grounds of the microorganism host. In one embodiment of the present invention in the vector included a gene that gives resistance to ampicillin, whereas in other plasmids included the gene, giving resistance to tetracycline, or a gene that gives resistance to chloramphenicol.

Such a breeding marker or the other is intended for partial assistance in the selection of transformants. In addition, the presence of such a breeding marker in the cloning vector may find application in the preservation of contaminating microorganisms aside from the process of reproduction in culture medium. In this embodiment, pure culture of transformed microorganisms of the owner can be obtained by cultivation of microorganisms under conditions which require the induced phenotype for survival.

The proposed operating elements can easily choose the experts in the art in light of prior knowledge and are contained in this description of the provisions. Basic examples of these operating elements are represented in the work B.Lewin, Gene, Wiley Son., New York (1983), which entered into this description by reference. Various examples of suitable operating elements can be found on the above vectors and can be found in the review of publications that reviewed the t main characteristics of the above vectors.

After synthesis and allocation of all necessary and desirable components in these vectors the vector gather well-known specialists in the field methods. The Assembly of such vectors is within the competence of professionals and can be carried out without burdensome experiments. For example, similar DNA sequences are ligated into appropriate cloning vectors, as indicated in Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratories (1984), which entered into this description by reference.

When designing cloning vectors of the present invention it should be remembered that each vector can insertitemat many copies of the DNA sequence and operational elements. This version of the body-the owner will produce a higher number is desirable inhibitor of IL-1 on a vector. The number of multiple copies of DNA sequences that can be insertitemat in the vector is limited only by the capacity of the received vector given its size, will be migrated, as well as replication and transcription in the corresponding cells of the host.

(b) Other microorganisms

Vectors suitable for use in organisms other than E. coli, are also addressed by the present invention. Such vectors are described in Table 1. In addition, the following are some the preferred vectors.

Table 1
OWNERREGULAR PROMOTERSThe INDUCTORTRANSCRIPTIONAL TERMINATORSTABILIZATION of mRNAThe SITE of the START of TRANSCRIPTION AND a LEADER PEPTIDEMARKERThe WEBSITE RIBOSOMAL BINDING
E.coliLac1, Tac2The lambda pL Trp5IPTG, temperature rise, adding indolylacetic acid (IAA) or the depletion of tryptophanrrn B6neg A8bla12< / br>
neg A12< / br>
phos
ampicillin14tetracycline14,15chloramphenicol16
Lambda
rrn C7int9
trp10
Bacillusalpha-amylase17IPTGE. coli rrnB.amy neutral protease

B.amy alpha-amylase22< / br>
B.subt subtilisin23
Kanr24B.amy neutral protease

B.amy alpha-amylase22
subtilisin18 rrn BT. T20Camr25
p-4319
PseudemonasTrp27(E.coli) Lac Tac (E.coli)The addition of IAA or depletion of tryptophan IPTGphospholipase C28exotoxin A29the sulfonamide30streptomycin30Trp (E.coli)
YeastThe depletion of glucose and galactose, the Depletion of glucose Depletion of phosphateCyc1Invertase36Acid phosphatase36Alpha-factorUra 337
UnaLeu 258
AlphaHis 3
FactorTap 1
Sac2
Unregulated (non-regulatory)

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(I) the Vectors Pseudomonas

Several vector plasmids can replicate autonomously in a wide range of gram-negative bacteria, are preferred for use as the systems of the body-master-cloning vector in the hosts of the genus Pseudomonas. Some of them are described Tait, R.C., Close, T.J., Lindquist, R.C., Hagiya, M., Redriquez, R.L. Kadoc in the journal Biotechnology, May, 1983, R-275; Panopoulos, N.J., in the journal of Genetic Engineering in the Plant Sciences, Praeger Publishers, New York, pp.163-185 (1981), as well as Sakagucki, K., Current Topic in Microbiology and Immunology 96: 31-45 (1982), and each of these works introduced in this description by reference.

One particularly preferred design includes plasmid RSF 1010 and its derivatives, as described by Bagdasarian, M., Bagdasarian, M.M., Coleman, S., and Timmis, K.N. in Plasmids of Medical, Environmental and Commercial Importance, Timmis, K.N. and Puhler, A. eds., Elsivier/North Holland Biomedical Press (1979).

Advantages plasmid RSF 1010 is that it is relatively small, has a large number of copies, easily transformed and stably maintained in E. coli, and Pseudomonas. In this system, it is preferable to use the expression of the speaker, as described for Escherichia because it turns out that the trp-promoter, E. coli easily recognized by RNA polymerase, Pseudomonas, as described in Sakagucki, K. in Current Topics in Microbiology and Immunology, 96: 31-45 (1982) and the work of Gray, G.L. McKeown, K.A., Jones, A.J.S., Seeburg, P.H., and Heyneker, K.L. in Biotechnology, Feb., 1984, R-165, both of which were put in this description as a reference. Transcriptional activity can be further maximized by replacing the promoters, for example, the trp promoter E. coli or .aeruginosa. In addition, lac1 gene of E. coli will also be included in the plasmids for the implementation of the regulation.

The broadcast may be linked to the initiation of translation for any of the proteins of Pseudomonas, as well as initiation sites for any highly expressed proteins of the type chosen for the implementation of the intracellular expression of inhibitor.

In cases where restriction negative strains of the species Pseudomonas host cannot be obtained, the efficiency of transformation of plasmid constructs, isolated from E. coli is very low. Therefore, the passage of the cloning vector Pseudomonas via r-m + strain of another species is desirable before the transformation required of the owner as specified in the work Bagdasarian, M., et al., Plasmids of Medical, Environmental and Commercial Importance, pp.411-422, Timmis and Puhler eds., Elsevier/North Holland Biochemical Press (1979), which entered into this description by reference.

(II) the Vectors of Bacillus

In addition to t the th, the preferred expression system in the hosts of the genus Bacillus includes the use of plasmid pu W as the body-master-cloned vector. As another system of vectors of the owner, there is the possibility of Bacillus to Express inhibitor IL-1 of the present invention as either intracellular or secreted protein. The proposed variants of the present invention include both systems. Vectors-shuttles that replicate in Bacillus and E. coli acceptable to design and test a variety of genes, as described by Dubnau, D., Gryezan, T., D, Contente, S., and Shivakumar, A.G., Genetic Engineering Vol.2, Setlov and Hollander eds., Plenum Press, New York, New York, pp.115-131 (1980), specific put into this description by reference. For the expression and secretion of an inhibitor of IL-1 from B.sultilis signal sequence of the alpha-amylase is preferably tied to the coding region relative to the protein. For intracellular synthesis inhibitor portable DNA sequence translational bind to the site of ribosomal binding leader sequence of alpha-amylase.

The transcription of any of these structures preferably direct alpha-amylase-promoter or its derivatives. This derivative contains a sequence recognition RNA polymerase native alpha-amylase-promoter, but also includes a lac-operator-about the art. Similar hybrid promoters, designed from the promoter of the gene penitsillinazy and lac operator, operating in Bacillus hosts an adjustable manner, as specified in the work Yansura, D.G. and Honner, Genetics and Biotechnology of Bacilli, Ganesan, A.T. and Hoch, J.A., eds., Academic Press, RR-263 (1984), which entered into this description by reference. Gene lac1 gene could also be included in a plasmid for carrying out the regulation.

(III) the Vectors Clostridium

One preferred construction for the expression in Clostridium is a plasmid pJU 12, described in the work of Squires, C.H. et al., in Journal of Bacteriology 159: 465-471 (1984), which specifically put into this description by reference. This plasmid transform in C.perfringens method Hufner, D.L. et al., J. Bacteriol 159: 460 to 464 (1984), which specifically put into this description by reference. Transcription is directed by the promoter of the gene, giving resistance to tetracycline. The broadcast associated with the sequences of the Shine-Dalgarno the same gene tetrexactly in accordance with the methodology described above with respect vectors suitable for use in other hosts.

(IV) Yeast vectors

Conservation of foreign DNA introduced into yeast, can be done in several ways, as described in Botstein, D. and Davis, R.W., The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Stranthern, Jones& Broach, pp.607-656 (1982), which is introduced in this op is a description by reference. One preferred expression system for use with organisms owners kind Saecharomyces carries the gene inhibitor of IL-1 to 2 μm plasmid. The advantages of 2 μm rings are a relatively high number of copies and stability during induction in strains cir0. These vectors preferably have a beginning of replication and at least one marker antibiotic resistance of the plasmid RvR to implement replication and selection in E. coli. In addition, the plasmid preferably has a 2 micron sequence and the yeast LEU2 gene for the same purpose in the LEU2-deficient mutants of yeast.

It is assumed that if the recombinant inhibitors of IL-1 in the end will be expressed in the yeast, preferably, the cloning vector was first transferred into Escherichia coli, where the vector can be replicated and where the vector can be cleaned after amplification. Then the vector can be transferred into the yeast for the final expression of the inhibitor of IL-1.

(C) mammalian Cells

cDNA for an inhibitor of IL-1 serves as a gene for the expression of inhibitor in mammalian cells. cDNA must be a sequence, which would be effective for the binding of ribosomes, as described, for example, Kozak, Nucleic Acid Research 15: 8125-8132 (1987), which is specifically introduced in the description to which the reference number, also must have a coding capacity for a leader sequence (see section 3 (a) (VI)) in order to direct the Mature protein from the cells in processed form. DNA restriction fragment carrying the complete cDNA sequence can be insertion in the expression vector, which has a transcriptional promoter and a transcriptional enhancer, as described in the works Guarante, L., Cell 52:303-305 (1988) and Kadonaga, J.T., et al. Cell 51: 1079-1090 (1987), which are introduced in this description as a reference. The promoter may be adjustable, as in the plasmid pMSG (Pharmacia Cat No. 27450601)if the constitutive expression of the inhibitor is detrimental to cell growth. The vector must have full signal polyadenylation, as described in Ausubel, F.M. et al., Current Protocols in Molecular Biology (1987), which specifically put into this description by reference; however, in order to properly process the mRNA transcribed from this vector. Finally, the vector should have a beginning replication and at least one antibiotic-resistance marker from the plasmid RvR to implement replication and selection in E. coli.

To select a stable cell line that produces an inhibitor of IL-1 expressing vector can carry the gene for breeding marker, such as marker of drug resistance, or to carry a complementary gene for definitelyyy cells, such as gene digidrofolyatreduktazy (dhfr) for the transformation of the cell line dhfr, as described in Ausubel et al., above. Alternatively, a separate plasmid, bearing breeding marker, can be transformed together with expressioni vector.

4. Cell host/Transformation

The resulting vector is transferred into the corresponding cell of the host. These cells-owners can be microorganisms or mammalian cells.

(a) Microorganisms

I believe that any microorganism having the ability to take exogenous DNA and to Express these genes and the accompanying operating elements can be selected for this purpose. After selection of the host body, the vector is transferred into a master-organism using methods commonly known to specialists in this field of technology. Examples of such methods are the descriptions presented in the work of the Advanced Bacterial Genetics, R.W. Davis et al., Cold Spring Harbor Press, Cold Spring Harbor, New York (1980), which specifically put into this description by reference. Preferably, in one embodiment, if the transformation occurred at low temperatures because temperature regulation is expected as a means of regulation of gene expression by means of operating elements above. In another embodiment, if the vector inserzioni osmolar the regulators, to ensure that the proper management of alien genes would need to adjust the salt concentration during the transformation.

Preferably, if the microorganism-host is optional gone anaerobic or aerobe. The hosts, which is preferred for use in this method include yeast and bacteria. Specific yeast include yeast of the genus Saecharomyces, especially Saecharomyces cerevisiae. Specific bacteria include bacteria of the genera Bacillus, Escherichia and Pseudomonas, especially Bacillus subtilis and Escherichia coli. Additional cells of the hosts listed in Table 1, above.

(b) Mammalian cells

The vector can be introduced into mammalian cells in culture using multiple methods, such as calcium-phosphate: DNA coprecipitation, electroporation (i.e. stun open cell foam) or protoplast merger. The preferred way is by coprecipitation with calcification, as described in Ausubel et al., above.

There are many types of resistant cells that are transformed and capable of transcribing and broadcast cDNA sequence, processing the precursor inhibitor of IL-1 and secretion of the Mature protein. However, the types of cells can vary depending on the glycosylation of secreted proteins and posttranslational modification of amino acid on Titkov. Thus, the ideal types of cells are those which produce recombinant inhibitor of IL-1, which is identical to the natural molecule.

5. Cultivation is made of cells

Cell owners are cultivated under conditions suitable for expression of the inhibitor of IL-1. These conditions are mostly specific for the host cell, and they are easily identified by experts in the light of the published literature related to the growing conditions of such cells, and also in the light of these provisions. For example, Bergey''s Manual of Determinative Bacteriology, 8thEd., Williams & Wilkins Co., Baltimore, Maryland, which is inserted into this description by reference, contains information about the conditions for culturing bacteria. Similar information regarding the cultivation of yeast and mammalian cells can be obtained from Pollack, R. Mammalian Cell Culture, Cold Spring Harbor Laboratories (1975)introduced in this description by reference.

When carrying out works on the stages of transformation and cultivation can be selected any conditions required for regulating the expression of a DNA sequence, depending on any operating elements, inserzioni in vector or present in it. In one embodiment of the invention, cells are grown to high density in the presence of suitable regulators the x terms which inhibit the expression of a DNA sequence. While achieving the optimal density of cells surrounding conditions change to those suitable for the expression of a DNA sequence. Thus, it is assumed that the production of an inhibitor of IL-1 occurs in the time interval after the growth of host cells almost to the optimum density and that received the inhibitor of IL-1 collected at a certain stage after the induction of regulatory conditions necessary for its expression.

6. Clean

(a) an Inhibitor of Il-1, obtained from microorganisms

In a preferred embodiment of the present invention the recombinant inhibitor of IL-1 is cleaned after collection and before you assume its active structure. This option is preferred because the applicant believes that removing the high output re-ordered protein is facilitated, if the protein is first purified. However, in one preferred alternative embodiment, the inhibitor of IL-1 can be re-ordered with the adoption of its active patterns before cleaning. In yet another variant, the preferred alternative, the inhibitor of IL-1 is present in his re-ordered, the active state after removal of the culture medium. Under certain circumstances, the inhibitor of IL-1 will accept him, Akti the structure after expression in a microorganism host and transport of the protein through the cell wall or membrane, or periplasmatic space. This usually occurs if the DNA encoding the appropriate leader sequence linked to DNA that encodes a recombinant protein. If the inhibitor of IL-1 does not accept it should, active structure, any educated disulfide bonds and/or any non-covalent interactions will be destroyed by the agents of denaturing and reducing agents, such as guanidine chloride and beta-mercaptoethanol before the inhibitor of IL-1 will take its active structure after dilution and oxidation of these substances under controlled conditions.

For cleaning before and after re-ordering, it is preferable to use a certain combination of the following operating steps: anion exchange chromatography (Mono Q or DEAE-Sepharose), gel filtration (superose), chromatofocusing (Mono P) and hydrophobic chromatography (octyl - or phenyl-sepharose). Especially valuable chromatography on affinity for the antibody with the use of an inhibitor of IL-1-specific monoclonal antibodies (described in Example 5).

(b) an Inhibitor of IL-1, obtained from mammalian cells

The inhibitor of IL-1 derived from mammalian cells, purified from the conditioned medium by a combination of the following stages: ion-exchange chromatography and immunoaffinity chromatography, with ispolzovanpokoleniya antibodies described in Example 3. The expert will understand that the processes and products in accordance with the present invention various modifications and variations provided that they all make up the volume of the attached claims.

It should be understood that the application of the principles of the present invention is in the competence of specialists in the art with consideration given to the application of the provisions. Examples of products of the present invention and exemplary methods for their isolation and below.

The following examples illustrate various preferred variants of the present invention. Publications listed in these examples, introduced as references.

EXAMPLES

Example 1 - Getting proteins

A. Materials

Balanced salt Hanks solution (HBSS) and RPMI supplied from Mediatech, Washington, DC. Lymphoprep get from Akyurt chemical & scientific Corp., Westbury, New York. Human immunoglobulin G (IHH), MTT, rabbit anticigarette to antiprostaglandin E2, ammonium bicarbonate, dithiothreitol, complete and incomplete stimulants's adjuvant, gipoksantin, aminopterin and thymidine were supplied from Sigma chemical Co., St. Louis, Missouri. Mouse SN/ / HeJ obtained from Jackson, laws, Bar Harbor, Maine. Mouse BAL b/C and P3 myeloma cells receive from the funds of Doctors John Ka is Lera and Philippa Marrack at the National Center of Jewish for the Study of Immunology and Respiratory Medicine (NJ C/IPM), Denver, Colorado. Recombinant human IL-1 derived from cistron Biotechnology, pine Office, new York. Purified phytohemagglutinin comes to Welkam Diagnostics, research Triangle Park, North Carolina. Fibroblasts from human foreskin from primary cultures receive from Dr. Richard Clark NJ C/IPM, Denver, Colorado. Mouse monoclonal protivoorechii IHH antibody reagents are shipped from AIA Aurora, Colorado. PPMI with a low content of methionine is obtained using equipment select-Amin from GEPCO Laboratories, Grand island, new York. /35S/-methionine, diphenyloxazole and /14With/-iodoxy acid obtained from DuPont-NEN, Chicago, Illinois. Amniotic calf serum obtained from Hanlon Laboratories, Logan, Utah. Column Mono Q and Superose 12 are supplied pharmacy, Inc., Piscataway, New Jersey. Columns with reversed phase C4 is obtained from Synchrom, Inc., Lafayette, Indiana. Columns with reversed phase C8 receive from Applid of Biosystems, Inc., Foster City, California. Acetonitrile and polyethylene glycol 8000 come Gutmaker chemical Co., Phillipsburg, New Jersey. Triperoxonane acid and guanidine hydrochloride is obtained from Pearce chemicals, Rockford, Illinois. Endoproteinase With Lys obtained from Boehringer Mannheim Biochemicals, Indianapolis, Indiana. Titration microplates, the use of which has been created for enzyme-linked immunosorbent assay RFE 2(RFE2ELISA), represent Nunc-Immuno, Plate 1, obtained from Intermountain of scientific Corporation, Bountiful, Utah. Cups used to obtain hybridoma receive from Costar, Cambridge, Massachusetts.

Century Generation inhibitor of IL-1 monocytes

Human leukocyte get from normal donors by leukophoresis, resuspended in a balanced salt Hanks solution (HBSS) at a concentration of 1 part condensed cells to 1 part HBSS, padlayat Lymphoprep and spin at 400 kg for 50 minutes at room temperature. Take mononuclear fraction (usually 4-5 109cells receive from a single donor), washed in HBSS without Ca++or M++suspended in serum-free R1 and plated on Petri dishes, covered with normal human IHH, which make bibliotekarinnen chromatography on a column of Sephadex S200 (6×107cells in 10 ml of 100 mm Cup). All reagents contain less than 10 PG/ml of lipopolysaccharide (LPS). Cells cultured for 24-48 hours, and the resulting air-conditioned environment is the untreated supernatant layer of an inhibitor of IL-1 (IL-1i). Typically, cells from a single donor can lead to 700-900 ml of the crude supernatant IL-1i.

C. analysis of the inhibitor of IL-1

Usually use two analyses of IL-1 to determine the IL-1i. The thymocytes (1×106cells of the t 4-6-week-old mice AWN/ / HeJ) are responsible for 1.0 unit/ml of recombinant human IL-1 plus 1 μg/ml of phytohemagglutinin when premaxillae proliferatio, as measured by the introduction of3H-thymidine or absorption of tetrazolium salt MTT (Mesmann, T., J. Immunol Method 65: 55-61 (1983)) three days after the start of stimulation. Untreated IL-1i completely inhibits this reaction to the proliferation at a dilution of 1/10. Human dermal fibroblasts (1×105cells per well in 96-well Cup) typically responsible for 0.5 units/ml recombinant human IL-1 when secretion by 6 h of stimulation, approximately 50,000 PG/ml PCE2that can be measured using ELISA method. This method is as sensitive to IL-1i, and analysis of thymocytes.

D. Metabolic labelling inhibitor of IL-1

IL-1i metabolic mark cultivation of mononuclear leukocytes within 48 hours on the IHH-coated tablets (as described in part b) without serum PPM1, containing only 0,75 μg/ml of cold methionine (15 µg/ml are the norm), and to which was added 0.5 ISM, inch35S-methionine (1151 kubum/mmol) for 107cells. Control tagging carried out similarly except that the tablets covered amniotic calf serum, and not the IHH. The analysis of such control supernatants shows that a very small amount of IL-1i is secreted when cells are cultivated on tablets covered with amniotic calf serum.

E. Purification of the tree of the inhibitor of IL-1

Untreated supernatant IL-1i adjusted to 1.0 M sodium chloride, incubated on ice for one hour and centrifuged at 10,000 rpm for 15 minutes. Supernatant that contain all inhibitory activity, but only 20% of the initial protein, then intensively cialiswhat at a temperature of 4°against 0.025 M Tris, pH of 7.6, containing 0.1% of sucrose (buffer A), for gradient fractionation of proteins by anion exchange column (Mono q After dialysis inhibitorsdisease solutions re-centrifuged at 10,000 rpm for 15 minutes and then passed through nylon filters by size of 0.22 μm. Supernatant usually combined with 10 ml of similarly prepared supernatant from metabolic labeling and passed through a column of Mono Q-Superose (liquid Express chromatography of proteins (IAHB) on the equipment of pharmacy)) with the volumes of the layers or 1.0 ml, or 8.0 ml), washed with buffer And to reach the value OD280exhaust flow returned to baseline, and carefully chromatographic using a linear gradient of sodium chloride (0.025 M-0.10 M) in buffer A. the Column fractions are collected and analyzed for radioactivity and bioactivity. Samples of each fraction analysed polyacrylamide gel electrophoresis in a 12.5% sodium dodecyl sulfate, silver paint, impregnate d is feniak Sashalom, dried and put on film to obtain autoradiographically data. Figa shows the protein profile of the Mono Q chromatography relative to 40 ml of the crude supernatant IL-1i, mixed with 5 ml of metabolically labeled supernatant IL-1i. Added the amount of radioactivity detected in 50 μl of each fraction, as well as the bioactivity of the IL-1i, measured when analyzing obtain PGE2. Shows two major and one smaller type of radioactivity, which completely coincide with the three peaks of bioactivity. Fig.1b shows similar chromatography 15 ml of the crude supernatant IL-1i, mixed with 5 ml supernatants from monocytes, metabolically labeled on tablets covered with amniotic calf serum (FCS), and not the IHH. The levels of the above-mentioned three types of radioactivity significantly reduced. Figa shows gels, stained with silver, the fractions of sites chromatography, is shown in Figa and 1b. It should be noted that the fraction of peak radioactivity and bioactivity on Figa (fraction 52 and 59) show a major band at 22 kilodaltons (marked by arrows) in polyacrylamide gel electrophoresis c by sodium dodecyl sulfate (SDS page-DDS Na). The third faction 48 Figa) shows the band at 20 KD during PAG-DDS Na). Gel-filtration experiments relative to untreated IL-1i show that the active molecule which has a molecular weight of 18 to 25 KD. Fig.2b is autoradiogram gels shown in Figa. It is easy to understand that protein bands at 20 and 22 KD are the main radioactive species in these fractions.

Summarizing these results, the applicant shows that metabolic labeling of monocytes, sown in Petri dishes, covered with IHH, leads to the production of radioactive species, which are poorly produced only when cells were seeded on plates coated with FCS. These induced radioactive species are well chromatographers together with several types of bioactivity of IL-1i on a column of Mono Q, and gels and received autoradiogram show that the three major induced molecules are proteins with a predicted molecular weight for the IL-1i.

Molecules IL-1, and subjected to further purification for sequencing in two ways. First, the fraction of Mono Q peak bioactivity and radioactivity load in column C4 reversed-phase and elute H2O/0.1% of TN: gradient acetonitrile/0.1% of TN. Because the molecule IL-1i mark isotopic indicators, samples of each fraction is subjected directly to the counting of the radioactivity level, and analyze PAG-DDS Na followed by autoradiography, Figa shows such a chromatography amended figure of radioactivity. Painted silver gels is spytyvayut relative to samples from each fraction (Fig.3b), and subsequent autoradiography gels (Figs) show that the molecule IL-1i is in the fractions 32-36. These fractions are dried and is sequenced. Alternatively, the maximum fraction of Mono Q is dried with Speed Vac, resuspended in 0.4 ml of 0.05 M NH4HCO3and directly chromatographic twice on the gel-filtration column Superose 12 (10×300 mm) (IAHB Pharmacia), equilibrated in the same buffer solution as shown in Figa and 4. Fractions are collected and the samples of each fraction are tested for radioactivity and toactively, and then analyzed by silver staining and autoradiography at PAG-DDS Na. The appropriate fractions are then dried on a Speed Vac and is sequenced.

Example 2

The proposed sequencing of the inhibitor of IL-1

Before sequencing, the samples are dissolved in 6 M guanidine-HCl, pH 8,6, recover for 4 hours at a temperature of 37°With N2100-fold molar excess dithiothreitol in comparison with protein, then alkylate within 1 hour 400-fold excess of14With-modokashii acid. In this case, the reaction mixture can be obsolete on a column with reversed phase C8, eluted and partially dried, N-terminal sequence determined using a sequencing machine proteins Applid of Biosystems. To obtain internal sequences recovered and alcaravan education is subjected to digest cyanogenmod or proteolytic enzymes using known methods. The reaction mixture is dried, dissolved in 0.1% TN/H2O, and peptizyde allocate using columns with reversed phase C8.

Example 3

Purification and sequencing of types of inhibitors of IL-1

A. Types of IL-1i-X, IL-1 and IL-1i-b

Mono Q purification of IL-1i breaks down biological activity into three main types, as shown in Figa and described in Example 1, where the peak fractions regarding this activity are 48, 52 and 59. PAG-DDS Na in the samples of these fractions, as shown in Figa, identifies species at 20 KD, 22 KD and 22 KD, respectively. Western analysis of these gels using mouse antisera, which is discussed in Example 4 below, the colors all three types. When the IL-1i is obtained from cells metabolically labeled with35S-methionine during growth on tablets covered with IHH, each of these bands is radioactive (as shown in Fig.2b, representing autoradiogram above the gel). On the basis of the doctrine presented in Example 1, namely that parallel cells, incubated in reinducing conditions, does not produce the bioactivity of IL-1i and not produce these radioactive bands, the applicant concludes that these three species are responsible for the biological activity. The applicant considered these types of IL-1i-X, IL-1i-a, and IL-1i-b, respectively.

C. Purification and sequencing of the IL-1i-X

Mono Q fractions, with the holding of the IL-1i-X and/or IL-1i-a, clear of liquid chromatography high-pressure reversed-phase column Synchromat PP-4 (C4), and radioactive species subjected to sequencing analysis. Failed numerous attempts to direct sequencing of the IL-1i-a and IL-1I-b, purified GHD with reversed phase, which indicates their chemical blocking by their N-ends. However, one drug IL-1i-a (IL-1i-AVR) results in the following sequence:

and subsequent preparations of IL-1i-X, similarly treated jhud with reversed phase C4, produce the same sequence:

No doubt, they are part of a sequence of detected during initial attempts to sequence the IL-1i. The applicant concludes that the data sequences are N-by the end of the 20 KD species called the IL-1i of Agriculture In these and all subsequent sequences underlined the position of the points or the inability to identify the remnant, or the existence of ambiguity concerning the identified residue. When two or more residues are placed in the same position, this indicates that it has detected more than one amino acid at the stage sequencing, and more correct balance is at the top.

C. Generation of cleaning sequencevalue peptides of IL-1i-a and IL-1i-b

Because IL-1 and IL-1i-b chemically blocked by their N-ends, peptides each of them generate digesting endoproteinase. Specifically, Mono Q fractions containing either IL-1i-a, or IL-1i-b, passed through the column obremenitve (C3) high-performance liquid chromatography size 4,6×250 mm (ZORBAX Protein Plus), which is an alternative speaker-4, used in all previous experiments. Very speed gradients (0.2% acetonitrile per minute at 0.5 ml/min) otscheplaut IL-1i-a (Piga, b) or IL-1i-b (Figa) from the main contaminating radioactive species, human lysozyme. The identity of the purified species confirm the presence of single, radioactive protein with a molecular mass of 22 KD on SDS page-DDS Na and subsequent autoradiograms (Figs, d) and (9b). Proteins are harvested manually in a test tube made of quartz glass, each of which is added 25 ml of 0.2% solution of tween-20. IL-1i-containing fractions then lead to a volume of 50 μl in a Speed Vac, adjusted to 300 μl by the addition of 1% NH4HCO3with the subsequent addition of 1 mg endoproteinase. In the case of IL-1i-use enzyme Endoproteinase LysC (Boehringer-Mannheim), whereas IL-1i-b split Endoproteinase AspN (Boehringer Mannheim). The splitting is carried out at a temperature of 37°C for 16 hours, after which the volume of the reaction mixture on iaut to 50 ml on a Speed Vac.

In the case of IL-1i-sample chromatographic directly, whereas the sample of the IL-1i-b first restore by adding 5 ml of 50 mm dithiothreitol 2 M Tris, pH 8.0, and subjected to interact for 30 minutes at a temperature of 37°With, then carboxymethylated by adding 1.1 µmol3H-modokashii acid in 10 ml of ethanol (interacting within 30 minutes at a temperature of 37°in the dark). Separation of peptides carried out on a column of narrow gauge however Akvapor RP-300 (C8) 2.1×250 mm when the velocity of the front of the solvent of 100 µl/min, using Ehud Beckmann, provided microcalibration equipment and microcalibration pumps. Use a linear gradient of 200 min, 0-100% (H2O/0.1% of TN - acetonitrile/0.1% of TN). Peptide selection is shown in Figure 10 and 11. Received the following information regarding sequences:

Two peptide sequences, apparently, associated in nature with that which you have previously received from the IL-1i-X. One of them, RaLysC-41, is a sequence of the IL-1i-a, and the other, RbAspN-51, is a sequence of the IL-1i-b that disputes the claim that three types of IL-1i are at least closely related proteins, if not chemically and/or physically modified forms only original m is likely IL-1i. If a given sequence of merge, it is possible to obtain the following compound sequence:

These composite sequences, apparently, are not present in any other known polypeptides presented in a recently published Database of the Resource Identified Proteins (PIR 16.0). The applicant believes that these sequences or their less significant options are a class of molecules that can act as inhibitors of IL-1.

Example 4

Antibodies specific inhibitor of IL-1

Mice BaL b/C at the age of ten weeks subcutaneously injected inhibitor of IL-1, partially purified (400-fold) from raw supernatants using Mono Q chromatography, Valitova against SFG (phosphate buffered saline) and emulsified full stimulator's adjuvant. Each mouse receives the IL-1i, purified from 5 ml of the crude supernatant layer. Mice booster subjected to immunization every two weeks equivalent amount of an inhibitor of IL-1, emulsifiable incomplete stimulator's adjuvant, and serum samples taken from the tailings within seven days after each booster. Antisera are tested for anti-IL-1i activity Western analysis transblot immunogen carried out on polyacrylamide gel ele is triarese with sodium dodecyl sulfate, as shown in Figa. Fig.5b shows that all mice produce antibodies to IL-1, and after three injections of an inhibitor of IL-1.

Because monoclonal antibodies will be of great value when cloning the gene inhibitor of IL-1 from the library of expression, purification of recombinant protein inhibitor of IL-1 and study biology molecules, the applicant began the process of making a battery of monoclonal antibodies specific for the inhibitor of IL-1. To obtain hybrid cells In the above mice injected intravenously with the use of the same amount of an inhibitor of IL-1 in saline solution for 24 hours before removing spleens. Splenocytes ofpreparing from spleens and placed in cold balanced salt solution (BSS), washed twice BSS, mixed with myeloma cells AG in the ratio of 2×107cells RH 108splenic cells and spin. Cells synthesize by pocataligo adding 1 ml heated, sahusilawane (5% CO2) PEG 6000 (40% polyethylene glycol 6000: 60% minimum support environment) to dry the precipitate. Synthesized cells washed with BSS and resuspended in 10 ml of enriched medium (10% FBS)containing 2×105peritoneal cells per 1 ml, and the precipitate carefully dispersed using a 10 ml pipette. The volume was adjusted to 20 ml by adding larger number is the number of peritoneal cells in the environment, cells were seeded in 96-well tablets at a concentration of 0.1 ml/well. The tablets are placed in the gas incubator and then treated as follows:

day 1 - addition of 3×GAT (gipoksantin, aminopterin, thymidine) in enriched medium to a final concentration of 1×

day 5 - changing environment by replacing 200 ál 1×GAT in an enriched environment

day 10 - begin checking on hybrid growth. Change the environment by replacing 200 ál 1×GAT in an enriched medium containing 1.5 x 106peritoneal cells / 1 ml

When hybrid cells almost confluently in the hole, the supernatant layers is transferred to the test cells and gently gather the pipette tip and transferred into the wells with 1 ml of culture containing 1×GAT in an enriched environment and 3×106peritoneal cells per 1 ml of the Supernatant layers of confluently holes examined for activity against inhibitor of IL-1 using enzyme linked immunosorbent assay (ELISA), in which partially purified inhibitor of IL-1 (Mono Q-purified substance, which is identical to that administered to mice) tied to the wells on the microtiter plate. Serum of normal mice and hyperimmune antisera used as negative and positive control substances, respectively. Positive supernatant layers re-examined using ELISA method N. the tablets, covered homogeneous purified inhibitor of IL-1, and the method thus cleared metabolically labeled inhibitor of IL-1. Positive cells then clone by limiting dilution and injected to mice treated with dock for the generation of ascites. Large amounts of IL-1i-specific antibodies can be obtained by tissue culture or mass generation and collection of ascitic fluid in mice. Purification of these antibodies and their adherence to the insoluble pellets produce affinity adsorbents for the purification of recombinant protein inhibitor of IL-1.

Example 5

Cloning of cDNA of an inhibitor of IL-1

It is shown that monocytes seeded on Petri dishes coated with immunoglobulin G, and cultured for 24 hours in the presence /35S/-methionine, produce /35S/-IL-1i, which can be identified by its chromatographic properties on a column of Mono q

To determine when (within a 24-hour period) IL-1i is produced with the maximum degree of inoculated monocytes exhibit K /35S/-methionine (pulse) for a short (2 hour) period, and during this time, add a large excess of unlabeled methionine and incubation continued for another two hours. Then Wednesday gather and analyze inhibitor of IL-1, labeled with a radioactive label. This technique is used is the ratio of monocytes at different time intervals after planting tablets, coated with immunoglobulin G, and found that monocytes exposed /35S/-methionine for 15 h after seeding, produce the maximum amount of an inhibitor of IL-1-/35S/, which suggests that mRNA inhibitor of IL-1 in monocytes has a maximum level after 15 hours after seeding on the immunoglobulin,

Then fresh monocytes plated on immunoglobulin G-free lipopolysaccharides as in example 1B. After incubation in media RPM1 for 15 hours at a temperature of 37°cells are washed with phosphate buffer saline, then are lysed with 4 M thiocyanate guanine, 25 mm sodium citrate, pH 7, 0.5% sarcasm, 0.1 M 2-mercaptoethanol. Then total RNA extracted from this lysate AGPC method in accordance with the work .Chomczynski and N.Sacchi, which appears in the journal Analytical Biochemistry, Vol 2, PP 156-159.

Poly And+RNA allocate chromatography on oligo-dT cellulose by the method of Aviv, H. and Leader, P. (1972) Proc. Natl. Acad. Sci. (USA) 69: 1408-1412, precipitating with ethanol and diluting to a concentration of 0.36 µg/µl. 1 µg poly And+RNA is used for cDNA in accordance with the work after Gubler, U. and Haffman (1983) Gene 25: 263-169.

cDNA incorporate in the library of expression of the lambda gt11 using linkers Eco RI from the catalogue numbers 988448 Boehringer Mannheim or No. 1070 new England Bio lab, as well as the instructions of the manufacturer is.

The resulting library, which contains 106individual clones are screened for E. coli Y1090rk-(Promega Biotec) with the appropriate polyclonal antibody to the inhibitor of IL-1, as described previously, using screening conditions described R.A.Young and R.W.Davis /(1983) PNAS 80: 1194-1198/. Positive signals will be determined using biotinylated second antibody, such as goat antimachine immunoglobulin G, Bethesda research Labza), followed by addition of conjugate streptavidin phosphatase (Bethesda research Labza), as described Bayer, A. and Wilchek, M. (1979) Methods in Biochemical and Guesdon, J.L.Ternynch. T. and Cevrameas, S. (1979) 27: 1151-1158, and in accordance with the manufacturers instructions.

Example 6

Obtaining and sequencing of the gene encoding the IL-1i

cDNA obtained in accordance with Example 5, incorporate into the cloning vector lambda GT10. This cDNA first was identified in using EcoRI-methylase, using s-adenosyl-methionine as a substrate, EcoRI linkers attached to the ligation reaction and excess linkers are removed by digestion with the endonuclease Ecor and chromatography on a column of CL6B-spin. Carry out a ligation reaction containing 0,124 mg of linkers selected according to the size of cDNA and 1 μg EcoRI-cut and phosphatase-treated vector lambda GT10 and products this is eacli ligating seal with the o extrato GIGAPACK GOLD (Stratagene). Get the library from 1×107members.

The purpose of the screening of this library GT10 synthesize oligonucleotide (antisense) probes on the basis of protein and peptide sequences shown in Example 3. The sequence of the probes and their corresponding peptide sequence is as follows:

Probe No. Ili-332R-phosphorylate the 5'-end and used for screening 3×105plaques of the library. The probe produces hybrids reproductive to three plaques, and one of them also it hybridises to the probe No. Ili-4. This plaque, GT10-Ili-2A, cultivate and DNA allocate using Lainbasorb (Promega) according to the manufacturer's instructions. GT10-Ili-2A deposited in the American type culture Collection (ATSS), Rockville, Maryland, under the Deposit number # 40488. DNA digested using EcoRI, divided into five equal aliquot and subjected to electrophoresis on 1%agarose gel.

After electrophoresis the gel is stained with bromide ethidium. The picture of this gel is shown in Figa. Tracks 6, 8, 10, 12 and 14 contain five aliquot in the digestion of EcoRI. Track 5 contains a mixture of lambda DNA wild-type, cut with Mind III, and X174 RF DNA cut with HaeIII (new England Biolabs), which are suitable in quality for the ve molecular markers, that is, markers of molecular weight. Rise shows that G10-Ili-2A contains an EcoRI fragment having a length of 1850 base pairs. In order to demonstrate more clearly that this 1850 BP fragment carries the coding sequence for an inhibitor of IL-1, perform southern blokirovanie as follows. DNA fragments in the gel shown in Figa, blueroot on the nitrocellulose using standard methods. The nitrocellulose is then cut lengthwise into five strips so that each strip contained DNA from tracks 6, 8, 10, 12 and 14. Then strips separately hybridizing to each of the five oligonucleotide probes (above)that have been labelled with 5'-end32P-phosphate. Oligonucleotide concentration is 1 pmol/ml, and the temperature of hybridization is as follows.

TRACKPROBETEMPERATURE
6No. ili-335°
8No. ili-442°
10No. ili-542°
12No. ili-640°
14No. ili-735°

After washing the strips align and connect with one another in order again clicks is to use the original nitrocellulose sheet. Sheet autoradiography in the presence of an intensifying screen at -70°C for 24 hours. Fig is a photograph of this autoradiography. It shows that all the probes hybridizing specific to the fragment length 1850 base pairs, providing that this fragment carries significant coding sequence for an inhibitor of IL-1.

To determine the DNA sequence GT10-Ili-2A DNA digested with EcoRI, subjected to electrophoresis on 1% agarose gel and isolated fragment 1850 base pairs. This fragment are ligated with EcoRI-digested M13 Mr and transform into E. coli strain JM109. Transformers are subjected to screening, watching those who do not have beta-galactosidase activity. There are five of these transformers get odnozadachnoy DNA sequencing is carried out in accordance with the method of Sanger et al. The DNA sequence of the three transformers corresponds to the 3'-end of mRNA, whereas two transformers produce the protein coding sequence. On Fig shows the DNA sequence, which is obtained for the protein-coding region cDNA.

Fig also shows the predicted amino acid sequence. Amino acid sequence of the first amino acid Alanine to 29 amino acids Proline and 79 amino acids Isoleucine up to the NCA is the hypothetical amino acid sequence. The predicted amino acid sequence of 30 amino acids Proline to 78 of the amino acid Proline is consistent with the peptide sequences described in Example 3.

Example 7

Sequencing G10-IL-1i-2A and IL-1i

Part G10-Ili-2A is sequenced and shown on Fig. DNA encodes a protein containing amino acid sequences that are hallmarks of an inhibitor of IL-1 (nucleotides 99-557). However, I believe that prior to secretion of the protein into the extracellular environment, you can make a few modifications to it. These modifications may or may not be significant to the protein so that it has activity as an inhibitor of IL-1.

GT10-Ili-2A encodes at least 32 amino acids, N-terminal (nucleotides 3-98) to aminobenzo the form of an inhibitor of IL-1, which is known as X. Suppose that in these 32 amino acids is a secretory leader sequence, which begins at M encoded by nucleotides 24-26, directs the nascent inhibitor of IL-1 in the extracellular environment, then deleted the leader peptidases, and possibly other peptidases. Currently unknown the extent to which this sequence is removed in the form of alpha - and beta inhibitor of IL-1, but N the end of these forms are believed to be close to the N end of the form X. Destruction of the secretory leader PEFC is the sequences, probably, it is necessary to ensure that the protein was an effective activity of an inhibitor of IL-1.

Nucleotides 349-351 GT10-Ili-2A encode N-balance, which is part of the site consistent N-glycosylation. On the basis of their susceptibility to digestion with N-glycanase believe that the form of alpha and beta inhibitor of IL-1 is glycosylated. Since the form X is not sensitive to digestion by this enzyme believe that it is not glycosylated, although there remains the possibility that this might happen, and the expert will easily be able to show this by sequencing of proteins taking into account the information here. I believe that glycosylation in this N-residue is not required for protein exhibiting effective activity of an inhibitor of IL-1.

Nucleotides 99-101 GT10-Ilia encode P (see Fig), but in this position (N-end) of the form X inhibitor of IL-1 was not detected neither one Works. it is Possible that this modified residue in the Mature protein. I believe that modification of this residue is not essential step for efficient activity of an inhibitor of IL-1.

Unknown at the present time the remains of the N-end forms alpha and beta cannot discover a completely splitting Admino and, in all likelihood, they modified after removing some of the residues of the N-Terminus of the protein encoded G10-Ili-2A. The floor is up, this modification is not essential for effective activity of an inhibitor of IL-1.

Example 8

The expression of the genes encoding inhibitor of interleukin-1 in animal cells

Expression of animal cells an inhibitor of IL-1 involves the following stages:

A. Designing expressing vector

. The choice of cell line-hosts

C. the Introduction of expressing the vector in a host cell

Manipulirovanie recombinant cell hosts with higher levels of expression of the inhibitor of interleukin 1

1. Vectors for the expression of an inhibitor of IL-1, intended for use in animal cells, can include several types, such as a strong constitutive expressing constructs induced gene construct, as well as those that are designed for expression in certain cell types. In all cases, the promoters and other gene regulatory areas, such as enhancers (induced or not) and polyadenylation signals, are placed in an appropriate position relative to the cDNA sequences of vectors based on the plasmid. Below are two examples of such structures: (1) Design using a strong constitutive promoter must be produced by applying control signals genome vacuolating virus monkey 40 (OB 40) in t is com order which can be found in the plasmid p SV2CAT described by Gorman et al., Mol. Cel. Biol 2: 1044-1051, 1982. This plasmid must be manipulated in such a way as to replace cDNA inhibitor of IL-1 coding sequences chloramphenicol-acyltransferase (CAT) using standard techniques in molecular biology (Maniatis et al., above), as shown in Fig.6. (2) Design-induced genes must be made using the plasmid R j, which contains the promoter region of the mouse metallothionein (MT-1) (Brinster et al., Cell 27: 228-231, 1981). This plasmid can be used as source material, and it must be manipulated, as shown in Fig.7, with obtaining metal-induced gene construct.

2. A number of lines of animal cells can be used for expression of the inhibitor of IL-1 using the vectors described above, to obtain active protein. Two potential cell lines, which are well characterized by their ability to promote expression of foreign genes, are cells of the mouse Ltk-and dhfr cells of the Chinese hamster ovary (CHO), although the expression of the inhibitor of IL-1 is not limited to these cell lines.

3. Vector DNA must be introductionat in these cell lines using a number of methods of gene transfer. Method used here includes m is todoku calcium-phosphate-DNA precipitation, described S.L.Graham & A.S. van der Eb (Virology) 52: 456-4670 (1973), in which expressive vector for an inhibitor of IL-1 Coosada with the second expressing vector coding for breeding marker. In the case of transfection of cells Ltk-breeding marker is a gene timedancing and selection carried out as described by Wigler et al. (Cell 16: 777-785, 1979), and in the case of cells CHO dhfr-breeding marker is dihydrofolic reductase (DHFR), selection of which is described by Ringold et al. J. Mol. Ap. Gen. 1: 155-175, 1981.

4. Cells expressing the gene inhibitor of IL-1, should be grown in conditions that increase the levels of reception of an inhibitor of IL-1. Cells bearing structures metallothionein promoter, you can now grow in the presence of heavy metals such as cadmium, which lead to 5-fold increase in the level of promoter used MT-1 (Mayo et al., Cell 29: 99-108), subsequently leading to increased levels of proteins of the inhibitor of IL-1. Cells containing the expression vectors of the inhibitor of IL-1 (or S-40, or MT-1) together with a DHFR expression vector can be taken from the Protocol of gene amplification, described by Ringold et al. (J. Mol. Appl. Genet 1: 165-175, 1981) using methotrexate, competitive antagonist digidrofolyatreduktazy. This leads to a greater number of copies of the DHFR genes present in the cells, and e is n, the number of gene copies inhibitor of IL-1, which, in turn, lead to more protein inhibitor of IL-1 produced by the cells.

Example 9

Purification of an inhibitor of IL-1 from rekombinantnyh animal cells

Since the inhibitor of IL-1 is secreted from cells as a natural substance, it is expected that the methods described above for the purification of natural protein can be used to make a similar purification and characterization of recombinant protein.

Example 10

The sequence of an inhibitor of IL-1

Aminobenzoic the rest of the inhibitor of IL-1 identified several times by direct protein sequencing as arginine. The result of this sequence is shown in Example 3. In contrast, aminobenzoic the rest of the inhibitor of IL-1, predicted by the cDNA sequence, is a Proline (P). This aminobenzoic the remainder correspond to nucleotides 85-87 on Fig and is surrounded by a ring on Fig and 15. This apparent discrepancy County cDNA sequence and direct the protein sequence can be resolved by the assumption that the error in the cDNA sequence was incorporated during synthesis catalyzed by reverse transcriptase from the mRNA. That is, the codon CGA (arginine), located on the mRNA, where he could encode this aminobenzoic balance could be changed during the reaction back the reverse transcriptase at codon CCA (Proline) in the cDNA. This type of concerns regarding reverse transcriptase described previously in the literature, for example in the work B.D.Clark et al., Nucleric Acids Research 14: 7897 (1986).

The applicant believes that the correct amino acid sequence of the protein is predicted cDNA, except that aminobenzoic amino acid is arginine instead of a Proline residue indicated on Fig-15. The applicant proposes that both DNA sequences and their corresponding peptide sequences fall in the scope of the present invention, although preferred, however, is the sequence aminoanisole arginine.

Example 11

In the scope of the present invention also includes a protein having the sequence:

where X denotes a cysteine, serine or alanine, and Z denotes arginine or Proline.

1. The selected DNA molecule encoding a polypeptide precursor inhibitor of interleukin-1 (IL-li)containing the nucleotide sequence comprising a) a nucleotide sequence encoding a N-terminal leader sequence that provides secretion of active IL-li cell, and (b) a nucleotide sequence encoding the Mature IL-li with the amino acid sequence

(X) P S G R K S S K M Q A F R I W D V N Q K T F Y L R N

N Q L V A G Y L Q G P N V N L E E K I D V V P I E P H A

L F L G I H G G K M C L S C V K S G D E T R L Q L E A V

N I T D L S E N R K Q D K R F A F I R S D S G P T T S F

E S A A C P G W F L C T A M E A D Q P V S L T N M P D E

G V M V T K F Y F Q E D E,

where X denotes R or P;

his fragment or homologue that retains the ability to inhibit the activity of interleukin-1.

2. The selected DNA molecule according to claim 1, where the nucleotide sequence of (b) encodes the Mature IL-li with the amino acid sequence

(X) P S G R K S S K M Q A F R I W D V N Q K T F Y L R N

N Q L V A G Y L Q G P N V N L E E K I D V V P I E P H A

L F L G I H G G K M C L S C V K S G D E T R L Q L E A V

N I T D L S E N R K Q D K R F A F I R S D S G P T T S F

E S A A C P G W F L C T A M E A D Q P V S L T N M P D E

G V M V T K F Y F Q E D E,

where X denotes R or R.

3. Recombinant expressing a vector containing a regulatory element operatively linked to a DNA molecule according to claim 1 or 2, is required for expression of the DNA molecule in the cell host, able to split the signal peptide, which is part of the product synthesized by the cell.

4. The vector according to claim 3, characterized in that a host cell is a bacterial cell or a cell of a mammal.

5. The method of obtaining biologically active inhibitor of interleukin-1 (IL-li), comprising the following operations:

a) transforming a suitable host cell expressing recombinant vector according to claim 3;

b) growing converted the Anna cells under conditions ensuring the expression of the DNA molecules contained in a vector, and the secretion of active IL-li

and

C) isolation of the final product from the culture medium.

6. The method according to claim 5, characterized in that the recombinant expressing the vector contains a promoter other than the promoter of the natural inhibitor of IL-1, operatively linked to a DNA molecule according to claim 1.

7. The polypeptide is a precursor of active secreted form of the inhibitor of interleukin-1 (IL-li), having the amino acid sequence represented by Fig, and amino acid residue corresponding to codon 99-101, represents an arginine or Proline.

8. Biologically active inhibitor of interleukin-1, produced by recombinant cell host, having the amino acid sequence

(X) P S G R K S S K M Q A F R I W D V N Q K T F Y L R N

N Q L V A G Y L Q G P N V N L E E K I D V V P I E P H A

L F L G I H G G K M C L S C V K S G D E T R L Q L E A V

N I T D L S E N R K Q D K R F A F I R S D S G P T T S F

E S A A C P G W F L C T A M E A D Q P V S L T N M P D E

G V M V T K F Y F Q E D E,

where X denotes R or P;

his fragment or homologue that retains the ability to inhibit the activity of interleukin-1.

9. Inhibitor of interleukin-1 by claim 8 having the amino acid sequence

(X) P S G R K S S K M Q A F R I W D V N Q K T F Y L R N

N Q L V A G Y L Q G P N V N L E E K I D V V P I E P H A

L F L G I H G GK M C L S C V K S G D E T R L Q L E A V

N I T D L S E N R K Q D K R F A F I R S D S G P T T S F

E S A A C P G W F L C T A M E A D Q P V S L T N M P D E

G V M V T K F Y F Q E D E,

where X denotes R or R.

10. Inhibitor of interleukin-1 on item 8 or 9, characterized in that a host cell is a bacterial cell.

11. Inhibitor of interleukin-1 by claim 10, characterized in that a host cell is an Escherichia coli cell.

12. Inhibitor of interleukin-1 on item 8 or 9, characterized in that a host cell is a cell of a mammal.

13. Inhibitor of interleukin-1 to 12, characterized in that a host cell is a cell SNO.

14. Inhibitor of interleukin-1 on any of PP-13, characterized in that type of glycosylation is different from the type of glycosylation of the human urinary IL-li, or is deglycosylated.

15. The DNA molecule encoding an inhibitor of interleukin-1, having the nucleotide sequence corresponding to the amino acid sequence of an inhibitor of IL-1, its fragment or homolog of claim 8.

16. The DNA molecule according to item 15, the nucleotide sequence which corresponds to amino acid sequence

(X) P S G R K S S K M Q A F R I W D V N Q K T F Y L R N

N Q L V A G Y L Q G P N V N L E E K I D V V P I E P H A

L F L G I H G G K M C L S C V K S G D E T R L Q L E A V

N I T D L S E N R K Q D K R F A F I R S D S G R T T S F

E S A A C P G W F L C T A M E A D Q P V S L T N M P D E

G V M V T K F Y F Q E D E,

where X denotes R or R.

17. Recombinant expressing a vector containing a regulatory element operatively linked to a DNA molecule according to item 15 or 16, and is required for expression of the indicated molecules in the cell host.

18. The vector according to 17, characterized in that a host cell is a bacterial cell or a cell of a mammal.

19. A method of producing an inhibitor of interleukin-1, including the transformation of animal cells expressing recombinant vector according to 17, cultivating the transformed cell under conditions enabling expression of the DNA molecules contained in a vector, and the allocation of the final product.

20. The method according to claim 19, characterized in that the inhibitor of interleukin-1 is of type glycosylation, non-type glycosylation of human urinary IL-li, or is deglycosylated.

21. The method according to claim 19, characterized in that the recombinant expressing the vector has a promoter other than the promoter of the natural IL-1 inhibitor, operatively associated with a DNA molecule according to item 15.



 

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10 cl, 4 dwg, 21 tbl, 12 ex

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

FIELD: immunobiotechnology.

SUBSTANCE: invention relates to soluble CTLA4, which represents mutant variant of wild type CTLA4 and conserves binding ability to CD80 and/or CD86. Molecules of soluble CTLA4 have the first amino acid sequence containing extracellular CTLA4 region, which includes some mutant amino acid residues in S25-R33 region and M97-G107 region. According the present invention mutant molecules also may include second amino acid sequence, enhancing solubility of mutant molecule. Nucleic acid (NA) molecules encoding said CTLA4 and including NA-vectors also are described. Invention also relates to method for production of mutant CTLA4 and uses thereof in controlling of interaction between T-cell and CD80 and/or CD86-positive cell; suppression of graft-versus-host reaction; and treatment of immune system diseases. Soluble mutant CTLA4 according to present invention binds to CD80 and/or CD86 antigen with higher avidity than wild type CTLA4 or non-mutant CTLA41g.

EFFECT: new preparation for treatment of immune system diseases.

65 cl, 19 dwg, 2 tbl, 2 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention relates to antibodies specifically binding to new human extracellular matrix polypeptides called as RGI; immunoconjugate containing the same and method for selective cell degradation; method for treatment of prostates cancer and metastasis in patients suffering from prostates cancer.

EFFECT: new method for treatment of prostates cancer.

28 cl, 7 ex, 7 dwg

FIELD: molecular biology, genetic engineering, polypeptides, medicine.

SUBSTANCE: in using the double-hybrid yeast system DNA sequences encoding polypeptides (55.1 and 55.3) have been found that elicit ability for binding with intracellular domain p-55 (p-55IC) of TNF-receptor. It has been established that these polypeptides represent fragments of amino acid sequences p-55IC, respectively, from 338 to 426 and from 277 to 426 residues. As result of insertion of DNA fragments with a sequence encoding polypeptide 55.1 or 55.3 into the structure of expressing vector and transformation suitable host-cells by this vector recombinant form of indicated polypeptides have been prepared. Using this invention provides the possibility for modulating the function of intact p-55 of TNF-receptor. Invention can be used in medicine in treatment of diseases associated with transfer of TNF-signal.

EFFECT: improved preparing method and valuable properties of polypeptide.

9 cl, 17 dwg, 3 tbl, 6 ex

FIELD: biotechnology, molecular biology, medicine.

SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.

EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.

23 cl, 71 dwg, 12 tbl, 17 ex

FIELD: gene engineering.

SUBSTANCE: the present innovation deals with transferring a mutant gene due to homologous recombination into animal embryo. The animal obtained is characterized by the capacity to express mutant protein of presenylin-1 and induction of beta-myeloid protein production that leads to the development of progressing nervous disease in hippocampus or peripheral department of cerebral cortex, It is, also, suggested to apply several plasmids carrying a mutated gene. It is, also, described the way to obtain primary cell culture or subcultivated cell out of obtained mutated animals. Moreover, several methods are, also, suggested for testing the substances for usefulness in therapeutic and/or prophylactic procedures at treating Alzheimer's disease. They deal with introducing a tested substance for mutated animal to evaluate the data obtained. The obtained mutated animals could be applied as model animals while studying Alzheimer's disease nature.

EFFECT: higher efficiency.

25 cl, 8 dwg, 10 ex

FIELD: genetic engineering, pharmaceutical and medical-biological industry.

SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.

EFFECT: improved preparing method, valuable biological properties of polypeptide.

23 cl, 67 dwg, 1 tbl, 35 ex

FIELD: medicine, genetic engineering.

SUBSTANCE: invention proposes a method that involves construction of bacteriophage library of random peptides based on oligonuleotide fragments encoding their, selection of bacteriophages binding with target-cells but not binding with cells of other types that can be involves in pathological process or able to show effect on its diagnosis and therapy, and confirmation of specificity of selected bacteriophages by using combination of different tests. Oligonucleotide fragments encoding random peptides are prepared by reaction of reverse transcription by using random primers and total RNAs isolated from indicated target-cells and cells of other types. Applying this invention provides preparing bacteriophages binding with target-cells with high degree of selectivity. Invention can be used in diagnosis, therapy and pharmaceutical industry.

EFFECT: improved preparing method.

3 cl, 2 dwg, 8 ex

FIELD: biotechnology, genetic engineering, immunology.

SUBSTANCE: invention proposes: isolated nucleic acid encoding feline ligand CD86; diagnostic oligonucleotide; cloning vector; vaccine for modulation of the immune response in cat; method for induction, enhancement and suppression of immune response in cats. Proposed group of inventions allows designing effective vaccines used in prophylaxis of immunodeficiency in felines and infectious peritonitis in domestic cats. Invention can be used in veterinary science.

EFFECT: valuable properties of nucleic acid.

27 cl, 13 dwg, 5 tbl, 8 ex

FIELD: biology, genetic engineering, biotechnology, medicine.

SUBSTANCE: invention relates to preparing glycosylated polypeptide (glycoprotein) as a component of human erythropoietin by using the technology of recombinant DNAs. This polypeptide shows ability to increase production of reticulocytes and erythrocytes, to enhance the level of hemoglobin synthesis and consumption of iron by marrow cells and characterized by the higher molecular mass as compared erythropoietin isolated from human urine. Invention describes variants DNA sequences encoding this polypeptide that comprise vector constructions with these sequences, a method for preparing transformed mammalian cell lines producing the recombinant human erythropoietin, and a method for its preparing and purification. Also, invention proposes pharmaceutical compositions comprising glycosylated polypeptide (glycoprotein) of erythropoietin as an active component. Applying this invention provides scaling the process for preparing active human erythropoietin useful for its using in medicine.

EFFECT: improved preparing method, valuable properties of polypeptide.

10 cl, 4 dwg, 21 tbl, 12 ex

FIELD: biotechnology, medicine.

SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.

EFFECT: valuable medicinal properties of allergens.

33 cl, 62 dwg, 10 ex

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