Fused protein or peptide with increased half lifetime in vivo, which is maintained due to slow release in vivo, and method for increasing half lifetime in vivo using it

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

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The technical field to which the invention relates.

The present invention relates to fused protein or peptide, which has an increased half-life in vivo, and to a method of increasing the time half-life in vivo protein or peptide with its use.

The level of technology

Protein and peptide drugs have excellent therapeutic effects in the case where it is not possible to treat using conventional synthetic chemical medicines, and therefore occupy an important position in medicine and pharmacology. For example, recombinant human growth hormone (hGH) is the only effective therapeutic tool for the treatment of growth hormone deficiency, and recombinant human erythropoietin (EPO) is used in the treatment of anemia resulting from chronic kidney disease, due to its ability to increase the level of red blood cells, and recombinant granulocyte colony-stimulating hormone (G-CSF) is used as the only drug to increase white blood cell count in patients with cancer after chemotherapy. In addition, detected in the body different types of cytokines, hormones and peptides are used as the sole therapy for a wide range of diseases for which there are no other is available at the present time alternatives.

Although protein or peptide drugs exhibit excellent therapeutic effects in vivo, they quickly lose their therapeutic activity and, thus, they have a short half-life periods in vivo, because immediately after the injection is their degradation under the action of proteinases of the blood or they are easily removed from the blood by the kidneys or liver. Therefore, their disadvantage is that they require frequent injections to maintain constant level in the blood or their permanent title. Patients less willing to agree to the admission medication entered using frequent injections, fear of injections and their pain or anxiety caused by repeated injections, if they are used over a long period.

Constantly doing a lot of research in order to increase the stability of protein and peptide drugs in the blood and to maintain the level of drug in the blood over a long period of time.

For example, were developed dosage forms with delayed release of drugs by creating compositions therapeutically active protein or peptide with a biodegradable polymer, which allows proteins or peptides slowly released from the injection site. If drug sredstvo slow release is injected with subcutaneous or intramuscular injection, the drug is slowly released to maintain a constant level of the drug over a certain period of time (M. Chasin & R. Langer, et al., Biodegradable polymer as drug delivery system. Marcel Dekker (1990); J.Heller, et al., Adv. Drug Del Rev., 10, 163 (1993)). Among the biodegradable polymers, are widely used PLGA (copolymer of lactic and glycolic acids). For example, was established dosage form with delayed release of the peptide agonist LHRH (luteinizing hormone releasing factor), and it was found that this product delivers the peptide in vivo within one or three months. Use of biodegradable polymers and together with high molecular weight proteins. For example, U.S. patent No..6,500,448 discloses a pharmaceutical composition for sustained release of human growth hormone, which comprises a biocompatible polymer and particles of human growth hormone in complex with metal cations. In another study in Korea patent No..10-0236771 and 10-0329336 described the use of hyaluronic acid to create microparticles with a slow release of protein drugs, including recombinant human growth hormone.

Although for slow release of drugs successfully used biodegradable polymers for discobolus what's peptides there are restrictions on their use for macromolecular proteins. The reason for this is that proteins are easily denatured during production of microparticles for sustained release and denatured amino acids reduce the activity of the protein, which may cause some unwanted immune responses in humans. In addition, the size of microparticles for sustained release of proteins or peptides is usually large, which requires the use of thick needles for syringe when introduced by injection into the body and causes pain at the injection site. Also, microparticles are disadvantageous from the point of view of the economy due to low outputs in the production of goods for commercial purposes.

In order to overcome the above problems, studies have been conducted aimed at slowing renal clearance of proteins or peptides. In General, proteins with a molecular mass equal to 60,000 daltons or less, pass through the kidneys without delay. Therefore, attempts have been made to increase low molecular weight peptide or protein therapeutic agents, in order to prolong the circulation time in the blood t vivo, thus reducing the frequency of injections. In accordance with these techniques, physiologically active proteins and peptides provide in the form of decreasing the major release, but rather in the form of a long-term effect.

One of the most popular strategies used to reduce the frequency of injections is joining soluble polymer, such as polyethylene glycol (hereinafter in this application is designated as "PEG") to the surface of pharmaceutically active proteins or peptides. PEG can be nonspecific attached to the amino group of the amino acids of proteins or peptides. Pegylation can provide solubility for hydrophobic drugs and proteins and increases the hydrodynamic size of the tool to extend circulation time in the blood after administration by injection into the body (Sada et al., J. Ferment Bioeng 71, 137-139, 1991).

Recently, there was a batch issue Paglinawan interferon-alpha to reduce the frequency of injections. In addition, Kinstler et al. showed that a single injection Paglinawan granulocyte colony-stimulating factor (G-CSF) per week (one cycle of chemotherapy) has such curative action, what produce injection of G-CSF, held three times a week (Kinstler et al., Pharm Res 12, 1883-1888, 1995). PEG-GCSF available for purchase under the trade name "Neulast".

Since Pegylation of the protein occurs as a result of non-specific covalent attach the PEG to the surface of the protein, in Paglinawan plot can be don is but the interaction of the protein with its receptor, that significantly reduces the activity of the protein in vivo. In addition, Pegylation is a labor-intensive procedure as proteins, Paglierani on physiologically active phase, due to be removed in the cleaning process, so that only such conjugates PEG-protein, whose activity would be reduced minimally. Therefore, in this process the yield of the desired conjugates of PEG-protein significantly reduced, which leads to adverse from the point of view of the economic situation. In addition, in the case of some proteins, which are unstable in aqueous solutions, attempts to konjugierte them with PEG failed.

In addition, the frequency of injections reduce using glycoengineering techniques currently used in commercial production. Elliot et al. reported additional glycosylation of erythropoietin (EPO) by substitution of amino acids in certain positions {Nat Biotechnol 21, 414-421, 2003; U.S. patent No..7,217,689). Erythropoietin modified glycoengineering methods currently available for purchase under the trade name "Aranesp", and it is known that by adding chains of Sugars with sialic acid at the end and increase the molecular weight slows down the circulation in the blood flow, metabolism and excretion of the modified erythropoietin. However, glycoengineering not use lsout widely for the introduction of additional glycosylation sites of proteins, since joining or adding chains of Sugars can lead to inactivation of the physiologically active protein, and its ability to maintain the stability of the t vivo has not been confirmed for many proteins. And the fabric physiologically active protein, which can be optionally attached chains of Sugars, is very small. In addition, glycoengineering difficult to apply in the case of peptides with low molecular weight.

The development of genetic engineering has allowed to increase the size of the physiologically active protein by merging it with the high molecular weight protein (Curr Opin Drug Discov Devel 12, 284-295, 2009). For example, gene physiologically active protein is drained from the genome of albumin human, and then Express in yeast cells to obtain the fused protein (international patent publication nos WO 93/15199 and WO 93/15200). Examples of physiologically active proteins, fused to albumin, include granulocyte colony-stimulating factor (Halpem et al., Pharm Res 19, 1720-1729, 2002), human growth hormone (Osborn et al., Eur J Pharmacol 456, 149-158, 2002), glucagon-like peptide-1 (Baggio et al., Diabetes 53, 2492-2500, 2004), and interferon-alpha (Osbom et al., J Pharmacol Exp Ther 303, 540-548, 2002).

Known also fused protein with transferrin obtained using the techniques of recombinant fusion. For example, U.S. patent No. 7,176,278 discloses fused molecule, in which the glucagon-like peptide-1 fused with natural what ransferring or deglycosylated by transferrin, and the time of its half-life in vivo is increased.

While the half-life of the protein in vivo may be extended by fusion with the Fc fragment of immunoglobulin (Ig) (U.S. patent No. 5,116,964 and No. 5,605,690). Fused gene fragment of the receptor of TNF-α and fragment of IgG1 Fc Express in animal cells (the cells of the Chinese hamster ovary, Cho), transformed with a gene coding for a protein, and protein is currently available for purchase (trade name: Enbrel) after approval by the USFDA as a therapeutic agent for the treatment of rheumatoid arthritis. In addition, Wang managed (Qinghua Wang; WO 2007/012188) to extend the half-life in vivo GLP-1 (t1/2<2 min) or basis-4 with a short half-life by merging with the Fc fragment of Ig.

Despite the fact that Ig Fc is widely used as a carrier for a fused protein with the aim of increasing the time half-life in vivo, IgG1 Fc retains its own antibody-dependent cellular cytotoxicity (ADCC) or complementation cytotoxicity (CDC). Thus, when introduced into the body by injection, protein physiologically active protein IgG1 Fc can cause complex immune responses. In addition, repeated administration of Fc-fused proteins over a long period of time can lead to the production of unwanted antibodies. Consequently, the use of proteins fused to IgG1 Fc has ogran is an increase in the application in medical practice.

The Korea patent No. 10-0725315 reveals protein complex with the use of a fragment of an immunoglobulin and a method thereof, in which the physiologically active protein is drained with IgG Fc through the PEG. "Protein complex", with the structure of the physiologically active protein-PEG-Fc, has a longer half-life in vivo compared to the physiologically active protein, according to the pharmacokinetic analyses. However, similar problems or issues, it is shown for a method of fusion with the Fc, you can also watch for "protein complex", because physiologically active protein and the Fc-fragment chemically bound PEG molecule.

Another example of the use of immunoglobulin for increasing the stability of peptide drugs in vivo is a fusion of the whole molecule IgG antibodies and low molecular weight peptide (Rader et al, Proc. Natl. Acad. Sci. USA 100, 5396-5400, 2003, Doppalapudi et al., Bioorg &Med Chew 17, 501-506, 2007). However, this technique, called "CovX-Body"cannot be applied to high molecular weight proteins, and its use is limited due to problems in obtaining Fc-fused proteins or PEG-fused proteins.

As described above, many attempts were made to merge biopolymer with a physiologically active protein or therapeutic peptide, but they can be applied only to a limited number of proteins is whether peptides for the following reasons: the existence in vivo is not long enough to develop a fused protein for medical purposes; very low output upon receipt, make the production economically unviable; unwanted immune responses when used for a long time; and unwanted residual presence of toxic chemical derivatives, when used for conjugation with proteins or peptides. Therefore, there is a need for new fused proteins or peptides that can prolong the half-life in vivo of physiologically active proteins or peptides, with minimal loss of activity in vivo.

Disclosure of inventions

Technical task

Leading to the present invention, intensive and thorough research of the fused proteins or peptides which have a longer half-life of t vivo and minimal loss of activity in vivo, conducted by the authors of the present invention, led to the discovery that alpha-1-antitripsin or option allow merged with it physiologically active protein or peptide to maintain a stable circulation in vivo and, thus, increase stability in vivo and the half-life in vivo (T1/2), compared to the private properties of the protein or peptide.

Technical solution

The present invention provides a protein or peptide with increased half-life in vivo with maintaining its stable circulation, and increase the straps half-life in vivo protein or peptide using the same.

Description of the drawings

Figure 1 is a graph showing the pharmacokinetic behavior of fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH]HGH/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH].

Figure 2 is a graph showing the pharmacokinetic behavior of fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α].

Figure 3 is a graph showing the pharmacokinetic behavior of fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF].

Figure 4 is a graph showing the pharmacokinetic behavior of the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: Basis-4/α1AT(P357N)].

Figure 5 is a graph showing in vivo activity (change of body weight in rats with remote pituitary) fused protein monovariant of human growth hormone/alpha-1-antitripsin [T: α1AT(P357N)/hGH].

6 is a graph showing in vivo activity (increase in the number of leukocytes) fused protein of granulate the ary colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/ trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF].

Fig.7 is a graph showing the results of intraperitoneal test glucose tolerance, conducted with the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: Basis-4/α1AT(P357N)].

Fig is a graph showing the influence of the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: Basis-4/α1AT(RN)] on the blood sugar levels in models of diabetes in mice.

Fig.9 is a graph showing the intracellular activity of the fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], fused protein of human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and fused protein of human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH].

Figure 10 is a graph showing the intracellular activity of the fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF].

11 is a graph showing the inhibitory activity of the fused protein human growth hormone/alpha-1-antitripsin [T109t: α1AT/hGH] and fused protein of human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] against trypsin.

Fig is a graph showing the inhibitory activity of the fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and fused protein of human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] against elastase human neutrophils.

Fig is a pictures showing the band fused protein human growth hormone/alpha-1-antitrypsin [T109wt: α1AT/hGH] and fused protein of human growth hormone/monovariant alpha-1-antitrypsin [T 109: α1AT(P357N)/hGH] and fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] after electrophoresis in SDS-polyacrylamide gel.

The best way of carrying out the invention

In accordance with an aspect of the present invention provides a protein or peptide with increased half-life in vivo, in which the physiologically active protein or peptide fused with alpha-1-antitripsin, resulting in a physiologically active protein or peptide may long to circulate in vivo.

In accordance with another aspect of the present invention provides a protein or peptide with increased half-life in vivo, in which the physiologically active protein or peptide fused with the variant alpha-1-antitrypsin, mutated at one or more amino acid residues, resulting physiologist who Cesky active protein or peptide may long to circulate in vivo.

In accordance with a further aspect of the present invention provide a way longer half-life in vivo in a physiologically active protein or peptide, including the merger of the physiologically active protein or peptide with alpha-1-antitripsin, or a variant of alpha-1-antitrypsin, having one or more mutated amino acids, resulting in a physiologically active protein or peptide may long to circulate in vivo.

The following is an implementation of the present invention.

Protein or peptide in accordance with the present invention is an example of the application of alpha-1-antitrypsin or variant alpha-1-antitrypsin to maintain a stable circulation of the physiologically active protein or peptide to increase the half-life in vivo by merging with him physiologically active protein or peptide.

As used in this application, the term "fused protein/fused polypeptide" means a new protein molecule, in which the physiologically active protein with high molecular weight fused to the N - or C-end of the alpha-1-antitrypsin or variant of alpha-1-antitrypsin deficiency. Similarly, the term "fusion peptide", as used in this application means a new peptide molecule, in which the physiologically active peptide, low molecular weight fused to the N - or C-to the CMA alpha-1-antitrypsin or variant of alpha-1-antitrypsin deficiency.

Physiologically active protein or peptide can be fused directly or through linker consisting of the amino acids, alpha-1-antitripsin or variant of alpha-1-antitrypsin, mutated at one or more amino acids.

Preferably, to merge the physiologically active protein or peptide with alpha-1-antitripsin or variant of alpha-1-antitrypsin, mutated at one or more amino acids, use the methods of genetic recombination. Alternatively, to merge the physiologically active protein or peptide with N - or C-end or a free group of alpha-1-antitrypsin or variant alpha-1-antitrypsin, mutated at one or more amino acids, can be applied linker, well known in this technical field.

Among the physiologically active proteins include hormones and their receptors, biological response modifiers and their receptors, cytokines and their receptors, enzymes, antibodies and antibody fragments. Specific examples of physiologically active proteins include human growth hormone (hGH), insulin, follicle-stimulating hormone (FSH), human chorionic gonadotropin, parathyroid hormone (PTH), erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony-stimulating factor(CSF), granulocyte-macrofamily colony-stimulating factor (GM-CSF), interfer the h-alpha, interferon-beta, interferon gamma, interleukins, macrophage activating factor, tumor necrosis factor, tissue plasminogen activator, coagulation factor VII, VIIa, VIII and IX, bone morphogenetic protein 2 (hBMP2), keratinocyte growth factor (KGF), platelet growth factor (PDGF), glucocerebrosidase, α-galactosidase And α-L-iduronidase, iduronate-2-sulfatase, lactase, adelaideans, butyrylcholinesterase, chitinase, glutamatdekarboksilazy, imiglucerase, lipase, uricase, acetylhydrolase of platelet activating factor, neutral endopeptidase, urokinase, streptokinase, myeloperoxidase, superoxide dismutase, botulinum toxin, collagenase, hyaluronidase, L-asparaginase, monoclonal antibodies, polyclonal antibodies, scFv, Fab, Fab', F(ab')2and Fd, but not limited to.

Examples of the physiologically active peptide include glucagon-like peptide-1 (GLP-1) and its analogues, basis and its analogues, somatostatin and its analogues, agonist and antagonist hormone releasing luteinizing hormone (LHRH), adrenocorticotropic hormone, a hormone that stimulates growth hormone, oxytocin, thymosin alpha-1, corticotropin-releasing factor, calcitonin, bivalirudin, vasopressin analogues and fragments of physiologically active proteins, but not limited to.

Alpha-1-antitripsin is a whey baie is OK mammals, approximately 50000 daltons, which is present in the blood in high concentrations, about 2 mg/ml (Robin W.C. et al., Nature 298, 329-334, 1982). Alpha-1-antitripsin also called inhibitor alpha-1-protease because it inhibits a wide range of proteases. However, in relation to known diseases, its main function is to protect the lung tissue from neutrophil elastase (Beatty et al., J Biol Chem 255, 3931-3934, 1980). In the absence of alpha-1-antitrypsin and neutrophil elastase freely destroys elastin, which contributes to the elasticity of the lungs that leads to breathing difficulties, such as emphysema. Disorders associated with protein include such a genetic condition as a deficiency of alpha-1-antitrypsin deficiency. Alpha-1-antitripsin, extracted from the serum, available for purchase as a therapeutic agent for the treatment of emphysema under the trade name "Prolastin"because it was approved by the FDA. The stability and security of Prolastin has been confirmed, and it is administered as an intravenous injection at a dose of 60 mg/kg per week. In addition, it is known that alpha-1-antitripsin itself has a half-life in vivo, approximately 5-6 days (Weweres, MD, et al., N Engl J Med 316, 1055-1062, 1987). This provides theoretical basis, according to which alpha-1-antitripsin, which is safe for the body, even if it is administered in large to the number, can be used to extend the half-life in vivo physiologically active protein or peptide by merging his and alpha-1-antitrypsin deficiency among themselves. The structure of alpha-1-antitrypsin and its role as a protease inhibitor is well known (Elliott, P. et al., JMB 275, 419-425, 1998). PI amino acid residue (position 358 from N-end) in alpha-1-antitripsin is a methionine residue critical for binding of elastase. It is also known that the protein inhibits a wide range of proteases, including trypsin, chymotrypsin, thrombin and elastase. Gene alpha-1-antitrypsin highly pleomorphic, with more than 100 identified to date, alleles, phenotypes installed using IEF (isoelectric focusing) and assigned a letter code (a to Z) (Stoller et al., The Lancet, 365, 2225-2236, 2005). The family of M-alleles, the most common among alleles, denoted as M, is further subdivided into subtypes, such as M1 (Val213), M1 (Ala213), M2 and M3, in accordance with mutations of amino acids in the sequence. Therefore, alpha-1-antitripsin, used in the present invention, is a specific subtype, belonging to the family of M-alleles, apply and also other subtypes with the same effect.

A variant of alpha-1-antitrypsin can be obtained using site-directed mutagenesis of one or a number of the x amino acids. For example, the variant alpha-1-antitrypsin deficiency may have an asparagine at position 357 in P2 instead of Proline. In addition to the substitution at the P2 Proline at the asparagine at position 357, a variant of alpha-1-antitrypsin deficiency may have one or more other mutant amino acids in other positions. In detail, the variant alpha-1-antitrypsin deficiency may have an asparagine instead of Proline at position 357 and, optionally, a threonine instead of serine at position 359 and/or serine instead of cysteine at position 232. A variant of alpha-1-antitrypsin used in the present invention may be selected from monovariant alpha-1-antitrypsin [α1AT(P357N)], divariant alpha-1-antitrypsin [α1AT(P357N, S359T)], divariant alpha-1-antitrypsin deficiency 2 [α1AT(P357N, C232S)] and trivariant alpha-1-antitrypsin [α1AT(P357N, C232S, S359T)].

Monovariant alpha-1-antitrypsin [α1AT(P357N)] get with the substitution of Proline (Pro) the asparagine (Asn) at position 357 in P2 with N-end. This variant of alpha-1-antitrypsin deficiency is characterized by the formation of the new site of N-glycosylation of Asn-X-Ser, which contributes to the neutralization of the inhibitory activity of alpha-1-antitrypsin as an inhibitor of proteases, and also to minimize the immunogenicity arising after the injection due to the replacement of amino acids.

Divariant alpha-1-antitrypsin [α1AT(P357N, S359T)] results in the substitution of Proline by aspartic acid in position 357 in P2 and serine threonine in position at variant of alpha-1-antitrypsin deficiency is characterized by the formation of the new site of N-glycosylation of Asn-X-Thr, which contributes to the neutralization of the inhibitory activity of alpha-1-antitrypsin as an inhibitor of proteases and minimize immunogenicity arising after the injection due to the replacement of amino acids

Divariant alpha-1-antitrypsin deficiency 2 [α1AT(P357N, C232S)] results in the substitution of Proline by aspartic acid in position 357 in P2 and cysteine by serine at position 232. This divariant alpha-1-antitrypsin 2 is characterized by the formation of the new site of N-glycosylation of Asn-X-Ser, which contributes to the neutralization of the inhibitory activity of alpha-1-antitrypsin as an inhibitor of proteases, minimize immunogenicity arising after the injection due to the replacement of amino acids, and additionally prevents dimer formation mediated by free cysteine.

Resulting from the substitution of Proline by aspartic acid in position 357 in P2, cysteine by serine at position 232 and serine threonine at position 359, trivariant alpha-1-antitrypsin [α1AT(P357N, C232S, S359T)] is characterized by the formation of the new site of N-glycosylation of Asn-X-Thr, contributes to the neutralization of the inhibitory activity of alpha-1-antitrypsin as an inhibitor of proteases, minimize immunogenicity arising after the injection due to the replacement of amino acids, and additionally prevents dimer formation mediated by free cysteine.

Among the fused proteins or peptides in the present invention can be called the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] (SEQ ID NO:1), the human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] (SEQ ID NO:2), human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] (SEQ ID NO:3), interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α] (SEQ ID NO:4), granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] (SEQ ID NO:5), granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] (SEQ ID NO:6) and on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)] (SEQ ID NO:7).

All fused proteins or peptides described in this application, the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH]HGH/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH]HGH/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH], interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α], granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF], granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] and on the basis 4/monovariant alpha-1-antitrypsin [T: the basis-4/α1AT(P357N], significantly increases the half-life in serum (t1/2), and he shows excellent stability in vivo, compared to the physiologically active protein or Pat the house itself.

With the introduction by injecting rats with a remote pituitary gland, it was found that the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] induces weight gain in animals. After injection fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] or fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] in rats increases the level of white blood cells. The group, which is injected protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)], showed a greater reduction in blood sugar levels, compared with teams that enter exendin-4, and this low level of sugar in the blood remained, at least within 24 hours after injection. Therefore, fused proteins or peptides in accordance with the present invention retain activity in vivo over an extended period of time.

In addition, fused proteins or peptides, human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH]HGH/monovariant alpha-1-antitrypsin [T 109: α1AT(P357N)/hGH]HGH/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH], granulocitary colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and granulocitary colony is stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF], have similar intracellular activity (EC50and, therefore, their activity is slightly differ depending on media type, ie, alpha-1-antitrypsin and variants of alpha-1-antitrypsin deficiency.

In addition, the protein of the present invention, the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], shows excellent inhibitory activity against trypsin and elastase human neutrophils, whereas the protein human growth hormone/monovariant alpha-1-antitrypsin [T 109: α1AT(P357N)/hGH], has a very low inhibitory activity against trypsin and elastase of human neutrophils. Therefore, the fact that the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] increase the half-life in vivo through sustainable circulation, not dependent on the intrinsic properties of alpha-1-antitrypsin deficiency.

As described above, fused proteins or peptides in accordance with the present invention increase the half-life in serum (T1/2through sustainable circulation and, thus, have a high stability in vivo, compared to actually physiologically active proteins or peptides. Therefore, fused proteins or peptides of the present invention can the be applied for development of dosage forms of the protein or peptide drugs with stable circulation.

The implementation of the invention

A better understanding of the present invention can be achieved by the following examples which are given for illustration, but should not be construed as limiting the present invention.

Example 1. Getting fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH]

1. Construction of expression vector, pSNAT

For the expression of human growth hormone fused with the C-end of the alpha-1-antitrypsin, construct the expression vector pSNAT bearing alpha-1-antitripsin. In detail, the gene for alpha-1-antitrypsin is obtained from the vector hMU001448 (KRIBB) using PCR, using a pair of primers ALT21 (SEQ ID NO:8) and ALT30 (SEQ ID NO:9), which were developed to merge human growth hormone with the end of alpha-1-antitrypsin deficiency. When developing in primer ALT30 was also included by the linker, which can create the mobility necessary to stabilize the fused proteins. Amplificatoare nucleotides cleaved in the presence of two restricts XhoI and BamHI and clone in pSGHVO (access code in the Bank of genes AF285183), resulting in the recombinant vector, named pSNAT.

2. The design vector for the human growth hormone/alpha-1-antitripsin [T109wt, α1AT/hGH]

The gene of human growth hormone (hGH) amplified from the vector IOH45734 ("Invitrogen) by PCR using a pair of primers DH22 (SEQ ID NO:10) and ALT12 (SEQ ID NO:11). The PCR product obtained t is thus, break down in the presence of two restricts BamHI and NotI, and clone in pSNAT by the same restriction site BamHI/NotI to create a recombinant expression vector, named T109wt (SEQ ID NO:1).

3. The expression of the fused protein human growth hormone/alpha-1-antitripsin (T109wt) protein of the human growth hormone/alpha-1-antitripsin (T109wt), obtained as described above in paragraphs 1-2, Express in the cells of the Chinese hamster ovary (Cho-K1). Cho-K1 support in DMEM (Wednesday Needle in the modification of Dulbecco), supplemented with 10% FBS (fetal bovine serum) and antibiotics at 37°C under 5%CO2. One day before the introduction of a gene human growth hormone/alpha-1-antitripsin (T109wt), cells inoculant in quantities of 1×106cells in 100 mm Cup for cultivation. To 800 μl of DMEM medium, free from FBS and antibiotics, add 5 µg fused protein of human growth hormone/alpha-1-antitripsin (T109wt), and the mixture is incubated at room temperature for 1 min, mixed with 20 μg PEI (polyethylenimine, linear, "Polysciences Inc. (catalog No.: 23966, MW ~25000)) and leave at room temperature for 10~15 minutes At this time, cells, inkubirovaniya in one day, washed with PBS and add 6 ml of fresh DMEM. Protein of human growth hormone/alpha-1-antitripsin (T109wt), left on for 10~15 min at room temperature, add in a Cup of coltivirus the deposits. The next day, cells are washed with PBS and added to them free from FBS IMDM medium (catalog No. 12200-028, "Gibco". environment Dulbecco, modified by the method of Claims) to identify the expression of the protein.

4. Purification of fused protein human growth hormone/alpha-1-antitripsin (T109wt)

Once in the cells of the Chinese hamster ovary (SNOOK) conducted an expression, as described above in paragraphs 1 to 3, protein T109wt cleaned as follows. In detail, since the protein human growth hormone/alpha-1-antitripsin (T109wt), is secreted into the medium, cell culture is centrifuged so that it was possible to collect the supernatant. This supernatant balance buffer solution (20 mm sodium phosphate, pH 8.0), applied to a column of Q-separate (GE Healthcare, USA), pre-equilibrated with a solution of equilibration buffer, and thoroughly washed with a solution of equilibration buffer, then elute in an increasing gradient of NaCl concentration (0~400 mm NaCl, 20 mm sodium phosphate, pH 8.0). The protein eluate is mixed with salt, are balanced on a column of phenyl-separate (GE Healthcare, U.S.A), and washed with a sufficient quantity of a solution of equilibration buffer, then elute in panyhose the concentration gradient of NaCl (2~0 M NaCl, 20 mm sodium phosphate, pH 6.8). Protein concentrate fraction using devices Vivaspin20" (GE Healthcare, USA) and get Vysokoe ewenny T109wt.

Example 2. Getting fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH]

1. Getting monovariant alpha-1-antitrypsin (vector for cloning pDHT3N)

Because the alpha-1-antitripsin, used as a media merger, the merged molecule possesses inhibitory activity inhibitory activity of alpha-1-antitrypsin deficiency significantly reduced, creating a variant of alpha-1-antitrypsin deficiency. In this regard, the gene for alpha-1-antitrypsin amplified from the vector hMU001448 (KRIBB) using PCR and clone in the vector ut&And to obtain a recombinant vector pDHT3. After this, carry out the replacement of the Proline residue at position 357 in P2 on the asparagine N-glycosylation, using a pair of primers ALT1 (SEQ ID NO:12) and ALT2 (SEQ ID NO:13) using a kit for carrying out mutagenesis (Stratagene, QuikChange II, No. 200523-5) and get the vector for cloning pDHT3N.

2. Construction of expression vector pSNATN

For the expression of human growth hormone fused with the end of inactivated alpha-1-antitrypsin, construct the expression vector pSNATN, bearing monovariant alpha-1-antitrypsin deficiency. In detail, the gene of monovariant alpha-1-antitrypsin is obtained from the vector pDHT3N using PCR, using a pair of primers ALT 14 (SEQ ID NO:14) and ALT30 (SEQ ID NO:9), clone in pSNAT cleaved in the presence of two restricts EcoRV and BamHI, and receive recombinant the initial vector, named pSNATN.

3. The design vector for the human growth hormone/monovariant alpha-1-antitrypsin [T, α1AT(P357N)/hGH]

The gene of human growth hormone, amplificatory in example 1-2 is inserted into the pSNATN the restriction site BamHI/NotI and receive recombinant expression vector, named T 10 (SEQ ID NO:2).

4. The expression of the fused protein human growth hormone/monovariant alpha-1-antitrypsin (T)

The expression of the fused protein of human growth hormone/monovariant alpha-1-antitrypsin (T) in the cells of the Chinese hamster ovary (Cho-K1) identify, using the same procedure as in example 1-3.

5. Purification of fused protein human growth hormone/monovariant alpha-1-antitrypsin (T)

Protein the human growth hormone/monovariant alpha-1-antitrypsin (T) is treated in the same manner as in example 1-4.

Example 3. Getting fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH]

1. Getting divariant alpha-1-antitrypsin (vector for cloning pDHT3NT)

To obtain inactivated alpha-1-antitrypsin by the introduction of a glycosylation site in the active center of the molecule, alpha-1-antitripsin mutate twice to reduce its activity and to obtain a homogeneous glycosylation. In this connection, carry out the replacement of the serine residue at position 359 on threonine for uniformity Glyco is helirovanie for inducing N-glycosylation pDHT3N vector for cloning, the carrier monovariant alpha-1-antitrypsin activity is reduced by mutation engine Proline at position 357 in P2 on asparagine, using a pair of primers ALT82 (SEQ ID NO:15) and ALT83 (SEQ ID NO:16) using a kit for carrying out mutagenesis (""Enzynomics"", EZchange catalog No. EM) and get the vector for cloning named pDHT3NT.

2. Construction of expression vector pSNATNT

For the expression of human growth hormone fused with the C-end of divariant alpha-1-antitrypsin, with homogeneous glycosylation and reduced activity, construct the expression vector pSNATNT. In this regard, gene divariant alpha-1-antitrypsin amplified from pDHT3NT using PCR. using a pair of primers ALT14 (SEQ ID NO:14) and ALT30 (SEQ ID NO:9), designed to merge human growth hormone with the end of divariant alpha-1-antitrypsin, and clone in pSNAT, pre-treated in the presence of two restricts EcoRV and BamHI, and receive recombinant expression vector, named pSNATNT.

3. Construction of a vector carrying the gene of human growth hormone/divariant alpha-1-antitrypsin [TT, α1AT(P357N, S359T)/hGH]

The nucleotide of human growth hormone, amplificatory in example 1-2, clone in pSNATNT the restriction site BamHI/NotI and get an expression vector, named TT (SEQ ID NO:3).

4. The expression of the fused protein of human growth hormone/divariant alpha-1-antitrypsin (TT)

The expression of the fused protein of human growth hormone/divariant alpha-1-antitrypsin (TT), in the cells of the Chinese hamster ovary (Cho-K1) identify in the same manner as in example 1-3.

5. Purification of fused protein of human growth hormone/divariant alpha-1-antitrypsin (TT)

Protein of human growth hormone/divariant alpha-1-antitrypsin (TT), cleaned in the same manner as in example 1-4.

Example 4. Getting fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α]

1. Construction of vector for interferon-alpha man/monovariant alpha-1-antitrypsin [T, α1AT(P357N)/IFN-α]

Gene interferon-alpha man (IFN-α) amplified from the vector MHS1010-98051913 ("Open biosystems") by PCR using a pair of primers ALT45 (SEQ ID NO:17) and ALT49 (SEQ ID NO:18). The PCR product thus obtained, split in two places in the presence of BamHI and NotI and then clone in pSNATN the restriction site BamHI/NotI and receive recombinant expression vector, named T (SEQ ID NO:4).

2. The expression of the fused protein interferon-alpha man/monovariant alpha-1-antitrypsin (T)

The expression of the fused protein interferon-alpha man/monovariant alpha-1-antitrypsin (T) in the cells of the Chinese hamster ovary (Cho-K1), identify in the same manner as in example 1-3.

3. Purification of fused protein interferon-alpha is the ne/monovariant alpha-1-antitrypsin (T)

Protein interferon-alpha man/monovariant alpha-1-antitrypsin (T), cleaned in the same manner as in example 1-4.

Example 5. Getting fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitripsin [T602S: α1AT(P357N, C232S)/G-CSF]

1. Getting divariant 2 alpha-1-antitrypsin (vector for cloning pDHT3NS)

For use in preparation of the protein or peptide therapeutic agents, divariant 2 alpha-1-antitrypsin get through mutation of alpha-1-antitrypsin to reduce the inherent activity of alpha-1-antitrypsin deficiency and eliminate the possibility of protein denaturation, resulting in the formation of dimers mediated by cysteine. In this context, the mutation resulting in the substitution of the cysteine residue at position 232 to threonine, hold on inducing N-glycosylation vector for cloning pDHT3N, which is monovariant alpha-1-antitrypsin deficiency with reduced by mutation of the Proline at position 357 in P2 on asparagine activity, using a pair of primers ALT52 (SEQ ID NO:19) and ALT53 (SEQ ID NO:20) using a kit for carrying out mutagenesis (Stratagene, QuikChange II No. 200523-5), and get the vector for cloning named pDHT3NS.

2. Construction of expression vector pSNATNS

For the expression of granulocyte colony-stimulating factor, merged with the end of divariant 2 alpha-1-antitripsin is, which has a reduced inhibitory activity, construct the expression vector pSNATNS. In this regard, gene divariant 2 alpha-1-antitrypsin amplified from pDHT3NS using PCR, using a pair of primers ALT14 (SEQ ID NO:14) and ALT30 (SEQ ID NO:9), designed to merge granulocyte colony-stimulating factor with the end of divariant 2 alpha-1-antitrypsin, and clone in the vector pSNAT, pre-treated in the presence of two restricts BstEII and BamHI, and receive recombinant expression vector, named pSNATNS.

3. Design vector granulocyte colony-stimulating factor/divariant 2 alpha-1-antitrypsin [T602S, α1AT(P357N, C232S)/G-CSF]

The gene for granulocyte colony-stimulating factor (G-CSF) amplified from IHS 1380-97652343 (Open biosystems) by PCR using a pair of primers ALT56 (SEQ ID NO:21) and ALT57 (SEQ ID NO:22). The PCR product thus obtained, split in two places using BamHI and NotI, clone in pSNATNS the restriction site BamHI/NotI and receive recombinant expression vector, named T602S (SEQ ID NO:5).

4. The expression of the fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin (T602S)

The expression of the fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin (T602S) in the cells of the Chinese hamster ovary (Cho-K1) identify in the same manner as in example 1-3./p>

5. Purification of fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin (T602S)

Protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin (T602S) is treated in the same manner as in example 1-4.

Example 6. Getting fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF]

1. Getting trivariant alpha-1-antitrypsin (vector for cloning PDHT3NST)

For use in preparation of the protein or peptide therapeutic agents, trivariant alpha-1-antitrypsin get through mutation divariant alpha-1-antitrypsin to achieve uniformity of glycosylation. In this regard, using a kit for carrying out mutagenesis ("Enzynomics", EZchange catalog No. EM) in the presence of a pair of primers ALT82 (SEQ ID NO:15) and ALT83 (SEQ ID NO:16), carry out the replacement of the serine residue at position 359 on threonine for homogeneity of glycosylation on inducing N-glycosylation pDHT3NS vector for cloning, the carrier divariant 2 alpha-1-antitrypsin deficiency, in which the inherent activity is reduced in the substitution of Proline at position 357 in P2 for asparagine, and the possibility of protein denaturation associated with mediated the cysteine formation of dimers, are excluded as a result of replacement of cysteine in position 232 to serine.

2 Construction of expression vector pSNATNST

For the expression of granulocyte colony-stimulating factor, merged with the end of trivariant alpha-1-antitrypsin, which has reduced activity, construct the expression vector pSNATNST. In this regard, gene trivariant alpha-1-antitrypsin amplified from pDHT3NST using PCR, using a pair of primers ALT14 (SEQ ID NO:14) and ALT30 (SEQ ID NO:9), designed to merge granulocyte colony-stimulating factor with the end of trivariant alpha-1-antitrypsin, and clone in the vector pSNAT, pre-treated in the presence of two restricts BstEII and BamHI, and receive recombinant expression vector, named pSNATNST.

3. Design vector granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST, α1AT(P357N, C232S, S359T)/G-CSF]

The gene for granulocyte colony-stimulating factor (G-CSF) amplified from the vector IHS1380-97652343 (Open biosystems) by PCR using a pair of primers ALT56 (SEQ ID NO:21) and ALT57 (SEQ ID NO:22). Amplificatoare nucleotides split in two places using BamHI and NotI, clone in pSNATNST the restriction site BamHI/NotI and receive recombinant expression vector, named T602ST (SEQ ID NO:6).

4. The expression of the fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin (T602ST)

The expression of the fused protein granulocyte colony-stimulating factor/trivariant and is LFA-1-antitrypsin (T602ST) in the cells of the Chinese hamster ovary (Cho-K1) identify in the same way, as in example 1-3.

5. Purification of fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin (T602ST)

Protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin (T602ST) is treated in the same manner as in example 1-4.

Example 7. Getting fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: Basis-4/α1AT(P357N)]

1. Construction of expression vector pSCAT

For expression of the basis 4, fused with the N-end of the alpha-1-antitrypsin, design vector pSCAT. In detail, alpha-1-antitripsin N-end which should be merged on the basis of 4, amplified from the vector hMU001448 (KRIBB) using PCR, using a pair of primers ALT21 (SEQ ID NO:8) and ALT5 (SEQ ID NO:23). Amplificatoare nucleotides cleaved in the presence of two restricts XhoI and NotI, clone in pSGHVO (access code in the Bank of genes AF285183) and receive recombinant vector, named pSCAT.

2. Construction of expression vector pSCATN

For expression of the basis 4, fused with the N-end of inactivated monovalent alpha-1-antitrypsin, design pSCATN. In this context, the gene encoding monovariant alpha-1-antitrypsin, N-end on the basis of 4 should be merged, obtained from vector pDHT3N, clone in pSCAT, using two restrictase EcoRV and NotI, and receive recombinant vector, named pSCATN.

3. Getting basis 4

E is sendin-4 gene was amplified using PCR, applying DH15 (sense codon, SEQ ID NO:24) and DH16 (antisense codon, SEQ ID NO:25).

4. Construction of the vector of basis 4/monovariant alpha-1-antitrypsin [T, the basis-4/α1AT(P357N)]

The gene of basis 4 amplified from the gene obtained in example 7-3 using PCR, using a pair of primers ALT44 (SEQ ID NO:26) and ALT41 (SEQ ID NO:27). This amplificatory nucleotide split in two places using Xhol and BamHI, clone in pSCATN the restriction site XhoI/BamHI and get a vector of basis 4/monovariant alpha-1-antitrypsin (T, SEQ ID NO:7).

5. The expression of the fused protein of the basis 4/monovariant alpha-1-antitrypsin (T)

The expression of the fused protein of the basis 4/variant alpha-1-antitrypsin (T), in the cells of the Chinese hamster ovary (Cho-K1) identify in the same manner as in example 1-3.

6. Cleaning the fused protein on the basis 4/monovariant alpha-1-antitrypsin (T) protein of basis 4/monovariant alpha-1-antitrypsin (T), cleaned in the same manner as in example 1-4.

Test example 1.

Enzyme-linked immunosorbent assay fused protein or peptide Enzyme-linked immunosorbent assay fused proteins or peptides of the present invention carried out as follows.

1. Enzyme-linked immunosorbent assay of human growth hormone (hGH), fused protein of human growth hormone/alpha-1-antitrypsin [T109wt: α1AT/hGH], fused protein human growth hormone/monovariant alpha-1-antari the Sina [T: α1AT(P357N)/hGH] and fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH]

Monoclonal antibodies to human growth hormone ("Medix Biochemica, Finland) was diluted to concentrations of 1~5 μg/ml in sodium phosphate buffer (PBS), breeding divided into aliquots of 100 μl/well in 96-well tablet ("Nunc, Denmark) and incubated at room temperature for 15~18 hours. After removal of remaining in suspension antibodies contribute PBS containing 1%bovine serum albumin, in the amount of 250 μl/well and incubated at room temperature for 2 hours. Tablets washed three times with washing buffer (0.05% of Tween 20, PBS) and the solution selected. Samples diluted in PBS containing 1%bovine serum albumin, and bring in 96-well tablets before incubation at room temperature for 2 hours. 96-well tablets washed five times with washing buffer in each well of 96-well plates add 100 μl conjugated with Biotin monoclonal antibodies to human growth hormone obtained by the use of sulfo-NHS-Biotin ("Pierce biotechnology, USA)and then incubated at room temperature for 2 hours. Tablets washed five times with washing buffer and incubated with streptavidin-HRP at room temperature for 30 minutes Again, tablets, washed five times with washing buffer and carry out the reaction with 100 μl of a mixture of TMB (3,3',5,5'-tetramethylbenzidine and peroxide of bodoro is as per well for 30 min in a dark place. To each well was added 100 μl of 1 M sulfuric acid to stop the reaction, then measure the absorbance at 450 nm using a reader for microplates "user's manual" ("Molecular Device, USA). Values for each sample calculated by regression analysis after constructing a standard curve for the control material.

2. Enzyme-linked immunosorbent assay of interferon-alpha man (IFN-α), fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α]

Enzyme-linked immunosorbent assay of interferon-alpha man (IFN-α) and fused protein interferon-alpha man/monovariant alpha-1-antitrypsin is carried out with compatible with IFN-α human pair antibodies for ELISA ("Bender Medsystems, Austria). Antibodies to IFN-α (10 μg/ml) are placed in 96-well plates to attach, then block them, as described in test example 1-1. At room temperature for 2 hours with shaking conduct the reaction between the sample at different dilutions with antibodies. Conjugates of anti-IFN-α-HRP was placed in the amount of 50 μl/well and carry out the reaction with antibodies at room temperature for 2 hours with shaking. Subsequent procedures are conducted in the same manner as in test example 1-1.

3. Enzyme-linked immunosorbent assay granulocyte colony-stimulating factor (G-CSF), fused protein granulocyte colonialstyle the ith factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF], fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF]

Repeat the same procedure as in test example 1, except that instead of antibodies to human growth hormone use monoclonal antibodies to granulocyte colony-stimulating factor (G-CSF) ("RND systems, USA), which was diluted to concentrations of 1~5 µg/ml and apply the conjugate polyclonal antibodies to granulocyte colony-stimulating factor-Biotin ("RND systems, USA), instead of the conjugate monoclonal antibodies to human growth hormone and Biotin.

4. Enzyme-linked immunosorbent assay of basis 4, fused protein on the basis 4/monovariant alpha-1-antitrypsin [T:basis-4/α1AT(P357N)]

Repeat the same procedure as in test example 1-1, except that the use of polyclonal antibodies basis 4 ("Peptron, Korea), instead of antibodies to human growth hormone, which is diluted to a concentration equal to 5~10 µg/ml in PBS and used a conjugate of a monoclonal antibody to the basis-4 and Biotin, instead conjugate monoclonal antibodies to human growth hormone and Biotin.

As for the fused protein on the basis 4/monovariant alpha-1-antitrypsin (T)obtained in example 7, to obtain antisera before injection to rats mixed with adjuvant's adjuvant (Sigma, USA). And is Titel cleanse, using the protein-O-sepharose (GE Healthcare, USA). Purified antibody was diluted to concentrations of 10~20 μl/ml in PBS, 96-well tablets are coated with antibodies, and conducting the reaction with the conjugate polyclonal antibody fused protein on the basis 4/monovariant alpha-1-antitrypsin (T) and Biotin, which was obtained by applying sulfo-NHS-Biotin conjugate ("Pierce biotechnology, USA), in the same manner as described in test example 1-1. Reaction image and the conjugate is carried out with shaking.

Test example 2. Pharmacokinetic analysis of fused proteins or peptides

For pharmacokinetic studies fused proteins or peptides, conduct the following experiments.

1. Pharmacokinetics of human growth hormone (hGH), fused protein of human growth hormone/alpha-1-antitrypsin [T109wt: α1AT/hGH], fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH], fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH]

As the use of experimental animals rats, Sprague-Dawley, three of them enter the human growth hormone, whereas the remaining rats are placed into groups, which enter the slit proteins, five rats in the group. Protein the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], a protein hormone human growth/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and fused white is the human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH], all obtained in example 1~3, is injected subcutaneously at a dose of 720 mg/kg of the corresponding group of rats Sprague-Dawley. Samples diluted in PBS before injection. Blood samples are taken through 0, 1, 2, 4, 8, 12, 16, 24, 30 and 48 hours after injection, and centrifuged to obtain serum. For control, Scitropin ("SciGen", Singapore), a variant of human growth hormone, injected subcutaneously by injection at a dose of 200 mcg/kg as a solvent used PBS. Blood samples are taken through 0, 0,33, 1, 2, 5, 8, 12, 18, 24, 30 and 48 hours after injection, and centrifuged to obtain serum. Each sample analyzed by enzyme-linked immunosorbent assay following the procedure described in test example 1.

Pharmacokinetics fused protein of human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and fused protein of human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH], shown in the graph presented in figure 1.

As shown in figure 1, the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] has a half-life in serum (t1/2), equal to 5.3 hours and the time at which its concentration in the serum reaches the maximum value (Tmax), is equal to 8 hours, the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] who meet the half-life in serum (t 1/2), equal to 5.4 hours, and Tmaxequal to 12 hours, and the protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] has a half-life in serum (t1/2), equal to 4.9 hours and Tmaxequal to 12.8 hours. On the other hand, the human growth hormone (hGH) has a half-life in serum (t1/2), is equal to 0.8 hours and Tmaxof 1 hour. Therefore, it was found all fused proteins, the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], a protein hormone human growth/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and the protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] have considerably increased in vivo stability compared to human growth hormone.

2. The pharmacokinetics of interferon-alpha man (IFN-α), fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α]

As the use of experimental animals rats, Sprague-Dawley, they are distributed on five rats in each test group. Protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α], obtained in example 4, is injected subcutaneously by injection at a dose of 200 mcg/kg to rats Sprague-Dawley one of the test groups, then through 0, 0,33, 1, 2, 5, 8, 12, 18, 24, 30, 48, 72 and 96 hours taken blood samples, centrif Giroud them to obtain serum. As a control, interferon-alpha man (IFN-α, "Intermax alpha, LG Life Sciences, Korea) was injected to rats subcutaneously by injection at a dose of 60 mcg/kg, then through 0, 0,33, 1, 2, 5, 8, 12, 18, and 24 hours is taken blood samples and centrifuged them to obtain serum. Each sample analyzed by enzyme-linked immunosorbent assay following the procedure described in test example 1.

Pharmacokinetics fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α] is shown in figure 2.

As shown in figure 2, protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α] has a half-life in serum (t1/2), equal to 18.5 hours and Tmaxequal to 12 hours, whereas interferon-alpha man has a half-life in serum (t1/2), equal to 3.4 hours, and Tmaxequal to 1.4 hours. Therefore, in vivo stability of the fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α] the present invention is significantly increased compared with interferon-alpha man.

3. The pharmacokinetics of granulocyte colony-stimulating factor (G-CSF), fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF], fused protein granulocyte colony-stimulating factor/trivariant alpha-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF]

As the use of experimental animals rats, Sprague-Dawley, and the three of them enter granulocyte colony-stimulating factor, whereas the remaining rats are placed into groups, cotrim enter fused proteins, five rats in the group. Protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF], all obtained in example 5~6, is injected subcutaneously at a dose of 340 mg/kg of the corresponding group of rats Sprague-Dawley. Blood samples are taken through 0, 1, 2, 4, 8, 12, 16, 24, 30, and 48 hours after injection, and centrifuged to obtain serum. For control, Filgrastim ("Gracin", "Jeil Pharmaceutical Co. Ltd.", Korea), available for purchase granulocyte colony-stimulating factor is administered subcutaneously by injection at a dose of 100 μg/kg of blood Samples taken through 0, 1, 2, 4, 8, 12, 18, 24, 30 and 48 hours after injection, and centrifuged to obtain serum. Each sample analyzed by enzyme-linked immunosorbent assay following the procedure described in test example 1.

Pharmacokinetics fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antidrip the ina [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] is shown in figure 3.

As shown in figure 3, protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] has a half-life in serum (t1/2), equal to 5.1 hours, and Tmaxequal to 13.6 hours and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] has a half-life in serum (t1/2), equal to 4.5 hours, and Tmaxequal to 16 hours. On the other hand, the half-life in serum (t1/2)equal to 1.8 hours, and Tmaxequal to 1.8 hours, it was determined in the group which was administered granulocyte colony-stimulating factor. Consequently, discovered that a protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] has significantly increased t vivo stability compared with granulocyte colony-stimulating factor.

4. Pharmacokinetics of basis 4, fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)]

As the use of experimental animals rats, Sprague-Dawley, and they are distributed on five rats in each test group. Protein on the basis 4/mon is a variant of alpha-1-antitrypsin [T: basis-4/α1AT(P357N)], obtained according to example 7, is injected subcutaneously by injection at a dose of 520 mg/kg to rats Sprague-Dawley in each of the tested grouppolicy blood taken through 0, 1, 2, 4, 8, 12, 16, 24, 30, 48 and 72 hours after injection, and centrifuged to obtain serum. As a control, on the basis of 4 injected subcutaneously by injection to rats at a dose of 40 µg/kg of blood Samples taken in heparinized tubes via 0, 10, 20, 30, 40, 60, 120, 180, 240, 300 and 360 min after injection, and centrifuged to obtain serum. Each sample analyzed by enzyme-linked immunosorbent assay following the procedure described in test example 1.

Pharmacokinetics fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N] is depicted in figure 4.

As shown in figure 4, a protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(RK)] has a half-life in serum (t 1/2)equal to 19.1 per hour, and Tmaxequal to 10.4 hours, whereas on the basis 4 has a half-life in serum (t1/2), is equal to 0.8 hours, and Tmaxequal to 0.4 hours. Consequently, the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(RK)] of the present invention has significantly increased in vivo stability, in comparison with basis 4.

Test example 3. Analysis of the activity of the fused protein or peptide in vivo

The following eksperimentirovat for research activity in vivo fused proteins or peptides in accordance with the present invention.

1. In vivo activity of human growth hormone (hGH), fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH]

As experimental animals used rats Sprague-Dawley remote pituitary gland, they are broken down into three groups of seven rats each. Rats Sprague-Dawley remote pituitary daily injected subcutaneously protein the human growth hormone/monovariant alpha-1-antitrypsin [T; α1AT(P357N)/hGH], obtained in example 2, and the human growth hormone (lutropin, LG Life Sciences Ltd., Korea) at a dose of 18 μg and 5 μg per rat, respectively. For control use PBS. After the injection the rats daily weigh.

The results of the analysis of in vivo activity (change of body weight in rats with remote pituitary) fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] is shown in figure 5.

As shown in figure 5, the rats with the remote pituitary almost no gain in weight, with the introduction of the PBS, while weight gain was observed approximately 10.2% and 9.4% on day 7 in rats with remote pituitary gland, which enter the human growth hormone and protein the human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH], respectively. Thus, it was found that the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] the present invention preserves ffektivnoe in vivo activity in rats with remote pituitary, like human growth hormone.

2. In vivo activity of granulocyte colony-stimulating factor (G-CSF), fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF]

As the use of experimental animals rats, Sprague-Dawley, they are distributed in five groups of five rats each. Protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF], obtained in example 5, is injected subcutaneously by injection in doses of 340 µg/kg 1700 mg/kg Other groups of rats injected subcutaneously protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF], obtained in example 6, and granulocyte colony-stimulating factor Filgrastim ("Gracin", "Jell Pharmaceutical Co. Ltd.", Korea) in the dose of 1700 mg/kg and 100 mcg/kg, respectively. Blood samples taken from the tail for 3 days before the experiment and at 1, 2, 3, 4 and 5 days after injection and determine the number of leukocytes using a Hematology analyzer Pentra 120).

The results of the analysis of in vivo activity (change in the number of leukocytes) fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitripsin the [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] is shown in Fig.6.

As shown in Fig.6, the number of cells reached a maximum level on day 1 and then decreased to the baseline level from day 2 in the group which was administered granulocyte colony-stimulating factor Filgrastim, and a maximum level on day 2 and then decreased from day 3 in the group, which was introduced protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] at a dose of 340 mg/kg In groups, which respectively have introduced protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] dose of 1700 mg/kg, the number of cells remained high until day 3, and mitigation was started on day 4. Therefore, it was shown that protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] prolong in vivo activity compared to granulocyte colony-stimulating factor.

3. In vivo Akti is the ability of the basis 4 and fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)]

For in vivo studies of the activity of the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)] of the present invention, spend intraperitoneal test glucose tolerance test to reduce blood sugar in the model in mice with diabetes, as described below.

3-1. Intraperitoneal test glucose tolerance

Eight of C57BL/6 mice fed for four weeks of food with high fat content for the induction of their obesity. Mouse fast 15 hours before intraperitoneal test glucose tolerance. In this context, a protein on the basis 4/monovariant alpha-1-antitrypsin [T: Basis-4/α1AT(P357N)]obtained in example 7, and the basis-4 injected with intraperitoneal injection of a single dose of 10 nmol/kg of the corresponding group of mice, then after 30 minutes, 12 hours and 24 hours using intraperitoneal injection in mice injected with glucose at a dose of 1.5 g/5 ml/kg of the sugar Level in the blood was determined using a glucometer ("Allmedicus", Korea) through 0, 10, 20, 30, 60, 90 and 120 min after injection of glucose.

Results intraperitoneal test glucose tolerance with fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(RN)] is shown in Fig.7.

As shown in Fig.7, in the group which was administered protein on the basis 4/monovariant alpha-1-antitrypsin [T: ex who nden-4/α1AT(RN)] were maintained in low glucose level over a longer period of time, compared with the group which was administered on the basis of 4.

3-2. The decrease in blood glucose in the model in mice with diabetes

As the use of experimental animals mice db/db at the age of 9 weeks, they are divided into three groups of six animals. Mice db/db contained in the conditions of free access to feed, injected subcutaneously protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)] obtained in example 7, or on the basis of 4 at a dose of 100 nmol/kg, and through 0, 1, 3, 6, 24, 43, 48 and 52 hours after that determine the concentration of sugar in the blood using a glucometer ("Allmedicus", Korea).

The effect of the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(P357N)] in the model of diabetes in mice is shown in Fig.

As shown in Fig, since 24 hours after injection, the group, which was introduced on the basis of 4, had a blood sugar level similar to that in the control group, whereas in the group, which was introduced protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis-4/α1AT(RN)], continued low blood sugar levels, demonstrating prolonged activity of the fused protein.

Test example 4. In vitro activity of fused proteins or peptides

To study the in vitro activity of fused proteins or peptides of the present invention conducted the following experiments.

1. In vitro activity of the mountains is she human growth (hGH), fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH]. fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH]

Line cell lymphoma rats (NB2 cells) support in RPMI1640 medium with addition of 10%HS (horse serum), 10%FBS, 2-mercaptoethanol and antibiotics at 37°C and 5%CO2. Within 24 hours before the experiment, NB2 cells incubated in medium RPMI 1640, with the addition of 10%HS. After that, NB2 cells washed once in 1×DPBS (environment Dulbecco: saline phosphate buffer), and placed in 96-well plates (Coming, USA) at a concentration of 2×104cells/100 µl/well in RPMI medium 1640 with the addition of 5%HS in the final volume of 100 μl. Series dilution of samples added to 20 μl in each well of 96-well plates, then incubated at 37°C for 48 hours in 5%CO2. Then, 20 μl of MTS solution (Promega, USA) is added to each well of 96-well plates and the reaction is performed at 37°C for 3 hours in a 5%CO2. The reaction is stopped by adding 20 μl of 10%SDS (sodium dodecyl sulphate) in each well. Measure the absorbance at 490 nm using a reader "user's manual" for microplates ("Molecular Device, USA). Size EU50(50%effective concentration of drugs i.e. the concentration at which survive 50% of the cells, determined on the basis of the results of measurement of absorption MTS method.

The results of the analysis of the in vitro activity of the fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] are summarized in table 1 and presented in Fig.9.

Table 1
SampleEU50(PM)
T109wt211,8
T181,1
TT217,1

As can be seen from the data presented in table 1 and figure 9, the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], a protein hormone human growth/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and the protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] slightly differ in magnitude EC50. Therefore, the protein human growth hormone/alpha-1-antitrypsin [T109wt: α1AT/hGH], a protein hormone human growth/monawar the Anta alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and the protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] indicate a relatively constant t vivo activity (EC50), regardless of amino acid mutations in the alpha-1-antitripsin.

2. In vitro activity of granulocyte colony-stimulating factor (G-CSF), fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF]

Cell myeloblastoma mice NFS-60 incubated in medium RPMI 1640, with the addition of 10%FBS, murine IL-3 and antibiotics at 37°C With 5%CO2. The absorption cell was measured at 490 nm in the same manner as in test example 4-1. Size EU50(50%effective concentration) of drugs, i.e. the concentration at which survive 50% of the cells, determined on the basis of the results of measurement of absorption MTS method.

Analysis of the results of the in vitro activity of the fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and fused protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] are shown in table 2 and figure 10.

Table 2
Sample EU50(PM)
T602S78,1
T602ST97,6

As can be seen from the data presented in table 2 and figure 10, protein granulocyte colony-stimulating factor/divariant alpha-1 antitrypsin fusion [T602S: α1AT(P357N, C232S)/G-CSF] and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] slightly differ in magnitude EC50. Therefore, protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] and protein granulocyte colony-stimulating factor/trivariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] indicate a relatively constant in vivo activity (EC50), regardless of amino acid mutations in the alpha-1-antitripsin.

Test example 5. Analysis of the inhibitory activity of fused proteins or peptides against trypsin

To study the inhibitory activity of the fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] against trypsin, conduct the following experiments.

Protein the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], the obtained p the example 1, and the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH], obtained in example 1, separately mixed with trypsin. Trypsin and fused proteins used at a concentration of 10 nm, respectively. After incubation at room temperature for 1 hour, spend the reaction mixture with 0.2 mm substrate N-benzoyl-Val-Gly-Arg-n-nitroanilide hydrochloride (Sigma, USA), and then measure the absorbance at 405 nm. Per unit of trypsin take the concentration of the substrate, resulting in the change of the absorption of 0.001, and the enzyme activity is expressed as unit/mg of trypsin. For comparison, in the quality control applied trypsin.

The results are shown figure 11.

As shown in figure 11, according to the calculations of Ka (the equilibrium constant of Association) between trypsin and alpha-1-antitripsin equal to about 7.5×108M-1for fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and approximately 8,0×106M-1for fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH]. Protein the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] showed excellent inhibitory activity against trypsin, whereas the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] is not a good inhibitor of trypsin. Therefore, the fact that what protein the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] increase the half-life in vivo through sustainable circulation, not dependent on the intrinsic properties of alpha-1-antitrypsin deficiency.

Test example 6. Analysis of the inhibitory activity of fused proteins or peptides against elastase human neutrophils

To study the inhibitory activity of the fused protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and fused protein human growth hormone/monovariant alpha-1-antitrypsin [T 109: α1AT(P357N)/hGH] against elastase human neutrophils conduct the following experiments.

Protein the human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], obtained in example 1, and the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH], obtained in example 1, are mixed separately with elastases human neutrophils. The elastase and fused proteins used at a concentration of 40 nm, respectively. After incubation at room temperature for 1 hour, spend the reaction mixture with the substrate 1 mm MeOSuc-AAPV-pNA (Santa Cruz Biotechnology, Inc.", USA), and then measure the absorbance at 405 nm. Per unit of elastase human neutrophils take the concentration of the substrate, causing the change of absorption of 0.001 and a lot of activity is ü enzyme is expressed as units/mg of elastase.

The results depicted in Fig.

As shown in Fig, it was found that the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] inhibited almost 100% of the elastase of human neutrophils, whereas human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] has Ka, equal to about 1.4×107M-1. Therefore, the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] serves as an excellent inhibitor of neutrophil elastase, whereas the human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] has a low inhibitory activity against elastase human neutrophils. Therefore, the fact that the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH] and the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] increase the half-life in vivo through sustainable circulation, does not depend on inherent actually alpha-1-antitripsin properties.

Test example 7. Analysis of the fused proteins or peptides by electrophoresis

To investigate the change in molecular weight associated with the introduction of an additional glycosylation site, the protein human growth hormone/alpha-1-antitripsin [T109wt: α1AT/hGH], a protein hormone human growth/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and merged the th protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] subjected to SDS-polyacrylamide gel electrophoresis.

The results are shown in Fig.

As shown in Fig, a protein hormone human growth/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH], which was attended by additional glycosylation site (Asn-X-Ser), migrates as a protein band with a higher molecular weight, compared with native fused protein human growth hormone/alpha-1-antitrypsin [T109wt: α1AT/hGH], which did not create additional glycosylation site. As for the fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH], it has a more extensive additional glycosylation at position 357 (Asn-X-Thr), and an increase in its molecular weight is even more obvious. Therefore, the protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH] and the protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH] have an increased molecular weight by introducing glycosylation site.

Industrial applicability

While in the forms stable during circulation in the blood, fused proteins or peptides of the present invention significantly increase the half-life in serum (T1/2and exhibit excellent in vivo stability, in comparison with the actual physiologist who Cesky active proteins or peptides. Therefore, fused proteins or peptides of the present invention can be applied to create a stable circulating dosage forms of the protein or peptide drugs.

1. A protein or polypeptide with increased half-life for therapeutic use, comprising a physiologically active protein or polypeptide, is fused with the variant alpha-1-antitrypsin, having at least one mutated amino acid residue, resulting in a physiologically active protein or polypeptide has an increased half-life in vivo by maintaining a steady circulation in the blood, where at least one mutated amino acid residue is a
the asparagine residue instead of a Proline residue at position 357; or
the asparagine residue instead of a Proline residue at position 357 and the residue threonine instead of serine at position 359; or
the asparagine residue instead of a Proline residue at position 357 and the residue serine instead of cysteine at position 232; or
the asparagine residue instead of a Proline residue at position 357, residue threonine instead of serine at position 359 and the residue serine instead of cysteine at position 232.

2. Protein or polypeptide according to claim 1, in which the physiologically active protein or polypeptide fused directly or through linker consisting of the amino acids, with a single variant is alpha-1-antitrypsin, having at least one mutated amino acid residue.

3. Protein or polypeptide according to claim 1, in which the physiologically active protein is selected from the group consisting of hormones and their receptors, modifiers of biological responses and their receptors, cytokines and their receptors, enzymes, antibodies and fragments of antibodies.

4. Protein or polypeptide according to claim 3, in which the physiologically active protein is selected from the group consisting of human growth hormone (hGH), insulin, follicle-stimulating hormone (FSH), human chorionic gonadotropin, parathyroid hormone (PTH), erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha, interferon-beta, interferon-gamma, interleukins, macrophage activating factor, tumor necrosis factor, tissue plasminogen activator, factor coagulation VII, VIIa, VIII and IX, bone morphogenetic protein 2 (hBMP2), keratinocyte growth factor (KGF), platelet growth factor (PDGF), glucocerebrosidase, α-galactosidase And α-L-iduronidase, iduronate-2-sulfatase, lactase, adelaideans, butyrylcholinesterase, chitinases, glutamatdekarboksilazy, imiglucerase, lipase, uricase, acetylhydrolase of platelet activating factor, athelney endopeptidase, urokinase, streptokinase, myeloperoxidase, superoxide dismutase, botulinum toxin, collagenase, hyaluronidase, L-asparaginase, monoclonal antibodies, polyclonal antibodies, scFv, Fab, Fab', F(ab')2and Fd, and their combinations.

5. Protein or polypeptide according to claim 1, in which the physiologically active polypeptide selected from the group consisting of glucagon-like peptide-1 (GLP-1) and its analogues, basis and its analogues, somatostatin and its analogues, agonists and antagonist releasing factor, luteinizing hormone (LHRH), adrenocorticotropic hormone, a hormone that stimulates growth hormone, oxytocin, thymosin alpha-1, corticotropin-releasing factor, calcitonin, bivalirudin, vasopressin analogues, fragments of physiologically active polypeptides and combinations thereof.

6. Protein or polypeptide according to claim 1, which is selected from the group consisting of fused protein human growth hormone/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/hGH](SEQ ID NO:2)fused protein human growth hormone/divariant alpha-1-antitrypsin [TT: α1AT(P357N, S359T)/hGH](SEQ ID NO:3), fused protein interferon-alpha man/monovariant alpha-1-antitrypsin [T: α1AT(P357N)/IFN-α](SEQ ID NO:4), fused protein granulocyte colony-stimulating factor/divariant alpha-1-antitrypsin [T602S: α1AT(P357N, C232S)/G-CSF] (SEQ ID NO:5), fused protein granulocyte colony-stimulating factor/three is Ariant alpha-1-antitrypsin [T602ST: α1AT(P357N, C232S, S359T)/G-CSF] (SEQ ID NO:6) and the fused protein on the basis 4/monovariant alpha-1-antitrypsin [T: basis 4/α1(P357N](SEQ ID NO:7).

7. Way longer half-life in vivo protein or polypeptide, including conjugation physiologically active protein or polypeptide variant of alpha-1-antitrypsin deficiency according to claim 1, resulting conjugate is characterized by stability during circulation in vivo.



 

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5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes compounds of labyrinthpeptins A1, A2, or A3 of formula (I) , where {A}, {B}, {C}, R1-R6, m and n have the values specified in the formula of the invention. Compounds are obtained at fermentation of Actinomadura namibiensis DSM 6313 strain under acceptable conditions in cultural environment till one or more compounds of formula (I) are formed. The invention proposes the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A2, and the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A1, as well as preprolabyrinthpeptins A1 and A2, and prolabyrinthpeptins A1 and A2. Labyrinthpeptins of formula (I) are used for therapy of infections caused by gram-positive bacteria, virus infections and/or neuropathic pain caused by inflammation.

EFFECT: improving therapy efficiency.

24 cl, 4 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: plasmid genetic structure pOL-DsRed2 is produced, being built on the basis of a plasmid vector pIRES (Clontech), where fragments of cDNA of human genes OCT4 and LIN28 are placed, being connected with a nucleotide sequence coding P2A-peptide and gene cDNA, coding fluorescent protein DsRed2.

EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a production method of recombinant protein through its hybrid precursor substance with natural decomposition site with enteropeptidase. The result is achieved by replacement in natural decomposition site with enteropeptidase Asp-Asp-Asp-Asp-Lys of amino-acid residue of lysine (Lys) with amino-acid residue of arginine (Arg) and further decomposition of hybrid precursor substance with light catalytic subunit of enteropeptidase of a human being or a bull.

EFFECT: improving quality and yield of target product under conditions when hybrid protein detects additional sites of decomposition with enteropeptidase.

3 tbl, 3 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.

EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.

39 cl, 8 dwg, 9 tbl, 46 ex

FIELD: biotechnology.

SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.

6 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: disclosed are anti-5T4 antibodies, nucleic acids which encode variable regions of such antibodies, antibody/drug conjugates, a method of delivering a drug using such a conjugate, as well as a method of treating a subject with cancer which is characterised by 5T4 antibody expression, by administering the disclosed conjugate.

EFFECT: present invention can further be used in therapy of 5T4-associated diseases.

42 cl, 8 ex, 15 tbl, 15 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions refers to biotechnology and concern hypoallergic fused proteins. A presented hypoallergic fused protein consists of one hypoallergic molecule originated from an allergen, fused with a second non-allergic protein originated from a pathogen, or a fragment thereof with the hypoallergic molecule originated from the allergen showing 50% decreased ability to bind IgE, 30% decreased T-cell responsiveness as compared with a wild-type allergen. A nucleic acid molecule coding the fused protein, an expression vector, a host cell expressing the same protein, and a vaccine composition containing the protein are also presented.

EFFECT: presented invention enables preparing therapeutic and prophylactic drugs for an allergy or diseases caused by pathogens with using no toxic adjuvants, showing no side effects.

14 cl, 23 dwg, 17 tbl, 27 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are presented versions of recovered monoclonal antibodies and an antigen-binding portion thereof specific to human IP-10. There are described: an immunoconjugate, bispecific molecule thereof, as well as versions of a composition of the antibody, immunoconjugate or bispecific molecule - for treating autoimmune and inflammatory diseases. There are also disclosed: a coding nucleic acid, an expression vector thereof and a host cell carrying this vector to produce the antibody. What is described is using the antibody or antigen-binding portion thereof for preparing a medicine for treating: either a viral or bacterial infection entailing undesired IP-10 activity, or autoimmune and inflammatory diseases caused by undesired IP-10 activity. What is presented is a hybridoma producing the antibody, derived from a transgenic mouse splenocyte cross-linked to an immortalised cell.

EFFECT: use of the invention provides the novel antibodies that can be find application in medicine to treat a variety of diseases associated with IP-10 activity.

22 cl, 30 dwg, 9 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: chicken egg albumin is mixed with an equal volume of a mixture 0.5 M aqueous trichloroacetic acid and an organic solvent in volume ratio 1:1.8-2.3. The formed residue is separated by filtering at 0-5°C. The filtrate is then mixed with an organic solvent in volume ratio of the organic solvent to the filtrate of 3-3.5:1. The supernatant fluid is then removed by decantation at 0-5°C. Low-molecular impurities are removed from ovomucoid through ultrafiltration. The ovomucoid is lyophilically dried. The organic solvent used is a mixture of ethanol and acetone in volume ratio 40-65:60-35.

EFFECT: high output of ovomucoid and high degree of purity.

1 tbl, 5 ex

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