Version of group i poaceae allergen characterised by lower allergenicity and maintained t-cell responsiveness (versions), coding dna molecule and application thereof

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

 

The present invention relates to the production and use of variants of group 1 allergens Roaseae (sweet grass sweet), which are characterized by reduced IgE reactivity compared with known allergens wild type and at the same time basically saved the reactivity with the T lymphocytes. These hypoallergenic variants of allergens can be used for specific immunotherapy (hyposensitization) patients with allergies to pollen or preventive immunotherapy allergies to pollen. The preferred embodiment of the present invention relates to variants of the main allergen PhI p 1 from Timothy grass pollen meadow (Phleum pratense).

Background of invention

Allergies type 1 are set on a world scale. Up to 20% of the population in industrialized countries suffer from diseases such as allergic rhinitis, conjunctivitis or asthma. These allergies are caused by allergens present in the air (aeroallergen), which are sources of different origin, such as plant pollen, mites, cats or dogs. Up to 40% of these patients suffering from allergies type 1, in turn, show specific IgE reactivity with pollen allergens (Freidhoff and others, 1986, J. Allergy Clin. Immunol. 78: 1190-2002). Substances that cause allergies type 1, before whom represent proteins, glycoproteins or polypeptides. After absorption through the mucous membranes of these allergens interact with IgE molecules attached to the surface of mastocytes in subjects with high sensitivity. Cross-linking of two IgE molecules with one another by means of allergen leads to release of mediators (such as histamine, prostaglandins and cytokines, effector cell and thus to the corresponding clinical symptoms.

Depending on the relative frequencies with which individual allergen molecules interact with IgE antibodies of patients suffering from allergies, make distinctions between major and minor allergens.

In the case of Timothy grass meadow (Phleum pratense), PhI p 1 (Petersen and others, 1993, J. Allergy Clin. Immunol. 92: 789-796), PhI p 5 (Matthiesen and Lowenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen and others, 1992, Int. Arch. Allergy Immunol. 98: 105-109), PhI p 6 (Petersen and others, 1995, Int. Arch. Allergy Immunol. 108, pp. 49-54). PhI p 2/3 (

Dominant major allergens of Timothy grass meadow (Phleum pratense) is a PhI p 1 and PhI p 5. As the major allergens of herbs of the family Roaseae are the Xia vysokomolochnye one another and therefore have very similar biochemical and immunological properties, these related proteins are grouped together as allergens group 1 and group 5. The group 1 allergens interact with IgE antibodies in more than 95% of patients suffering from pollen Allergy, and so are dominant major allergens of plant pollen.

The group 1 allergens are glycoproteins, which have a molecular weight of approximately 32 kDa and are localized in the cytoplasm of the pollen grains. As the contact of pollen grains with the mucous membrane of the upper respiratory tract and hydration of these pollen grains rain leads to rapid release of these allergens. The rapid release of the group 1 allergens in the form of subcellular particles provides penetration into the lower Airways, which can lead to the initiation of severe asthma attacks.

Were identified by cDNA-s group 1 allergens from Phleum pratense (Laffer and others, 1994, J. Allergy din. Immunol. 94: 689 - 698), Lolium perenne (Perez and others, 1990, J. Biol. Chem. 265: 16210-16250), Holcus lanatus (Schramm and others, 1997. J. Allergy Clin. Immunol. 1999: 781-787), Poa pratensis (Sturaro u. Viotti, 1998. NCBI GenBank Ace. No. A J131850), Cynodon dactylon (Smith and others, 1996, J. Allergy Clin. Immunol. 98: 331-343), Phalaris aquatica (Suphioglu and others, 1995, Clin. Exp. Allergy 25: 853-865) and Oryza sativa (Xu and others, 1995, Gene 164: 255-259).

In addition to these first descriptions of the sequences in the databases were published more sequences of group 1 allergen, which differ from the IP is adnych sequences in separate provisions. These isoforms also known for other pollen allergens.

Due to their homology to the group 1 allergens sweet grass sweet (Roaseae) have a high cross-reactivity with IgE antibodies person (Laffer and others, 1996, Mol. Immunology 33: 417-426). This immunological cross-reactivity based on a very similar amino acid sequences, as shown by comparing the sequence PhI p 1, group 1 allergen from Timothy grass meadow (Phleum pratense), with molecules of group 1 of the selected species in figure 1.

Homologous region sequence in the other group 1 allergens Roaseae exist for both regions of sequence deletions PhI p 1 amino acid sequences described herein in the structure of hypoallergenic variants and their flanking sequences. Besides, there are both the numbers and the surrounding region sequence cysteine allergen group 1 Roaseae. Due to the sequence homology of the group 1 allergens Roaseae classified in the protein family of β-expansins (Cosgrove D. J., 2000 Nature 407: 321-6).

The classical approach to effective therapeutic treatment of allergies is specific immunotherapy or hyposensitization (Fiebig, 1995, Allergo J. 4 (6): 336-339: Bousquet and others, 1998, J. Allergy Clin. Immunol. 102 (4): 558-562), which extracts natural allergen is injected subcutaneously to the patient with increasing doses. However, in this method there is a risk of allergic reactions or even anaphylactic shock. In order to minimize these risks, use the latest drugs in the form of allergoids. They are chemically modified allergen extracts, which have significantly reduced IgE reactivity, but is the same T-cell reactivity, comparable to the untreated extract. These T-cell epitopes are crucial for therapeutic action of allergenic preparations in hyposensitization (Fiebig., 1995, Allergo J. 4 (7): 377-382).

A greater degree of optimization of therapy would be possible with allergens obtained using recombinant methods. Certain mixtures of high-purity allergens obtained using recombinant methods, if desired, consistent with individual sensibilization models of patients could replace the allergen extracts of natural origin, as the latter, in addition to various allergens contain a relatively large number of immunogenic, but non-allergenic associated proteins.

Real prospects that can lead to safe hyposensitization using products of recombinant expression proposed specifically mutated recombinant allergens, in which IgE epitopes specifically removed without damage the statement of T-cell epitopes, which are important for therapy (Schramm and others, 1999, J. Immunol. 162: 2406-2414).

Another concept for hyposensitization is based on the fact that the protective immune response is called, in particular, IgG4 antibodies with blocking action. In accordance with this hypothesis have been described fragments of recombinant PhI p 1, which, as they say, must be appropriate to call protective IgG4 response (Ball and others, 1999, FASEB J. 13: 1277-1290).

This concept is completely different from the concept of hypoallergenic variants of allergens with reduced IgE reactivity and preserved T-cell reactivity.

Another possibility to influence the balance of T helper cells in patients suffering from allergies, using therapeutic methods is treatment with ekspresinvest DNA, which encodes the relevant allergens (immunotherapy DNA vaccination). Experimental confirmation of allergen-specific effect on the immune response was obtained in rodents by injection allergen-encoding DNA (Hsu and others, 1996, Nature Medicine 2 (5): 540-544).

The purpose of the present invention is to provide new variants of group 1 allergens from Roaseae on the protein and DNA level, which have reduced IgE activity with substantial preservation of T-cell reactivity, and therefore approaching the for treatment and prevention of specific immunotherapy immunotherapy DNA vaccination.

Figures

Figure 1: Block lined up PhI p 1-homologous amino acid sequences (sequences of Mature proteins derived from cDNA sequences from Roaseae species: Genus pratensis (Poa p), Holcus lanatus (Hol I), Lolium perenne (Lol p), Cynodon dactylon (Cyn d), Oryza sativa (Ory s) and Phalaris aquatica (Pha), protein sequences derived from cDNA sequences from Gen-Bank database of the National Center for Biotechnology Information (National Center for Biotechnology Information (NCBI, Bethesda, USA), numbering: the position of the amino acids of the Mature protein is marked with an underscore: amino acids that differ from PhI p 1 sequence, a "black box": cysteine.

Figure 2: Block lined up amino acid sequences processed PhI p 1 protein wild-type and variant PhI p 1 NoCys, PhI p 1 Wt (wild type): the protein sequence derived from the cDNA sequence (registration Z27090 in "GenBank" database of the National Center for Biotechnology Information (NCBI), Bethesda, USA), numbering: the position of the amino acids of the Mature protein, highlighted in black: amino acid substitution of cysteine for serine in the protein PhI p 1 NoCys.

Figure 3: Block lined up hypoallergenic amino acid sequence variants of PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220, shown by example, the numbering: the position of the amino acids marked with an underscore: d is the lecture can be found.

Figure 4: SDS-PAGE and comparing the identity of the recombinant variants PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220

A: SDS-PAGE

In: Western blotting with aPhI p 1 antibodies (Allergopharma)

1. Marker proteins

2. nPhI p 1*

3. rPhI p 1 Wt (-His-tag)*

4. Marker proteins

5. PhI p 1 NoCys (+His-tag)

6. PhI p 1 NoCys D 213-220 (+His-tag)

7. PhI p 1 NoCys D 1-6, 115-119, 213-220 (+His-tag)

8. Marker proteins

* Samples recovered (dithiothreitol)

Figure 5: Strip test for comparison of IgE binding ability PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 in adenocarinoma conditions.

1) rPhI p 1 Wt

2) PhI p 1 NoCys

3) PhI p 1 NoCys D 213-220

4) PhI p 1 NoCys D 1-6, 115-119,213-220

5) rPhI p 1 Wt

TP: total protein staining

P: serum of patients suffering from clinically definite Allergy to pollen.

Figure 6: Determination of reduced IgE reactivity PhI p I NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 with EAST test inhibition with four typical four sera of patients allergic to pollen (P).

Figure 7: Definition of hypoallergenicity PhI p 1 NoCys using test activation of basophils with basophils four patients suffering from allergies to pollen (P).

Figure 8: Definition of hypoallergenicity PhI p 1 NoCys Δ 213-220 using test activation of basophils with basophils four patients suffering from allergies to pollen (P).

Figure 9: Determining the s of hypoallergenicity PhI p 1 NoCys Δ 1-6, 115-119, 213-220 using test activation of basophils with basophils four patients suffering from allergies to pollen (P).

The work leading to the found variants was performed using a PhI p 1 as a model allergen. From built of blocks of sequences shown in figure 1, it becomes clear that due to the high homology within group 1 were obtained the same results if the starting point to take a different allergen groups 1.

Thus, it is also necessary to assume that the results presented above and below, can also be applied to the Sec with 1 of Secale cereale or could be obtained using Sec with 1, while the sequence is still unknown for this group 1 allergen.

In addition, the present invention relates to variants of group 1 allergens from Roaseae, which are characterized by reduced IgE reactivity compared with known allergens wild type and at the same time saved the reactivity with the T lymphocytes. These group 1 allergens are mostly PhI p 1, PoA p 1, Hol p 1, Lol p 1, Cyn d 1, Ory s 1 and Pha a 1 from Phleum pratense, Lolium perenne, PoA pratensis, Holcus lanatus, Cynodon dactylon, Oryza saliva and Phalaris aquatica. More preferred, PhI p 1, PoA p 1, Hol p 1, Lol p 1 or Pha a 1, and particular preference is given to PhI p 1.

The starting point for construction of hypoallergenic variants of group 1 allergens is cDNA PhI p 1 dick is the second type, you allocate using specific primers (seed) by polymerase chain reaction (PCR) from total cDNA of pollen from Phleum pratense ("GenBank" registration Z27090; NCBI, Bethesda, USA) (SEQ ID NO 1).

Amino acid sequence of SEQ ID NO 2 is obtained from the cDNA sequence of wild-type PhI p 1.

PhI p 1, which consists of 240 amino acids is glycosylated in a natural form, like all the group 1 allergens (see figure 1), is characterized by the existence of seven cysteines in the Mature molecule. Except Cyn din Ory s 1 these provisions of the amino acids are numbered 41, 57, 69, 72, 77, 83 and 139 all of the group 1 allergens (Petersen and others, 1995, J. Allergy Clin. Immunol 95: 987-994).

PhI p 1 was expressed in E. coli as a non-glycosylated protein. Recombinant wild-type protein (rPhI R 1 wt) interacts with IgE antibodies of patients suffering from allergies to pollen, which have reactivity with purified natural PhI p 1 (nPhI p 1) (Petersen and others, 1998, Clin. Exp.Allergy 28: 315-321).

A detailed description of the invention

Fabrication and characterization of hypoallergenic variants PhI p 1

Proceeding from the described rPhI R 1 wt cDNA receive recombinant variants PhI p 1, which is modified through genetic engineering.

Amino acid sequence of the recombinant variant PhI p 1 NoCys (SEQ ID NO 4) has seven serine residues instead of seven cysteines found in wild type (figure 2). Variant PhI p 1 NoCys logic as a starting point to build different variants with deletion. Them in each case were removed separate parcels that have a length of from 15 to 90 bp or a combination of these areas cDNA coding for PhI p 1 NoCys, which leads to the corresponding deletions of amino acids 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 in the polypeptide chains of proteins expressed in E. coli: PhI p 1 NoCys Δ 1-6 (SEQ ID nos 5 and 6), PhI p 1 NoCys Δ 1-30 (SEQ ID NO 7 and 8), PhI p 1 NoCys Δ 92-104 (SEQ ID NO 9 and 10), PhI p 1 NoCys Δ 115-119 (SEQ ID NO 11 and 12), PhI p 1 NoCys Δ 175-185 (SEQ ID NO 13 and 14), PhI p 1 NoCys Δ 213-220 (SEQ ID NO 15 and 16), so e and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 (SEQ ID NO 17 and 18).

Recombinant proteins were expressed as histidine-hybrid proteins in Escherichia coli. Immunological characterization was performed with a hybrid proteins of this type.

First, after immobilization on nitrocellulose membrane recombinant variants were investigated for the ability to bind IgE antibodies typical serum pool and IgE antibodies individual sera of patients allergic to pollen (strip test). In this way unexpectedly observed reduced binding of IgE antibodies with PhI p 1 NoCys. This result is confirmed using IgE test inhibition (EAST), which explores the IgE binding capacity neimmunizirovannah protein to IgE antibodies in solution.

Moreover, the present invention relates, in particular, to the cases of group 1 allergen, in which cysteine at amino acid positions 41, 57,69, 72, 77, 83 and 139 in accordance with Mature PhI p 1 protein is removed or replaced by another amino acid. Particular preference is given in this description appropriate options PhI p 1, PoA p 1, Hol p 1, Lol p 1 or Pha a 1, especially PhI p 1.

Reduced binding of IgE antibodies is achieved if at least two of the 7 cysteines deleted without replacement or replaced by another amino acid. Preferably, however, if all 7 cysteine replaced by serine.

Effects reduced IgE binding capacity PhI p 1 NoCys on the functional action during the cross-linking of membrane-associated IgE effector cells and their activation in vitro was investigated using a test activation of basophilic granulocytes of patients suffering from allergies to pollen. PhI p 1 NoCys showed significantly lower activation of basophilic granulocytes in this test compared to rPhI R 1 wt and thus functionally reduced allergenicity.

Various mutants with deletions derived from PhI p 1 NoCys were investigated in the IgE binding capacity (strip test, EAST) and functional activity (activation of basophils) by the same method. Unexpectedly, the mutant with a deletion showed particularly strong allergenic properties.

Therefore, the present invention also relates to variants of group 1 allergens, which are optional in addition to the above is the option with the remote or is substituted by Cys, at least one section or combination of sections that correspond to amino acids 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 primary sequence of Mature PhI p 1 protein lacking compared to the wild-type allergen.

Particular preference is given in this description of the respective mutants with the deletion of the group 1 allergens PhI p 1, Poa p 1, HoI p 1, Lol p 1 and Pha a 1. Even more particular preference is given to the relevant PhI p 1 variants.

Particular preference is given, and the present invention therefore also relates to variants of group 1 allergens, lacking only amino acids 213-220, or both amino acids 1-6 and 115-119 in accordance with Mature PhI p 1 sequence. Preference is again given in the description of allergens PhI p I, Poa p I, Hol p 1, Lol p 1 and Pha a 1, where PhI p 1 is especially preferred.

T-cell reactivity hypoallergenic variants PhI p 1, which forms the basis for the effectiveness of specific immunotherapy was tested in vitro by the test on the proliferation of using PhI p 1-specific T-lymphocytes of patients suffering from allergies to pollen. Modified allergens showed significantly preserved T-cell reactivity, which allows immunotherapy of application of hypoallergenic variants PhI p 1.

Hypoallergenic variants according to the present invention can be derived from cloned DNA sequences using methods of genetic engineering. In principle, however, can also be used and chemical modification of natural allergen extract (Fiebig, 1995, Allergo J. 4 (7), 377-382). Additional modifications at various positions, for example, in order to increase hypoallergenic, are, of course, also possible, in addition to variations of group 1 allergens described in this patent application. These modifications may, for example, be an amino acid inclusion, deletion and substitution, the decomposition of the protein into fragments, and fusion protein or its fragments with other proteins or peptides. Moreover, this invention relates to a DNA molecule that encodes a variant of the allergen described above, expressing recombinant vector containing the DNA molecule, and the body-master, transformed using the selected DNA molecule, or a specified expressing vector. Suitable organisms-owners can be Pro - or eukaryotic, single - or multi-celled organisms, such as bacteria or yeast. The body-master, which is preferred in accordance with this invention is E. coli.

Moreover, the present invention relates to a method for producing a variant of the allergen in accordance with this invention by culturing specified the host body and the selection of the corresponding option is Llerena of culture.

The present invention additionally relates to variants of the allergen, DNA molecules and expressing the vectors described above in their property as medicines.

Moreover, the present invention relates to pharmaceutical compositions that include at least one of these options allergen or the corresponding DNA molecule or the corresponding expressing vector and optional additional active compounds and/or ancillary medicinal substance for treatment of allergies in the triggering of which involved the group 1 allergens from Roaseae, or for immunotherapy vaccination of patients with allergies in the triggering of which involved the group 1 allergens from Roaseae, and/or for the prevention of such allergies.

If the pharmaceutical compositions are of the second type (which include at least one DNA molecule or expressing vector), these compositions preferably include aluminum hydroxide, immunostimulating CpG-containing oligonucleotide, or a combination of these two as a subsidiary of drugs.

For the purposes of this invention the pharmaceutical composition can be used as therapeutic agents in the treatment of human or in veterinary medicine. Appropriate order what indicators are organic or inorganic substances, suitable for parenteral use and do not interact with variants of group 1 allergens in accordance with this invention. Suitable for parenteral application are, in particular, solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants. Options allergens in accordance with this invention can also be lyophilized and the resulting lyophilizate used, for example, to get injectables. These compositions can be sterilized and/or may include ancillary medicinal substances, such as lubricants, preservatives, stabilizers and/or wetting agents, emulsification, salts for modifying the osmotic pressure, buffer substances and/or multiple additional active compounds.

In addition, drugs with a slow release can be obtained by appropriate receptionarea options allergens in accordance with this invention, for example by adsorption on aluminum hydroxide.

Finally, the present invention relates to the use of at least one variant of the allergen in accordance with this invention or a DNA molecule in accordance with this invention or expressing the vectors in accordance with this invention DL is receiving medications for treatment of allergies, in the initiation which involved the group 1 allergens from Roaseae, or for immunotherapy vaccination of patients with allergies in the triggering of which involved the group 1 allergens from Roaseae, and/or for the prevention of such allergies.

Receiving options PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 (figure 3) and their immunological characteristics described below using an example to hypoallergenic variants PhI p 1 with modifications of genetic engineering, described above.

Expression and purification of recombinant variants PhI p 1

Recombinant proteins were expressed as histidine-hybrid proteins (expressing vector pProExHT; Invitrogen, Carlsbad, USA) in Escherichia coli (strain JM109). rPhI R 1 wt and variants were first cleaned by specific binding N-terminal his-tag residues with Ni2+-chelate matrix (affinity chromatography with immobilized metal ion IMAC) and then by preparative gel chromatography (size-exclusion chromatography, SEC). Purity ruiruima proteins was controlled by SDS-PAGE and analytical SEC (figure 4,a). The identity of the purified proteins showed by linking PhI p 1-specific monoclonal antibody (figure 4,b).

Demonstration of reduced IgE binding of recombinant variants PhI p 1

A simple test method to determine the IgE reactivity of specific IgE is C serum of patients suffering from allergies, membrane-attached test proteins is a strip test.

For this purpose, the test substance is attached in parallel one to another in the same concentration and number on a strip of nitrocellulose membrane under non-denaturing conditions. A series of such membrane strips can be incubated in parallel with different sera of patients suffering from allergies. After the stage of washing is specifically associated IgE antibodies do visible on the membrane using a color reaction, promoted by using anti-human IgE / alkaline phosphatase conjugate.

The results of the test strip for PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220, using the serum of individual patients suffering from pollen Allergy, shown in this description using the example described modified PhI p 1 molecules (figure 5). Were used only serum of patients suffering from allergies, have a strong IgE titer against natural PhI p 1. IgE antibodies in these patients also interact with recombinant equivalent rPhI p 1 wt.

It is clear that PhI p 1-specific IgE antibodies in the serum of all patients bind recombinant variant PhI p I NoCys reduced in degree, but not allergen PhI p 1 wild-type.

Even greater reduction in IgE binding capacity is achieved PU is eat additional removal of certain sections of the sequence, shown with reference to variant PhI p 1 NoCys Δ 213-220. Variant PhI p I NoCys Δ 213-220 shows strongly reduced IgE binding capacity with all tested sera of patients suffering from allergies, compared to the unmodified recombinant protein of the wild type. Additional reduction in IgE binding capacity PhI p 1 IgE antibodies from specific serums can be achieved by a combination of several deletions, which are apparent from the test result for the variant PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 serum R patients suffering from pollen Allergy (figure 5).

Thus, it becomes clear that the displacement of cysteine and deletion of specific sections of the sequence reduces IgE binding capacity PhI p 1 molecule.

In contrast to the test strip EAST test inhibition (enzyme allergosorbent test) allows you to conduct a study allergen / IgE interactions in solution, providing a disturbing influence on the masking of epitopes of the test substance in order to largely exclude them by immobilization on the membrane. EAST test for inhibition carried out as follows. Microtitre tablets covered with allergens, in this description nPhI p 1. After removal of unbound molecules of the allergen by washing the tablet block, using the bullish saw retacnyl albumin, in order to prevent later nonspecific binding. IgE antibodies of patients suffering from allergies, as a common pool of individual sera (serum pool) or as individual serum, incubated with the appropriate dilution of the allergen-coated microtitre tablets. The number of allergen-bound IgE antibodies determined photometrically using an anti-hlgE / alkaline phosphatase conjugate by reaction of the substrate with obtaining a colored end product.

The binding of IgE antibodies inhibit substance specifically using soluble allergens or substances that you want to test (recombinant modified allergen) as a function of concentration.

The test results for PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 shown in this description by example for modified PhI p 1 molecules described in comparison with a standard molecule nPhI p 1.

Typical IgE tests of inhibition, shown in Fig.6, with four individual sera of patients suffering from allergies to pollen, show that there was only about 20-50% of the maximum inhibitory effect of unmodified natural allergen nPhI p1, even with high concentrations variant PhI p 1 NoCys (up to 5 µg/ml). Lower maximum effective deactivatable loss of IgE epitopes.

Curves for variants PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 demonstrate even lower IgE binding capacity of these PhI p 1 variants. The inhibitory effect could not be determined with individual variations or only in a very small degree (0-20% of the maximum inhibitory effect). In accordance with the test strip therefore can be proven that the inclusion of additional specific deletions further reduces IgE binding capacity PhI p 1.

The definition of hypoallergenicity recombinant variants PhI p 1 using the test activation of basophils

Functional reduction in allergenicity was determined in vitro using a test activation of basophils. To test the activation of basophils heparinized whole blood of patients suffering from allergies to pollen, incubated with different concentrations of the test substances. Allergenic substances that specifically bind with FcεRI-bound IgE antibodies basophils, resulting in cross-linking FcεRI molecules. This is caused by allergen IgE-promoted FcεRI cross-linking leads to the activation of basophils. Activation represents the first phase in allergic reactions of these effector cells. Subsequent signal transduction results in the degranulation of effector cells and thus it is iniciirovanii allergic reactions in vivo.

In vitro, caused by allergen activation of basophilic granulocytes, can be determined by quantitative analysis of the expression of surface protein (CD203c), which is combined with the signal transduction IgE receptor cross-linking (Kahlert and others, 2003, Cli., Exp. Allergy 33: 1266-72). The number downregulation of CD203c proteins on the cell and the percentage of activated cells cell pool is measured with high sensitivity by using svyazyvaniya fluorescently-labeled monoclonal antibodies with surface marker and subsequent analysis by fluorescently-activated flow cytometry. The standard substances used in this study consisted of purified natural PhI p 1 (nPhI p 1) in parallel with the test substances. Results for PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 shown in this description using the example described modified PhI p 1 molecules.

Typical test results for a variant PhI p 1 NoCys with basophils four patients suffering from clinically certain allergies, shown as curves in Fig.7. The reduction in allergenic efficiency options PhI p 1 NoCys towards nPhI p 1 wild type becomes apparent offset in the curves of activation.

In accordance with the results of the test strip and IgE test for inhibition of the test results for Varian is s PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1-6, 115-119, 213-220 show even greater reduction in the relative allergenic efficiency, as shown in Fig and 9 on the basis of typical curves.

While the largest proportion of basophils activated with natural allergen in the concentration range of test substances 100-1000 PM, not determined or no identified only a very low activation of basophils using modified allergens PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220.

Allergenic efficiency PhI p 1 NoCys Δ 213-220 was, as can be calculated using the A50 values of the curves decreased in ~ 100-1000 times and allergenic efficiency options PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 was reduced by more than 1000 times in comparison with standard nPhI p 1 (A50: the concentration of allergen in 50% of the maximum number of activated basophils).

T-cell reactivity hypoallergenic variants PhI p 1

T-helper lymphocytes interact with the peptide fragments of the allergen (approximately 12-25 amino acids), which are obtained by enzymatic degradation of antigen-presenting cells (APC) and injected into T-cells after activation of suitable peptides in individual MHC molecules of class II on the surface of APC. This allergen-specific activation of T-helper lymphocytes is necessary for proliferation and functional differential and (TN and TN). Effect on allergen-specific T-lymphocytes by treatment with allergen or derivative of allergen within hyposensitization is seen as the key to therapeutic efficacy.

In order to investigate T-cell reactivity, oligoclonal T-cell lines of patients suffering from allergies to pollen, established by well-known methods with stimulation using nPhI R 1 or rPhI p 1 wt molecules. In the test on the proliferation of various T-cell lines stimulated the standard allergens nPhI R 1 and rPhI p 1 wt and modified recombinant PhI p 1 variants. The rate of proliferation was determined by incorporation of [3H]-thymidine well-known methods.

The results of the test on the proliferation of PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 shown in this description using the example described modified PhI p 1 molecules.

Results from T-cell lines eight patients suffering from allergies to pollen, are presented in table 1, show that you can stimulate T-lymphocytes for proliferation using recombinant variants of allergens. T-cell reactivity PhI p 1 NoCys, PhI p 1 NoCys Δ 213-220 and PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 only slightly reduced compared to non-modified natural allergens and recombinant wild-type allergens, which shows the saving to eticheskikh T-cell epitopes.

Construction of hypoallergenic variants PhI p 1 using genetic engineering

Example 1:PhI p 1 NoCys

To build a variant PhI p 1 NoCys (SEQ ID NO 3 and 4) conduct six PCR stages, starting with cDNA rPhI p 1 wt ("Gen-Bank check Z27090; NCBI, Bethesda, USA). Point mutations introduced using specific PCR primers that contained codons encode the series instead of primers for cysteine (sequences of primers see table 2).

Stage 1 - Receipt of the N-terminal DNA fragment "PhI p 1 [C41S, C57S, C69S] (bp 1-212)": a DNA fragment containing the mutation C41S, C57S, C69S, generated by amplification of long overlapping oligonucleotides (P 1-63, P 49-111, P 97-158 and P 144-212) using PCR.

Stage 2 - Getting C - terminal DNA fragment "PhI p 1 [C69S, C72S, C77S, C83S] (bp 193-720)": PCR PhI p 1 wt - cDNA with primers P 193-261 and P 703-720 HindIII.

Stage 3 - Getting DNA coding for "PhI p 1 [C41S, C57S, C69S, C72S, C77S, C83S] (bp 1-720)": overlapping PCR fragments "PhI p 1 [C41S, C57S, C69S] (bp 1-212)" and "PhI p 1 [C69S, C72S, C77S, C83S] (bp 193-720)with primers P 1-63 and P 703-720 HindIII.

Stage 4 - Getting the N-terminal DNA fragment "PhI p 1 [C41S, C57S, C69S, C72S, C77S, C83S, C139S] (bp 1-428)": PCR cDNA "PhI p 1 [C41S, C57S, C69S, C72S, C77S, C83S] (bp 1-720)with primers P 1-63 and P 406-428 as.

Stage 5 - Obtaining the C-terminal DNA fragment "PhI p 1 [C139S] (bp 406-720)": PCR cDNA rPhI p 1 wt with primers P 406-428s and P 703-720 HindIII.

Stage 6 - Obtaining a full DNA kodirovaniyadlya PhI p 1 NoCys: overlapping PCR fragments "PhI p 1 [C41S, C57S, C69S, C72S, C77S, C83S, C139S] (bp 1-428)" and "PhI p 1 [C139S] (bp 406-720) with primers P 1-63 and P 703-720 Hindlll".

DNA coding for PhI p 1 NoCys process, using the restriction enzyme HindIII, and include in expressing vector pProExHT (Invitro-gen, Carlsbad, USA) using the restriction enzymes cut sites EheI and HindIII and then set the sequence completely.

Example 2: PhI p 1 NoCys Δ 213-220

A DNA sequence encoding a variant with a deletion of PhI p 1 NoCys Δ 213-220 (SEQ ID NO 15 and 16), was obtained using PCR DNA PhI p 1 NoCys using 5'-primer R 1-18 and 3'-primer R 613-720 (Δ 637-660) HindIII, specifically shorter region of the sequence that you want to delete.

cDNA encoding process using the restriction enzyme HindIII, and include in expressing vector pProExHT (Invitro-gen, Carlsbad, USA) using the restriction enzymes cut sites EheI and HindIII and then set the sequence completely.

Example 3: building a PhI p I NoCys Δ 1 to 6, 115-119, 213-220 using genetic engineering

A DNA sequence encoding a variant with a deletion of PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 (SEQ ID NO 5 and 6)was obtained by three stages using PCR, using oligonucleotides, specifically shorter region of the sequence that you want to delete.

Stage 1 - Receipt of the N-terminal DNA fragment "PhI p 1 NoCys Δ 1-6 (bp 1-300)": PCR cDNA PhI p 1 NoCys with primeraly-63 (A1-18) and P 250-318.

Stage 2 - Getting C-terminal DNA fragment "PhI p 1 NoCys Δ 115-119, 213-220 (bp 283-663)": PCR PhI p 1 NoCys with primers P 301-384 (Δ 343-357) and P 613-720 (Δ 637-660) HindIII.

Stage 3 - Getting the complete DNA coding for PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220: overlapping PCR fragments "PhI p 1 NoCys Δ 1-6 (bp 1-300)" and "PhI p 1 NoCys Δ 115-119, 213-220 (bp 283-663)with primers P22-63 (Δ 1-18) and P 703-720 HindIII.

DNA coding for PhI p 1 NoCys Δ 1 to 6, 115-119, 213-220 process using restriction enzyme Hindm, and include in expressing vector pProExHT (Invitro-gen, Carlsbad, USA) using the restriction enzymes cut sites Ehel and HindIII and then set the sequence completely. DNA variants PhI p 1 NoCys Δ 1-6 (SEQ ID nos 5 and 6), PhI p 1 NoCys Δ 1-30 (SEQ ID NO 7 and 8), PhI p 1 NoCys Δ 92-104 (SEQ ID NO 9 and 10), Phlp 1 NoCys Δ 115-119 (SEQ ID NO 11 and 12), PhI p 1 NoCys Δ 175-185 (SEQ ID NO 13 and 14) was obtained, cloned and determined their sequence, respectively.

1. Variant of the group I allergen of Roaseae with substitutions Cys41Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser in the sequence of the Mature protein PhI p1, characterized by amino acid sequence SEQ ID NO:4 and reduced IgE reactivity compared with a known allergen wild-type and largely saved by the reactivity with the T lymphocytes.

2. Option group 1 allergen from Roaseae with substitutions Cys41Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser and absence of fragments, which correspond to amino acid residues 1-6, 1-30, 92-104, 115-119, 17-185 and 213-220 in the primary sequence of the Mature protein PhI p1, characterized by amino acid sequence SEQ ID NO:16 and a reduced IgE reactivity compared with a known allergen wild-type and largely saved by the reactivity with the T lymphocytes.

3. Option group 1 allergen from Roaseae with substitutions Cys41Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser and absence of fragments, which correspond to amino acid residues 1-6, 115-119 and 213-220 in the primary sequence of the Mature protein PhI P1, characterized by amino acid sequence SEQ ID NO:18 and a reduced IgE reactivity compared with a known allergen wild-type and largely saved by the reactivity with the T lymphocytes.

4. Variant of allergen according to claims 1 to 3, characterized in that it is obtained using recombinant genetic engineering techniques.

5. Variant of allergen according to any one of claims 1 to 3 for the prevention or treatment of Allergy, the development of which is mediated by group I allergens from view Roaseae.

6. A DNA molecule encoding a variant of the allergen according to any one of claims 1 to 3.

7. The DNA molecule according to claim 6 for the prevention or treatment of Allergy, the development of which is mediated by group I allergens from view Roaseae.

8. The use of at least one variant of allergen according to any one of claims 1 to 3 to obtain a medicinal product intended for the prevention and treatment of allergies, the development of the which mediate the group I allergens from view Roaseae.



 

Same patents:

FIELD: medicine.

SUBSTANCE: biologically active peptide is proposed with the common formula: , where OBu - residue of 2-oxobutyric acid, Abu - residue of 2-aminobutyric acid, Dha - residue of 2,3-didehydroalanine, Dhb - residue of 2,3-didehydroaminobutyric acid, besides residues are in sequence linked with amide links, residues Abu3-Ala7, Abu22-Ala27 and Abu24-Ala31 are also linked pairwise with thioether links with formation of three residues of 3-methyllanthionine, residues Ala11-Ala21 are linked with thioether link to form residue of lanthionine. This peptide has antimicrobial effect at gram-positive bacteria Bacillus subtilis, strains L1 and 1621; Bacillus pumilus, strain 2001; Bacillus globigii, strain I; Bacillus amyloliquefaciens, strain I; Bacillus megaterium, strain VKM41; Mycobacterium smegmatis, strain 1171; Mycobacterium phlei, strain 1291; Micrococcus luteus, strain B 1314; Staphylococcus aureus, strain 209p; Rhodococcus sp., strain SSI.

EFFECT: increased efficiency of peptide antimicrobial action.

4 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: as feedstock for fractioning used is anticytomegalovirus plasma with activity index not less than 70%. Process of production includes: ultrafiltration of immunoglobulin at ion force not higher than 0.02 and pH value 4.0-5.7 with further diafiltration against distilled water with pH value 3.5-6.0. Immunoglobulin is incubated with maltose of 2-11% concentration at temperature 32-40°C and pH value 4.0-4.8 for 10-36 hours, or with pepsin in dose 1:10-6-1:10-5 in presence of 2-11% maltose. After that inactivation/removal of viruses is carried out by immunoglobulin incubation at pH value 4.0-4.4 and/or with application of depth filtration, and/or nanofiltration. Afterpurification of immunoglobulin is performed by methods of: ultrafiltration and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography, and/or dialysis. Immunoglobulin stabilisation is carried out with application of sodium chloride and/or maltose, and/or proline, and/or glucose and/or glycerol, whose content provides preparation osmolarity within 270-400 mOcm/kg. In obtained immunoglobulin preparation pH value 5.0-7.5 and protein content 4.5-10.5% are installed.

EFFECT: invention allows to obtain highly avid immunoglobulin preparation against cytomegalovirus for intravenous introduction with improved and stable qualitative quaracteristics.

7 cl, 5 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a pharmaceutical composition which contains TAT-HOXB4 protein as an effective component. Said composition has stimulating effect on production of hematopoietic stem cells. More specifically, the recombinant protein TAT-HOXB4 enhances acceptance of intramedullary transplants, hematopoietic reconstruction, repopulation and number of circulating stem cells, specifically after chemotherapy or exposure.

EFFECT: higher protein output and stability.

24 cl, 11 dwg, 2 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention can be used in medical and biologic industry for preparing antineoplastic drugs. Plasmid DNA pFK2 providing synthesis of recombinant analogue of human kappa casein fragment, in Escherichia coli cells is designed; and a method for preparing a recombinant product with using it is described. The recombinant analogue of human kappa-casein fragment recovered from Escherichia coli cells transformed by recombinant plasmid DNA pFK2 has molecular weight of approximately 16 kDa; consists of residual methionine, human kappa-casein fragment with 24 on 134 amino acid residue and C-terminal histidine path and exhibits apoptotic activity in relation to malignant cells.

EFFECT: higher anticancer activity of the compounds.

3 cl, 6 dwg, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

3 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.

EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.

17 cl, 1 dwg, 7 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: in dissolvent, which contains from 55% to 70% of water (wt/wt), precursor of insulin or precursor of insulin derivative is exposed to fermentative splitting at alkaline values of pH. In process of fermentative splitting, they use tripsin or lysil-specific protease, preferably Achromobacter lyticus protease I. Then without separation of intermediate product from reaction mixture, mentioned intermediate product is fermentatively complemented with nucleophilic compound, which represents aminoacid ether, aminoacid amide, peptide, peptide ether or peptide amide in reaction mixture, having water content in the range from 10% to 50% of water (wt/wt), at acidic values of pH, close to neutral pH value. If required, protective group (s) is/are removed.

EFFECT: preparation of insulin compound from its precursor by efficient improved method.

24 cl, 5 ex

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: murine benign spontaneous breast tumour cells are cultivated. At first, the cultivation procedure is in vitro in the DMEM/RPMI-1640 medium with 10 % of bovine embryo serum up to 20 passages. Then, in vivo up to 22 passages. Then, once again in vitro up to 25 passages. Then, 10 thousand-1 million produced cells are introduced in a connective tissue capsule formed in the Balb/c mice after the introduction of a biocompatible gel. The tumour growth is enabled. The encapsulated tumour is aseptically removed and trypsinised to produce primary epithelial cell cultures. The produced cells concentrated 10 thousand-1 million are introduced into the capsule subcutaneously gel-formed in the Balb/c mice. The tumour growth is enabled for vaccination. In the in vitro culture, the cells are transplanted once a week while being disseminated in the ratio 1:3 - 1:4, with a monolayer twice washed with EDTA solution and removed with 0.25% trypsin during 2-3 min at room temperature, and the trypsinisation process is terminated by adding 10 % of fetal serum "к.р.с.".

EFFECT: invention allows producing an anticancer vaccine application of which is based on a principle of transplantation of a live pathological material to a person from an animal with similar disease.

3 dwg

FIELD: medicine.

SUBSTANCE: there are offered versions of a monoclonal antibody specific to GPVI polypeptide, peptide or its naturally occurred version. A based antithrombotic composition and a method for preparing thereof are described. The versions of methods for inhibition and treatment of various thrombocyte aggregation mediated diseases are disclosed. An antithrombotic set and hybridoma for producing the monoclonal antibody are described.

EFFECT: use of the invention provides the antibodies inhibiting thrombocyte aggregation that can find the further application in medicine for treating various thromboses.

13 cl, 16 dwg, 9 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention refers to chlitin derivative photoproteins and to application thereof both as intracellular calcium indicators, and in cellular studies. Said proteins are produced by mutagenesis of a coding sequence of chlitin. Also, there are offered nucleic acids coding said protein, a vector containing said nucleic acids and a host cell carrying the vector. They can find application in genetic communication technologies for monitoring the cellular events associated with signal transmission and gene expression. Besides, photoproteins of the present invention can be used as intracellular calcium indicators in diagnostic techniques based on calcium concentration measurement in response to the various effects.

EFFECT: produced proteins exhibit enhanced bioluminescence, high affinity to calcium and prolonged light emission.

19 cl, 16 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology and biotechnology. Claimed are versions of antibodies or their functional fragments, which are bound with receptor of human insulin-like growth factor I IGF-IR, and/or natural binding of its ligands IGF1 and/or IGF2 and/or are capable of specific inhibition of tyrosine kinase activity of said IGF-IR. Antibodies contain respective CDR sections of light and heavy chains. Described is mouse hybridoma I-3193 for production of antibodies. Composition for prevention or treatment of cancer, based on antibody application. Described is application of antibodies and/or composition for obtaining respective medication. Claimed is method of diagnostics in vitro of diseases, caused by over-expression or insufficient expression of receptor IGF-I based on antibodies..

EFFECT: invention application ensures antibodies able to bind with isophorms A and B insulin, insulin/ IGF-1 of hybrid receptors which can be applied in medicine for tumour treatment.

15 cl, 8 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to radiation synthesis nanotechnology for the creation of a low-immunogenicity agent to increase the reserved stem cell number in an organism, and can be used in regenerative medicine. The agent represents polymer immobilised hyaluronidase introduced in the dissolved low-molecular water-soluble polymer pre-exposed to ionising radiation in dosage 1-5 Mrad, to final concentration 70 UN in 1 ml.

EFFECT: invention allows producing immobilised hyaluronidase which is characterised by substantially higher ability to increase the reserved stem cell number in an organism and effective both in parenteral, and in oral introduction.

4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: there are offered specific antibodies linked at least with KIR2DL1, KIR2DL2, KIR2DL3 human receptor, neutralise KIR-mediated NK cytolergy inhibition in relation to Cw3+ or Cw4+ target-cells. There are described: B-lymphocyte hybrid cell for producing the antibodies, versions of the method for producing the antibody, as well as a method for detecting a NK-cell, a method for purifying the NK-cells with the use of the antibody and versions of the pharmaceutical antibody composition. Using the antibody for preparing a medicinal agent is offered.

EFFECT: use of the invention provides producing the antibody which controls NK cytolergy of various types, intensifies cytotoxicity, increases NK cytolergy or cytotoxicity in individuals.

63 cl, 13 dwg, 3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention can be used for screening of anti-tumour preparations in vivo, in particular for screening synthetic and natural substances with potential anti-tumour activity.

EFFECT: claimed invention makes it possible to create stable cell line of human ovarian adenocarcinoma.

2 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: recombinant plasmid pFastBac-B17R DNA carries a cowpox virus genome fragment. BvB 17RG recombinant baculovirus strain is produced with the use of recombinant plasmid pFastBac-B 17R DNA and deposited in the Microorganism Cultures Collection of the Federal State Research Institution 'State Research Centre for Virology and Biotechnology 'Vector' of Federal Service for Supervision of Consumer Rights Protection and Human Welfare' (FGUN GNC VB 'Vector' of Rospotrebnadzor) under No. V-388, characterised as a producer of a soluble analogue protein of cowpox interferons type 1 cell receptor.

EFFECT: extended spectrum of preparations of new generation.

2 cl, 5 dwg, 8 ex

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