Isolated polypeptide and using it for treating malignant disease and stimulating immune system, pharmaceutical composition containing such polypeptide and method of treating cancer

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns an isolated polypeptide, a pharmaceutical composition containing such polypeptide, as well as a method of treating cancer. The presented polypeptide contains an amino acid sequence corresponding to SEQ. ID. NO: 2 or SEQ ID NO: 4. There are also characterised fragments or versions of the presented polypeptide.

EFFECT: group of inventions may be used to stimulate the immune system in treating malignant diseases and for diagnosing loss of immunologic activity.

7 cl, 7 dwg, 1 tbl, 6 ex

 

The technical FIELD

The present invention relates to a new protein and its use in therapy.

The LEVEL of TECHNOLOGY

Diseases that afflict people can be classified depending on the mechanism that underlies these diseases. For example, diseases including immunological component or aetiology, include infectious diseases, acute and chronic inflammatory diseases, cancer, complications of transplantation and autoimmune diseases.

Examples of autoimmune diseases include multiple sclerosis (PC), autoimmune uveitis, autoimmune uveoretinitis, autoimmune thyroiditis, Hashimoto's thyroiditis, insult, Sjogren syndrome, spontaneous abortion (miscarriage), experimental autoimmune myocarditis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease, lupus (systemic lupus erythematosus (SLE), psoriasis and diabetes, in particular type I diabetes.

Additional examples of autoimmune diseases include acute necrotizing hemorrhagic leukoencephalitis, Addison disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia alopecia, amyloidosis, ankylosing spondylitis (Bechterew's disease), Anti-GBM/Anti-TVM nephritis, antiphospholipid syndrome (APLS), autoimmune hypoplastic the th anemia, autoimmune autonomic dystonia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune disease of the inner ear (CBA), autoimmune myocarditis, autoimmune thrombocytopenic purple (ATP), axonal and neuronal neuropathy, a disease Bal (Bal''s disease), a disease Bennett (Behnet''s disease), bullous pemphigoid, cardiomyopathy, a disease of Castlemaine, celiac disease-sprue (non-tropical enteropathy), Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinizing the polyneuropathy (hwdp), syndrome Cerca-Strauss, scar pemphigoid /benign pemphigoid mucosa syndrome Kogan, illness cold agglutinins, congenital heart block, myocarditis Coxsackie syndrome Tiberia-Eisenbach, the basic mixed cryoglobulinemia type demyelinating neuropathies, dermatomyositis (illness Wagner-Unverricht-Happa), a disease), discoid lupus, Dressler syndrome, endometriosis, eosinophilic fasciitis, nodoso erythema, experimental allergic encephalomyelitis syndrome Evan, fibromyalgia, fibrosing alveolitis, giant cell arteritis diagnostics (temporal arteritis diagnostics)syndrome?, graves ' disease (graves disease), Guillain-Barre syndrome, hemolytic anemia, purple (illness) Seleina's disease, herpes pregnant, GI is agammaglobulinemia (deficiency syndrome antibodies), hemorrhagic purple (idiopathic thrombocytopenic purple), primary IgA nephropathy-type immunoregulatory lipoproteins, myositis enabled cells, insulin-dependent diabetes (type 1), interstitial cystitis, juvenile juvenile arthritis, juvenile (juvenile) diabetes, Kawasaki syndrome, syndrome of Eaton-Lambert leukocytoclastic vasculitis, vitiligo Wilson, lichen lichen, ligneous conjunctivitis, IgA-dependent linear dermatosis (LAD), liskow disease, Meniere's disease (dropsy of the labyrinth of the inner ear), the microscopic disorder, syndrome Sharpe (mixed lesions of the connective tissue), corroding ulcer of the cornea acute lichenoides ostanovochnyy parapsoriasis, heavy pseudoparalysis the gravis, myositis, narcolepsy, agranulocytosis (neutropenia), scar pemphigoid eyes, osteoarthritis, migratory arthritis, paraneoplastic cerebellar degeneration, night the paroxysmal hemoglobinuria (APG), a syndrome Parsonage and Turner, Pars planitis (peripheral uveitis, pemphigus (common bladderwort), peripheral neuropathy, paravenously encephalomyelitis, pernicious anemia, POEMS syndrome (polyneuropathy (polyneuropathy) organomegaly), polyarteritis polyarteritis, autoimmune pluriglandular syndromes types I, II and III, rheumatic rheumatica, polymyositis (more than the HB Wagner-Unverricht-Happa), postinfarction syndrome, complications of pericardiotomy, progesterone dermatitis, primary biliary cirrhosis, psoriatic arthritis, idiopathic pulmonary fibrosis, the gangrenous pyodermia, true red cell aplasia, the phenomenon Raynaud's syndrome, reflex sympathetic dystrophy, a triad of Reiter (ureterocutaneostomy syndrome), recurrent polyhedric, tired leg syndrome, rheumatic fever (acute rheumatic fever), benign Wegener syndrome (Besnier-Beck-Soumana)syndrome Schmidt, scleritis, scleroderma, autoimmunity against sperm and testes syndrome bound man, subacute bacterial endocarditis (PSE), metastatic ophthalmia, aortic arch syndrome (Takayasu's arteritis), temporal arteritis diagnostics /giant cell arteritis diagnostics, thrombocytopenic purple (TTP), autoimmune thyroid disease, syndrome, Tolosa-hunt, transverse myelitis and necrotic myelopathy, ulcerative colitis, undifferentiated connective tissue disease (NDCT), vasculitis (inflammation of blood vessels), vesiculate-bullous dermatosis, Vitiligo syndrome and Wegener (systemic necrotizing granulomatous vasculitis).

Non-limiting examples of the types of cancer include cancer of the adrenal cortex (adrenal cortical); malignant melanoma; not elenany skin cancer; T-cell lymphoma of the skin; Kaposi's sarcoma; bladder cancer; colorectal cancer; colorectal cancer; rectal cancer; neuroectodermal cancer and cancer of the pineal gland; the glioma of the brain stem in children; cerebellar astrocytoma of childhood; astrocytoma of the brain in children, medulloblastoma in children; a glioma of the optic paths of childhood; meningioma; mixed glioma; oligodendroglioma; astrocytoma; the ependymal glioma, pituitary adenoma; metastasized adenocarcinoma; acoustic neuroma; paravertebral malignant teratoma; breast cancer; ductal (Doctorow) carcinoma; tumors of the breast; ovarian cancer; carcinoid the tumor; cervical cancer (cervical cancer); cervical cancer; endometrial cancer; cancer of the vagina; vulvar cancer; gestational USSR cancer; cancer of the fallopian tubes; sarcoma cancer; leukemia; lymphoma (Hodgkin's and non-Hodgkins lymphoma; neuroblastoma; retinoblastoma; soft tissue sarcoma; nephroblastoma (Wilms tumor); anemia, Fanconi; Wegener of Langerhans cells; malignant rod-like tumor of the kidney; liver cancer; neuroblastoma; retinoblastoma; horiokartsinoma; cancers of the endocrine glands; endometrial cancer; esophageal cancer; Ewing sarcoma; cancer of the eye; cancer stomach; cancer zheludochno the gastrointestinal tract; cancer of the urinary organs; glioma; cancers of the female genital organs; head and neck cancer; liver cell carcinoma; podletochnyh cancer; cancer of the islet cells; kidney cancer; larynx cancer; lung cancer; lymphoma; breast cancer in men; melanoma; mesothelioma; ploskokletochnyi myeloma; nasopharyngeal cancer; non-melanoma skin cancer; esophageal cancer; osteosarcoma; ovarian cancer; pancreatic cancer; cancer of the hypophysis; prostate cancer; renal cell cancer; retinoblastoma; rhabdomyosarcoma; sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; cancer of the testis, cancer of the thymus; thyroid cancer; transitional cell cancer; USSR cancer; uterine cancer; acute lymphocytic leukemia; acute myeloid leukemia; adenomatosnuu carcinoma; cancer of the anal canal; cancer of the bone; colon cancer; ductal (Doctorow) carcinoma, liposarcoma; neuroblastoma; neuroblastoma and osteosarcoma.

Inflammatory diseases include sepsis, endotoxicosis, pancreatitis, uveitis, hepatitis, peritonitis, keratitis, SIRS (Systemic Inflammation Response syndrome systemic inflammatory response and inflammation caused by injury.

Diseases associated with fertility (reproductive ability) include male infertility and female infertility.

Male infertility can be caused by a number of problems. Below are presented the EN number of the most frequent violations.

Low production of sperm: Ninety percent of male infertility is caused by the failure to produce enough sperm. Azoospermia is diagnosed with a complete lack of viable sperm in the semen, while the oligospermia diagnosed at a very low concentration of sperm in the semen, and, since most of the sperm is destroyed before reaching the egg, the greater the number of sperm in the semen, the greater the chance that one of them will successfully fertilize the egg. However, low sperm count or the total number of sperm in less than 5 million/ml, does not necessarily mean that the man is infertile, if his sperm are healthy, well-formed and active.

- Varicocele (varicose veins of the spermatic cord): Varicose veins around one of the two spermatic cord causes stagnation of blood in the testes; this, in turn, causes an increase in temperature in this area. Increasing temperature reduces the production of sperm and can cause infertility. Fortunately, this problem is eliminated by surgical intervention.

Other violations: Other disorders that can cause male infertility include abnormal development or damage to the testicles (call is by endocrine disorders or inflammation), violations extension (adventitious) glands, disorders in coitus, the impact of diethylstilbestrol (DES), a synthetic estrogen used in the 1950's and 1960's, which causes the formation of cysts in the male genital tract, cryptorchid testes, and, in rare cases, genetic disorders, such as chromosomal abnormalities.

Female infertility can also be caused by a number of problems. Here are some of the most common violations.

- Polycystic ovaries: This disease is the most common cause of ovulation disorders in women and is characterized by the presence of multiple small cysts in the ovaries, excessive production of androgens and rare menstruation (obligatoria (optimizarea)) or the absence of menstruation (amenorrhea). Failure to ovulate is the most common cause of infertility in women and can occur for unknown reasons or due to stress, hormonal imbalance and various disease and disorders of the reproductive system (some of which are described below).

Inflammatory pelvic disease: The infection of the genital tract can lead to blockage or damage to the fallopian tubes and is usually caused by sexually transmitted disease, miscarriage, abortion, childbirth, or the presence of NR is timetochange contraceptives.

- Ovulatory disorders: These disorders occur when the ovaries women do not produce eggs or produced fewer eggs due to age, hormonal imbalances or other problems.

- Uterine fibroids: benign tumors of the uterus have 40% of women and can interfere with embryo implantation or growth of the fetus.

- Endometriosis: This violation occurs when the tissue lining the uterus (endometrium) to form outgrowths or violations outside the uterus (usually on the ovaries, fallopian tubes, and ligaments that support the uterus; the area between the vagina and the anus; on the outer surface of the uterus; on the tissues lining the cavity of the pelvis; urinary bladder, bowel, vagina, cervix, vulva and scar tissue remaining after surgery in the abdominal cavity). This tissue grows, breaks down and separates each month in phase with the menstrual cycle, but unfortunately, she is unable to leave the body. In turn, this leads to internal bleeding, bleeding and tearing of tissues from affected areas, and often causes inflammation accompanied by pain, infertility, scar tissue, adhesion and problems with the intestines.

- Immunological infertility: This violation occurs when a woman's body produces sperm and is tetela, which destroy the sperm of her partner.

Disorders of carbohydrate metabolism have many forms. The most common disorders - acquired. Acquired or secondary disorders of carbohydrate metabolism, such as diabetic ketoacidosis, hyperosmolar coma and hypoglycemia, affect the Central nervous system. In diabetes there are also many forms and variants of peripheral nerve diseases. Other disorders of carbohydrate metabolism are rare congenital forms of metabolic disorders (i.e. genetic defects).

Acquired disorders of carbohydrate metabolism are common, both in the U.S. and around the world. Hypoglycemia is a common cause of neurological diseases, in particular acute mental disorders, memory loss, disorientation, blunting of pain sensitivity and coma, as among alcoholics, and among patients with diabetes who take insulin. Hyperinsulinemia with other reasons, is rare; however, it can cause cancer of the pancreas. The most common violation in adult patients is diabetes and its various neurological complications. Diabetic ketoacidosis still occurs, although knowledge in this area and careful medical h is the observation makes it more rare complication, than a few decades ago. Hyperosmolar coma is also currently represents less of a problem than at a time when attention was first attracted to the classical monograph of internists Plum and Posner: Diagnosis of Stupor and Coma. Hyperosmolar coma still occurs, and it should be paid attention in the examination of patients suffering from dulling pain sensitivity.

Hereditary disorders of carbohydrate metabolism are rare. Serious defects in complex piruvatdegidrogenazy (PDH) and benign chemical anomaly called pentosuria were found only a few (2-6) patients.

Hypoglycemia, diabetic ketoacidosis and hyperosmolar coma are potentially fatal, but also potentially curable condition.

BRIEF description of the INVENTION

Currently, on the basis of a new cDNA, was discovered a new protein, named KTPAF50. The peptide encoded by this cDNA contains 74 amino acids and includes a signal peptide, containing 24 amino acids in the N-terminal end group. The cDNA sequence (SEQ. ID. NO:1) and amino acid sequence (SEQ. ID. NO:2) for KTPAF50 presented below:

atgccaggc cattctagg cttctgtct atcctggtt tctggtctg tgcgttgtg ggtagcagc attggcgta ttacgccgg agggagcag gctgagcga ggctccaga aggtgcgca atagccgga gaggaaagg gcgatgctg tcacctagc cccctccct gagactcca ttcagccca gaaaaagga gctgctttc tcccccatc taccctagg agaaaa (SEQ. ID. N:1)

MPGHSRLLSILVSGLCVVGSSIGVLRRREQAERGSRRCAIAGEERAMLSPSPLPET PFSPEKGAAFSPIYPRRK (SEQ. ID. NO:2)

Thus, the present invention relates to a nucleic acid molecule corresponding to SEQ. ID. NO:1, and the peptide corresponding to SEQ. ID. NO:2. The polypeptide corresponding to SEQ. ID. NO:2, in the present description is called "full KTPAF50 peptide".

Full KTPAF50 peptide also includes a signal sequence that is considered consists of 24 amino acids. Thus, the invention also relates to a peptide comprising the sequence of the full KTPAF50 peptide that does not contain a signal peptide, and consisting of the following sequence (SEQ ID. NO:4):

LRRREQAERGSRRCAIAGEERAMLSPSPLPETPFSPEKGAAFSPIYPRRK (SEQ ID. NO:4)

KTPAF50 peptide not containing the signal sequence (SEQ. ID. NO:4), in the present description is called "KTPAF50 peptide" or "KTPAF50".

The invention also relates to a nucleic acid molecule comprising a sequence encoding a KTPAF50 peptide. It includes the following sequence (SEQ. ID. NO:3);

ttacgccgg agggagcag gctgagcga ggctccaga aggtgcgca atagccgga gaggaaagg gcgatgctg tcacctagc cccctccct gagactcca ttcagccca gaaaaagga gctgctttc tcccccatc taccctagg agaaaa (SEQ. ID. NO:3)

The invention also relates to a modified nucleic acid molecules corresponding to SEQ. ID. NO:1 or SEQ. ID. NO:3, and modified peptides corresponding to SEQ. ID. NO:2 or SEQ. ID. NO:4 in which one or more nucleotides or amino acid residues that corresponds to the public, added, removed, or replaced without significant changes in biological characteristics of the modified molecules compared to the unmodified molecule.

In the present description, the term "peptide" means a peptide, polypeptide or protein. The peptide can be obtained by the synthetic method, the methods of genetic engineering, expression in the cell-the owner or any other suitable method. Unless otherwise stated, the peptide consists mainly of L-amino acids occurring in nature.

The term "biological characteristics"is related to the molecule, peptide, refers to the ability of the peptide to provide at least either in vitro or in vivo exposure, which can provide full KTPAF50 peptide or KTPAF50 peptide, and non-limiting examples of such impacts include the biological activity described in the present description. For example, biological characteristics include the ability to treat cancer, diseases associated with the immune system, viral infections and diseases associated with inflammation. The term "biological characteristics"is related to the molecule of nucleic acid, means its ability to encode a peptide having the biological characteristics similar to the characteristics of the full KTPAF50 peptide or KTPAF50 peptide, and relates in particular: (i) to the molecule of nucleic acid, Kotor which has the sequence different from the sequence SEQ. ID. NO:1 or SEQ. ID. NO:3, but, thanks to the redundancy of the genetic code, encodes the full KTPAF50 peptide or KTPAF50 peptide, respectively; and (ii) molecule nucleic acid molecule which encodes the amino acid sequence differing from the sequence of full KTPAF50 peptide or KTPAF50 peptide, but has biological characteristics similar to the characteristics of the full KTPAF50 peptide or KTPAF50 peptide, respectively.

The term "without substantial changes in biological characteristics of the modified molecules compared to the unmodified molecule" means that the modified molecule retains the biological activity, qualitatively similar to the activity of the unmodified molecule. In respect of the modified peptide, this means that it stores one or more of the biological characteristics of the peptide corresponding to SEQ. ID. NO:2 or SEQ. ID. NO:4, non-limiting examples of which include its diagnostic and therapeutic properties, discussed below, as well as its activity in vitro and in vivo, are considered in the present description. To determine whether the peptide biological activity, qualitatively similar to the activity of unmodified molecules, can be carried out one or more tests such as in vitro, in vivo or clinically the cue experiment, in which the modified peptide is compared with the corresponding unmodified peptide (namely, with full KTPAF50 peptide or KTPAF50 peptide), analyzing in parallel; or an experiment in which the modified peptide analyze, to understand, does he biological effects similar to those of unmodified peptide determined in a separate experiment conducted. Such an experiment may be conducted, for example, by a method similar to that described below in the Examples. In relation to the modified nucleic acid molecules, the term "without substantial changes in biological characteristics of the modified molecules compared to the unmodified molecule" refers to the ability to encode a modified peptide having any of the characteristics mentioned above.

The modified peptide can be a peptide that includes a contiguous sequence of at least 8, 12, 15, 20, 25, 30, 35, 40 or at least 45 amino acid residues, having a degree of identity with the corresponding sequence consisting of at least 8, 12, 15, 20, 25, 30, 35, 40 or at least 45 amino acid residues included in KTPAF50 peptide comprising at least 70%, preferably at least 80%, more preferably at least 90% and in particular, less is th least 95%.

The invention also relates to a peptide containing a part of a continuous sequence contained in the full KTPAF50 the peptide comprising at least 8 amino acid residues, and the specified continuous sequence included in a continuous sequence specified full KTPAF50 peptide. In the present description, such a peptide is called "partial KTPAF50 peptide". Non-limiting examples of partial KTPAF50 peptides include SEQ. ID. NO:7 and SEQ. ID. NO:8, described below in Example VI.

The invention also relates to a protein or polypeptide comprising the amino acid sequence of the full KTPAF50 peptide, KTPAF50 peptide, modified peptide or partial KTPAF50 peptide (such protein or polypeptide in the present description is called the "protein comprising KTPAF50"). Protein, including KTPAF50 may, for example, be a hybrid protein, which includes a full KTPAF50 peptide, KTPAF50 peptide, modified peptide or partial KTPAF50 peptide; he may be a conjugate of a protein or another peptide or polypeptide containing the full KTPAF50 peptide, KTPAF50 peptide, modified peptide or partial KTPAF50 peptide; etc

The invention also relates to an oligonucleotide that includes at least 24 nucleotides, which represents: (i) an oligonucleotide that encodes a partial continuous after outermost, contained in KTPAF50 the peptide comprising at least 8 amino acid residues, which may include a continuous sequence of 24 nucleic acids contained in SEQ. ID. NO:1; (ii) the nucleotide sequence, which may in harsh environments subjected to hybridization with the nucleotide sequence corresponding to SEQ. ID. NO:1; (iii) an oligonucleotide that includes a sequence consisting of at least 24 contiguous nucleotides having a degree of identity with the corresponding contiguous sequence of nucleotides contained in SEQ. ID. NO:1, comprising at least 70%, preferably at least 80%, more preferably at least 90%, particularly at least 95%.

The invention also relates to a nucleic acid molecule, such as vector transfer or expression vector comprising any of the above nucleic acid molecules.

The invention also relates to modified peptides derived from any of a peptide as defined above, for example, modified peptides in which one or more amino acids are substituted by other amino acids by a conservative substitution. Used in the present description, the term "conservative substitution" means the replacement of amino acids of the same class am what nomikoto, corresponding to the same class, and the class is determined by the General physico-chemical properties of the side chains of amino acids and a high frequency of substitution in homologous proteins found in nature. It was divided into six General classes of side chains of amino acids, which include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of Asp by another residue class III, such as Asn, Gln or Glu is a conservative substitution.

In one example implementation, the amino acid sequence has only one substitution. In another embodiment, the amino acid sequence has two substitution. In yet another embodiment, the amino acid sequence has three substitutions. The maximum number of substitutions shall not exceed such number of amino acids, in which the unsubstituted sequence retains at least 70%, preferably at least 80%, preferably at least 90%, most preferably at least 95% amino acids. In one of the preferred embodiments, the substitution of up to 3, sometimes up to 6 amino acid residues, substituted by other amino acid residues are conservative substitutions.

In d the natives example implementation of one or more amino acids may be substituted for D-amino acids, preferably the corresponding D-amino acids. In the preferred embodiment, all amino acids are D-amino acids.

In another embodiment, the invention also includes a sequence having the reverse order in relation to the above sequences.

Thus, the invention also applies to full KTPAF50 peptides having the sequence of SEQ ID NO:2, or preferably to KTPAF50 peptides having the sequence of SEQ ID NO:4, or a partial KTPAF50 sequences of these peptides are modified by one or more conservative substitutions.

Thus, the invention also relates to a peptide comprising at least 10, or 15, or 20, or 25, or 30, or 35, or 40 amino acid residues, or the full sequence KTPAF50 peptide.

The invention also includes methods of treatment, diagnostics and pharmaceutical compositions, in which the used KTPAF50 peptide, full KTPAF50 peptide, partial KTPAF50 peptide, modified peptide or protein, including KTPAF50, or any nucleic acid molecule mentioned above. Methods of treatment, diagnostics and pharmaceutical compositions can be applied to one or more of the diseases and disorders listed above in the introductory section.

The pharmaceutical composition p is edlagaemoe according to the present invention, includes KTPAF50 peptide, full KTPAF50 peptide, partial KTPAF50 peptide, modified peptide or protein, including KTPAF50, or any nucleic acid molecule, referred to above, and pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" refers to any inert non-toxic material that does not react with the active ingredient. Sometimes the media selected in accordance with desired form of the composition. In some cases, the media can improve the delivery or penetration of the active ingredient to the target tissue, to increase the stability of medicines, to slow down the rate of clearance, to give the properties required for slow release, to reduce unwanted side effects and so the Media can also be a substance that stabilizes the composition (e.g., preservative), providing food features composition, etc. Media can be any conventionally used carriers; impose restrictions only physico-chemical parameters, such as solubility and reactivity with respect to the polypeptide, and the method of administration of the composition. The media may include additives, colorants, diluents, buffering agents, substances that regulate raspadaemost, humectants, preservatives, flavorings and farmacologiche the key compatible media. In addition, the media can be an auxiliary tool, which by definition is a substance that is known to affect the action of the active ingredient. Typical examples of the carriers include: (a) liquid solutions, in which an effective amount of the active substance dissolved in diluents, such as water, saline, natural juices, spirits, syrups, etc.; (b) capsules (e.g., normal, hard or soft gelatin capsules, containing, for example, surfactants, slip agents (lubricants and inert fillers), tablets, lozenges (in which the active substance is in the flavor basis such as sucrose and the Arabian gum or tragacanth gum, or the active substance is inert basis such as gelatin and glycerin), and pills, and each of them contains a certain number of active funds in the form of solids or granules; (C) powders; (d) suspensions in an appropriate liquid; (e) suitable emulsions; (f) liposomal formulations; and other media.

Potential diagnostic and therapeutic applications KTPAF50 peptide may include one or more of the following applications:

1. KTPAF50 can serve as a tool to diagnose the absence of immunological activity (immunological competence) after subcutaneous injection the injection of toxins, obtained from different organisms.

2. Definition of levels KTPAF50 in the blood can serve as an indicator of the level of activity of the immune system.

3. Level KTPAF50 may serve as an indicator of autoimmune diseases.

4. KTPAF50 can serve as a stimulator of the immune system. For example, KTPAF50 can be used to treat insufficient immunological activity against bacteria, parasites and viral toxins.

5. KTPAF50 can be used as a therapeutic tool to reduce allergic and inflammatory responses. For example, KTPAF50 can be used to reduce the frequency of asthma attacks or symptoms.

6. KTPAF50 can be used as a therapeutic tool for the treatment of immunodeficiency diseases such as AIDS and combined immunodeficiency.

7. KTPAF50 can be used as a therapeutic tool for the treatment of impaired glucose metabolism.

9. KTPAF50 can be used for treatment of some other diseases. For example, KTPAF50 can serve as a suppressor of the immune system, used in the treatment of autoimmune pathologies, such as inflammatory bowel diseases, heavy pseudoparallelism gravis, multiple sclerosis, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic auto is munney hemolytic anemia, colitis and Crohn's disease, etc.

10. KTPAF50 may serve as an immune system stimulant used to treat cancers, such as lung cancer, carcinoma of the pharynx, cancer of the head and neck and breast, Hodgkin's lymphoma, non-jackinsky lymphoma, breast cancer, hepatic cell carcinoma, melanoma.

11. KTPAF50 may enhance the immune response in younger or older patients or patients with compromised immune systems.

12. KTPAF50 can be used as a therapeutic tool for the treatment of male or female infertility.

13. KTPAF50 can serve as a General stimulator or inhibitor of a variety of immune responses and may also directly or indirectly affect other organs such as the heart, lungs, etc.

14. KTPAF50 can serve as a model for the identification of certain immune system cells and their use in cell therapy.

15. The sequence of nucleic acids encoding KTPAF50 or part thereof, can serve as a model for the identification of specific DNA sequences and cDNA person.

For the above diagnostic and therapeutic purposes can also be used full KTPAF50 peptide, partial KTPAF50 peptide or protein, including KTPAF50, or modified peptide.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

For the understanding of the invention and its implementation in practice, the following are your options for using non-limiting examples, accompanied by graphics, in which:

Figure 1 presents a graph showing the secretion of IL-17 (PG/ml) peripheral leukocytes of man, treated with the indicated concentrations (ng/ml) KTPAF50;

Figure 2 presents a graph showing the secretion of INF-γ (PG/ml) peripheral leukocytes of man, treated with the indicated concentrations (ng/ml) KTPAF50;

Figure 3 presents a graph showing the secretion of TNF-α (PG/ml) peripheral leukocytes of man, treated with the indicated concentrations (ng/ml) KTPAF50;

Figure 4 presents a histogram showing the dependence of the % of viable cells (normirovannogo control number) from the concentration KTPAF50 (µg/ml);

Figure 5 presents a histogram showing the degree of induction KTPAF50 mRNA (messenger RNA) in the cell types involved in immune cytotoxic response;

Figure 6 presents a graph which shows the dependence of U937 tumor volume (mm3) naked mice (nude mice)treated (▲) and not treated (■) (control) from time to time; and

Figure 7 presents a histogram showing the state of the dependence of the % of viable cells (normalized by control number) concentration (µg/ml) KTPAF50 peptide and its fragments.

DETAILED DESCRIPTION of embodiments of the INVENTION

Example I

From the libraries of human cDNA was isolated new cDNA.

For the analysis of PCR with reverse transcription, we used the following primers:

5' - GCT TCT GTC TAT CCT GGT TTC TGG - 3' (SEQ. ID. NO:5)

5' - TTT CTC HUNDRED GGG TAG ATG GG - 3' (SEQ. ID. NO:6)

We used the following PCR conditions:

95°C for 2 min

40 the following cycles:

95°C for 45 sec

59°C for 45 sec

72°C for 5 min

Destination cycles:

72°C for 5 min

The PCR product sequenced.

After PCR analysis on an agarose gel and staining Cybar Green (Invitrogene), the intensity of the PCR product was evaluated by means of an analyzer BioRad ChemiDoc. The results are presented below:

4678
The cDNA librarySignalG3PDH(Signal/G3pdh)the minimum ratio
Heart367554340,6762971,209034
The brain334059710,559371,000001
Placentae is* 602946681,291562,308954
Easy292941160,7116131,272169
Liver480960020,8012331,432385
Skeletal muscles584962730,9324091,666891
Kidney*827240692,0329323,634324
Pancreas*838438982,1508473,845123
Embryonic brain372155830,6664881,522944
Embryonic lung459255540,8267921,88943
Embryonic liver442455250,8007241,829678
Embryonic kidney*463537291,2429612,840202
Embryonic heart229152350,4376311,000001
Embryonic spleen384568270,5632051,28694
Embryonic thymus gland301351330,5869861,341281
Embryonic skeletal muscles282147540,5933951,355926
Spleen5476221160,2476041,179064
The thymus gland200380,2334561,111697
The prostate gland4685196620,2382771,134652
Egg*5710190030,3004791,430852
Ovary4435180720,2454071,168606
Small intestine3247154240,2105161,002458
Rectum2779118470,2345741,11702
* - significant results
Resting CD141185111650,1061351,061352
Resting CD8*1132100420112727 1,127265
Resting CD4194689320,2178682,178683
Mononuclear*86982040,1059241,059239
activated CD8*240685350,2818982,818981
activated CD4197990650,2183122,183122
activated mononuclear*169570820,2393392,393392
resting CD19*266863650,4191674,191673
activated CD19*163571400,2289922,289916
* - significant results the ATA

It can be noted that the main fabric, which is expressed cDNA represent kidney, pancreas, testes, and placenta. It is interesting to note that the product is also expressed in cells, and its expression varies with cell activation.

Example II

To determine the potential impact KTPAF50 various diseases have incubation KTPAF50 with peripheral leukocytes of man (pWBC), and measured the number of panels of cytokines.

KTPAF50 was chemically synthesized in the Company Anaspec Inc.

All human leukocytes were cultured in a medium containing RNA (phytohemagglutinin) (Biological Industries INC - catalog number - 01-201-1) (2 million cells/well in 2 ml medium). Cells were treated KTPAF50 for 3 days at concentrations KTPAF50 indicated on the figures. Control cells were not treated.

Over 3 days the medium was collected and analyzed by the ELISA method using a set of e-Bioscience for analysis of the human IL-17 (catalog number: 88-7176), human INF-γ (catalog number: 88-7316) and human TNF-α (catalog number: 88-7346). The results are presented in figures 1, 2 and 3.

It may be noted that adding KTPAF50 stimulates the secretion of all three defined cytokines by leukocytes pWBC. The secretion of IL-17 shows that KTPAF50 may have a proinflammatory effect. The secretion of INF-γ p is found, what KTPAF50 may have antiviral, anticancer and anti-inflammatory action. Secretion of TNF-α shows that KTPAF50 may play a role in stimulation of the immune system.

Example III

Also, to determine the effect of KTPAF50 on cancer conducted incubation KTPAF50 cancer cell lines.

The U937 cells acute myeloid leukemia and cancer cells RS prostate cancer were grown separately in medium with 10% FCS (calf serum) + RPMI and inoculable 4 samples on a 96-well plate at 20,000 cells/well.

Incubation KTPAF50 cells was carried out for one day and then viable cells were detected using resazurin (R&D System) and spectrophotometer. The results are presented in figure 4.

It may be noted that KTPAF50 causes a significant decrease in the number of viable cells of these two types of cancer.

Example IV

To study the role KTPAF50 in the immune response, was defined by the presence KTPAF50 in various cytotoxic immune cells.

Libraries of human cDNA, obtained from the following cells were purchased from Clontech Ltd:

1. mono - resting (R) and activated (A) monocytes (cells were activated by LPS action (liposaccharide) or RNA (phytohemagglutinin));

2. CD8 - R and cytotoxic CD8 T-cells;

3. CD19 - R and A CD19 B-cells;

4. CD4 - R and CD4 cells T-helper cells.

Quantitative analysis KTPAF50 mRNA in these cells was performed by means of PCR with reverse transcription, using specific primers KTPAF50. The results are presented in figure 5.

It can be noted that the activation of monocytes and cytotoxic T-cells leads to a significant increase in the expression of KTPAF50, while activation of b-cells leads to a decrease in the expression of KTPAF50. Activation or deactivation of cells T-helper cells has no effect on the expression of KTPAF50. Thus, KTPAF50 can be used as a marker for activation of the cellular immune response and to identify TN depending on ways Th2.

Example V

Action KTPAF50 on cancer cells was also tested in vivo.

14 females naked (mutation gene "nude"Nude mice by the age of 8-9 weeks were purchased from the Company Harlan Biotech Israel. Mice were injected subcutaneously 15×106the U937 cells. The tumor began to grow, and on the 9th day, the mice were divided into 2 groups:

The control group received injections of saline solution.

The group treated with injections KTPAF50 (25 ultragamma/mouse).

On the 20th day 4 mice of the control group killed for ethical reasons, because their tumors were huge. The results are shown in Fig.6.

Amazing results indicate that KTPAF50 completely prevents tumor growth.

Example VI

To determine the whole l is KTPAF50 peptide required to achieve the activity, the experiment described above in Example III was repeated, using U937 cells, and full KTPAF50 peptide and its fragments.

We used the following peptides KTPAF50:

A - KTPAF50 peptide (50 amino acids);

In - N-terminal 36 AA peptide KTPAF50

(LRRREQAERGSRRCAIAGEERAMLSPSPLPETPFSP) (SEQ. ID. NO:7);

With the C-terminal 14 AA peptide KTPAF50 (EKGAAFSPIYPRRK) (SEQ. ID. NO:8).

The results are shown in Fig.7.

It can be noted that the fragments KTPAF50 show anticancer activity similar to that KTPAF50 peptide.

1. An isolated polypeptide that is suitable for use for the treatment of cancer selected from leukemia; pancreatic cancer; prostate cancer; acute lymphocytic leukemia, and acute myeloid leukemia, stimulation of the immune system or stimulate the secretion of the cytokines IL-17, INF-γ and TNF-α, comprising an amino acid sequence corresponding to SEQ. ID. NO: 2 or SEQ. ID. NO: 4.

2. An isolated polypeptide that is suitable for use for the treatment of cancer selected from leukemia; pancreatic cancer; prostate cancer; acute lymphocytic leukemia, and acute myeloid leukemia, stimulation of the immune system or stimulate the secretion of the cytokines IL-17, INF-γ and TNF-α, comprising at least 20 amino acid residues of SEQ. ID. NO: 2.

3. An isolated polypeptide that is suitable for use for the treatment of cancer selected from leukemia; cancer POG lodochnoy gland; prostate cancer; acute lymphocytic leukemia, and acute myeloid leukemia, stimulation of the immune system or stimulate the secretion of the cytokines IL-17, INF-γ and TNF-α, comprising SEQ. ID. NO: 7 or SEQ. ID. NO: 8.

4. An isolated polypeptide that is suitable for use for the treatment of cancer selected from leukemia; pancreatic cancer; prostate cancer; acute lymphocytic leukemia, and acute myeloid leukemia, stimulation of the immune system or stimulate the secretion of the cytokines IL-17, INF-γ and TNF-α, comprising a modified sequence of SEQ. ID NO: 2 or SEQ. ID. NO: 4 in which each of from one to three residues replaced with another amino acid residue by conservative substitution without providing significant effect on the biological characteristics of the modified molecules compared to the unmodified molecule.

5. Pharmaceutical composition for treating a cancer selected from leukemia; pancreatic cancer; prostate cancer; acute lymphocytic leukemia, and acute myeloid leukemia, stimulation of the immune system or stimulate the secretion of the cytokines IL-17, INF-γ and TNF-α, comprising an isolated polypeptide according to any one of claims 1 to 4 as an active agent.

6. A method of treating cancer selected from leukemia; pancreatic cancer; prostate cancer; acute lymphocytic leukemia, and acute myeloid the th leukemia, includes introduction to the individual in need an effective amount of the polypeptide according to any one of claims 1 to 4.

7. The effective amount of the polypeptide according to any one of claims 1 to 4 as an active agent pharmaceutical composition for the treatment of cancer selected from leukemia; pancreatic cancer; prostate cancer; acute lymphocytic leukemia, and acute myeloid leukemia.



 

Same patents:

Hmo synthesis // 2517602

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and particularly to a bacterial cell stably cultured in a medium, where the cell is adapted to produce oligosaccharides. The cell is transformed such that it contains at least one nucleic acid sequence which encodes an enzyme involved in oligosaccharide synthesis. The cell is further transformed such that it contains at least one nucleic acid sequence which encodes a protein of the sugar efflux transporter family, or functional homologue thereof. The invention also relates to a method of producing oligosaccharides which involve said cell.

EFFECT: invention enables to obtain oligosaccharides with high efficiency.

13 cl, 3 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed are versions of an isolated monoclonal antibody, specific to hGM-CSF, where each version is characterised by a heavy and light chain. Each of the versions is characterised by the fact that it contains six appropriate CDR. Described are: a pharmaceutical composition, and a set, representing medication, based on the antibody application. Disclosed are: a coding isolated nucleic acid, an expression vector, containing it, and a vector-carrying host cell, used for the antibody obtaining. Described is a method of obtaining the antibody with the cell application.

EFFECT: claimed inventions can be applied for treating disease or disorder, associated with superexpression of hGM-CSF.

25 cl, 9 dwg, 14 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. What is presented is a method for generating an antibody and its functional fragments against a tumour antigen expressed on the tumour surface resistant to at least one anti-tumour compound by applying ground homogenate, and/or suspension, and/or cell lysate originated from the same tumour for immunisation. There are also disclosed using the method according to the invention for producing the monoclonal antibodies and their functional fragments, the monoclonal antibodies produced by the method, nucleic acids coding them, an expression vector, a host cell, and a method for preparing the antibody with using them, as well as hybridomes secreting these antibodies and their functional fragments for preparing a drug, the anti-tumour composition and using it as a drug.

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42 cl, 7 dwg, 4 ex, 6 tbl

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

FIELD: chemistry.

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21 cl, 2 dwg, 6 ex

FIELD: chemistry.

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30 cl, 4 dwg, 3 ex

FIELD: medicine.

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45 cl, 27 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a recombinant cell of Ralstonia eutropha, designed to obtain 2-hydroxyisobutyric acid. The cell is transformed by a plasmid with the sequence SEQ ID NO: 2.

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4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology. Described are antibodies against VEGF, one on which contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 4, 6 and 7, another contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 5, 6 and 7, disclosed in description. Also described are polynucleotides, coding said antibodies; espression vectors, containing said polynucleotides, and host cells, intended for obtaining antibodies in accordance with the claimed invention. Claimed is method of obtaining antibodies against VEGF, which includes expression of vector in host cell and separation of antibody. Disclosed is method of obtaining immunocongugate of antibody against VEGF, which includes conjugation of antibody with drug or cytotoxic agent. Described is method of VEGF identification, which includes identification of complex VEGF-antibody against VEGF in biological sample. In addition, described are compositions for treatment of VEGF-associated disease, one of which contains efficient quantity of antibody against VEGF, and another - efficient quantity of polynucleotide, coding said antibody. Also disclosed are methods of: 1) treating tumour, cancer or VEGF-associated cell proliferative disease; 2) inhibition if angiogenesis in subject and 3) inhibition of vascular permeability; consisting in introduction to subject of efficient quantity of antibody against VEGF in accordance with claimed invention.

EFFECT: invention makes it possible to obtain antibodies against VEGF and apply them for treatment of VEGF-associated diseases.

41 cl, 16 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a method for specific collection of DNA molecules (DNA aptamers) with high affinity for a recombinant protein target. Said method involves synthesis of a single polypeptide chain of a recombinant protein containing a fragment of glutathione S-transferase, a protein target, a peptide sequence split by the B. Anthracis lethal factor, a peptide which is biotinylated in vitro under the action of an E.coli biotin-ligase enzyme, binding the obtained recombinant polypeptide with an oligonucleotide library and immobilising the protein on paramagnetic particles bearing glutathione, washing the paramagnetic particles with the immobilised polypeptide from unbound oligonucleotides in a liquid stream, splitting the protein target with the bound DNA aptamers from the surface of paramagnetic particles with the B. anthracis lethal factor, separating and amplifying the DNA sequence with affinity to the recombinant protein target in a polymerase chain reaction and obtaining a set of single-chain DNA aptamers that are specific to the protein target.

EFFECT: invention provides efficient production of DNA aptamers with high affinity for recombinant protein targets.

4 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

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Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.

EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.

9 cl, 15 dwg, 11 tbl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a method for obtaining recombinant DNAse I of a human or its mutein, as well as their conjugates with polyethylene glycol, using a bacterium belonging to Escherichia class, transformed with expression plasmid, containing a promoter functioning in a bacterial cell, DNA fragment coding a hexahistidine cluster, a fragment coding enterokinase recognition sequence amalgamated in frame with human DNAse I or its functionally active mutein containing replacements of asparagine with cysteine, transcription termination section, vector pET28a(+) fragment containing initiation section of replication of bacteriophage fl, sequence coding aminoglycoside-3'-phosphotransferase, area of beginning of plasmid pBR322 replication, gene RNA-organising protein Rop, sequence coding lactose operon repressor.

EFFECT: invention allows obtaining recombinant human DNAse I or its mutein with high yield.

18 cl, 7 dwg, 1 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: isolated peptide having cytotoxic T lymphocyte (CTL) inducing capacity in the presence of an antigen-presenting cell bearing HLA-A*2402, is used to obtain antigen-presenting cells and therefore CTL. The obtained CTL are used for targeted action against CDCA1-expressing cancer cells.

EFFECT: invention provides an effective vaccine for inducing anti-tumour immunity in a subject.

16 cl, 4 dwg, 1 tbl, 1 ex

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.

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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: 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: biotechnology.

SUBSTANCE: invention relates to biotechnology, and represents a method of producing a recombinant mutein [C112S] of human enterokinase light chain, using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a mutein [C112S] inactive precursor of human enterokinase light chain, comprising a sequence encoding a cleavable N-terminal peptide comprising a hexahistidine cluster and the enterokinase recognition sequence, and a sequence fused with it in frame encoding a precursor of mutein [C112S] of human enterokinase light chain with non-cleavable C-terminal hexahistidine cluster under control of a promoter operating in the bacterial cell.

EFFECT: invention enables to produce the recombinant mutein of human enterokinase light chain with a high yield.

11 cl, 5 dwg, 1 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention represents a method of production of precursor of a recombinant fragment of human tissue plasminogen activator (tPA) (reteplase) using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a precursor of the recombinant fragment of human tissue plasminogen activator (tPA) (reteplase), comprising a sequence encoding a cleavable N-terminal peptide comprising decahistidine cluster and sequence of enterokinase recognition fused in frame, or a fragment of DNA encoding the [-1] methionyl of fragment of human tPA (reteplase), under the control of a promoter operating in the said bacterial cell.

EFFECT: invention enables to obtain a recombinant fragment of human tissue plasminogen activator with a high yield.

10 cl, 6 dwg, 1 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

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