Polypeptide with antiviral, antiproliferative and/or immunomodulating activities, separated polynucleotide

FIELD: bioengineering.

SUBSTANCE: novel polynucleotide is invented which is produced from the nucleotide sequence of the IFNα-17 gene, containing the single nucleotide polymorphism SNP g771c. Also, the novel polynucleotide is invented which is derived from the natural protein IFNα-17 of the wild type containing SNP G45R.

EFFECT: can be used for producing effective therapeutic agent with antiviral, antiproliferative and/or immunomodulating activity.

13 cl, 5 dwg, 6 ex

 

Related applications

The present invention has the priority of French application 0105516 filed April 24, 2001 and entitled "New polynucleotide and polypeptides of the gene IFNα-17".

Prior art

The scope to which the invention relates.

The present invention relates to new polynucleotides, derived from the nucleotide sequence of a gene IFNα-17, comprising new SNPS, and new polypeptides derived from the natural protein wild-type IFNα-17 containing mutations caused by these SNP, and to their therapeutic application.

Prior art

Gene interferon alpha 17, hereinafter referred to as IFNα-17 described in the following publications:

- Olopade et al.: "Mapping of the shortest region of overlap of deletions of the short arm of chromosome 9 associated with human neoplasia"; Genomics; 14: 437-443; 1992.

- Lawn R. et al.: "DNA sequence of two closely linked human leukocyte interferon genes"; Science 212 (4499), 1159-1162 (1981).

The nucleotide sequence of this gene is available in Genbank under the registration number V00532.

IFNα known for its antiproliferative effect on cells and their participation in antiviral and antiparasitic responses.

It is also known that IFNα inhibit the expression of other cytokines at the level of hematopoietic stem cells, and inhibit cell proliferation of some tumors.

It is also known that IFNα induce the expression of tumor-specific antigens on the surface of tumor cells and induce genes under the control of the promoter regions of type ISRE (item response stimulated by interferon), by affecting specific transcription factor specified ISRE.

It is known that IFNα cause various disorders and/or diseases in humans, such as various types of cancer, such as carcinoma, melanoma, lymphoma, leukemia, and cancer of the liver, neck, head and kidneys, cardiovascular disease, metabolic disease, not associated with the immune system, such as obesity, infectious diseases such as hepatitis b and C and AIDS; pneumonia, ulcerative colitis, diseases of the Central nervous system such as Alzheimer's disease, schizophrenia and depression; graft rejection of tissue or organs; healing RAS; anemia in patients subjected to dialysis; allergies; asthma; multiple sclerosis; osteoporosis; psoriasis; rheumatoid arthritis; Crohn's disease, autoimmune diseases and disorders; R is stroisch gastro-intestinal tract or even disorder, associated with treatment with chemotherapy.

IFNα used, in particular, to treat certain types of leukemia, metastatic carcinoma of the kidney and tumors caused by immunodeficiency, such as Kaposi's sarcoma in AIDS cases. IFNα is also effective against other types of tumors and against some viral infections. IFNα also approved by the FDA (office of quality control of food, drugs and cosmetics) as a treatment for genital warts or venereal diseases.

However, IFNαand in particular IFNα-17, their use in pharmaceutical compositions, give a lot of side effects, such as acute reactions hypersensitivity (urticaria, bronchospasm, anaphylactic shock and the like), cardiac arrhythmia, low blood pressure, seizures, thyroid gland, syndromes, flu-like (fever, sweating, myalgia), etc.

In addition, patients undergoing treatment with interferon IFNαmay produce antibodies that neutralize these molecules, resulting in his effectiveness reduced.

The authors of the present invention have been found new polypeptide and new polynucleotide analogs of IFN geneα-17, having functions different from the natural protein IFNB1; -17 wild-type.

These new polypeptides and polynucleotide can be, in particular, used for the treatment or prophylaxis of the above disorders or diseases, and they do not have all or part of those side effects that are typically associated with their use.

Brief description of the invention

The first object of the present invention are new polynucleotide that differ from the nucleotide sequence of the original gene IFNα-17 wild type in that they contain one or more SNPS (polymorphism, single nucleotide).

The nucleotide sequence of SEQ ID NO:1 of the original human gene IFNα-17 wild type consists of 1873 nucleotides and contains the coding sequence of 570 nucleotides from nucleotide 639 (start codon) and to nucleotide 1208 (stop codon).

The applicant identified 2 SNPS in the nucleotide sequence of the original gene IFNα-17 wild-type.

Such SNP are: DS, 808Ins(a).

It should be noted that in the description of the present invention, the numbering corresponding to the SNP position, defined above, refers to the numbering of the nucleotide sequence of SEQ ID NO:1.

The letters a, t, c and g correspond to the nitrogenous bases of the adenine, Cimino, cytosine and guanine, respectively.

The first letter corresponds to the nucleotide of the wild type and Poslednyaya letter corresponds to the mutated nucleotide.

For example, SNP DS corresponds to the mutation by substitution of guanine (g) at position 771 nucleotide sequence of SEQ ID NO:1 of the original gene IFNα-17 wild type cytosine (C).

SNP 808Ins(a) corresponds to the insertion of adenine (a) at position 808 of the nucleotide sequence of SEQ ID NO:1 of the original gene IFNα-17 wild-type.

These SNP were identified by the applicant in accordance with the method of determination described by the applicant in patent application FR 00 22894, entitled "Process for the determination of one or several functional polymorphism(s) in the nucleotide sequence of a preselected functional candidate gene and its applications" and filed December 6, 2000, the description of which is introduced into the present description by reference.

The method described in this patent application, allows you to identify one (or more) existing SNP, at least in one individual randomly selected from this population.

In accordance with the scope of the present invention, the fragment of the nucleotide sequence of the gene IFNα-17, including, for example, the coding sequence was isolated from different individuals, randomly selected from this population.

Then, after analysis using DASH ("denaturing high performance liquid chromatography)was carried out by sequencing these fragments, some of these samples with heteroduplexes profile (e profile different from the profile of the sequence of the original gene IFNα-17 wild type).

Then the fragment sequenced by this method was compared with the nucleotide sequence of a fragment of the original gene IFNα-17 wild-type and SNP identified in accordance with the present invention.

Thus, these SNPS are natural, and each of them is present in some individuals of the population all over the world.

The original gene IFNα-17 encodes wild-type immature protein of 189 amino acids corresponding to the amino acid sequence of SEQ ID NO:2, which can be turned into a Mature protein of 166 amino acids by cleavage of the signal peptide, including the first 23 amino acids.

Coding SNP of the present invention, namely DS and 808Ins(a), leads to modifications at the level of the amino acid sequence of the protein encoded by the nucleotide sequence of the gene IFNα-17. These modifications are:

- SNP DS causes a mutation of the amino acid glycine (G) at position 45 in the immature protein of the gene IFNα-17 corresponding to the amino acid sequence of SEQ ID NO:2, and replaced with arginine (R) in position 22 of the Mature protein. In the description of the present invention the mutation encoded by the SNP are indicated G22R and G45R, regardless of whether they are in the Mature protein or to the immature protein;

- SNP 808Ins(a) causes the mutation of the amino acid histidine (H) at position 57 in the immature protein of the gene IFNα-17 corresponding to the amino acid sequence of SEQ ID NO:2, with the substitution for glutamine (Q) in position 34 of the Mature protein. In addition, the insertion of an adenine at position 808 nucleotide sequence leads to a shift of the reading frame during translation of the protein, resulting in a stop codon is present at position 58 the amino acid sequence. Thus, SNP 808Ins(a) leads to a stop the transmission immediately after glutamine 57. As a consequence, received the immature protein is shortened and only consists of 57 amino acids. This polymorphism is also called "57Q frame 57". In the description of the present invention, a polymorphism, i.e. the mutation encoded by the specified SNP, indicate "34Q-frame 34 and 57Q frame 57", regardless of whether it is in the Mature protein or to the immature protein.

Amino acid sequence of SEQ ID NO:3 is mutated immature protein ("57Q frame 57")encoded by nucleotide sequence SEQ ID NO:1, containing SNP 808Ins(a).

Each SNP of the present invention leads to modifications to the spatial conformation of the polypeptides of the present invention in comparison with the polypeptide encoded by the nucleotide sequence of the original gene IFNα-17 wild-type.

These modify the tion can occur when computer molecular modeling, carried out by methods well known in the art, using, for example, simulation de novo (for example, SEQFOLD/MSI), homology (for example, MODELER/MSI), minimize force fields (e.g., DISCOVER, DELPHI/MSI) and/or molecular dynamics (for example, CFF/MSI).

One example of such models is given below in the description of the experimental part.

Computer molecular modeling showed that the mutation G22R in mutated Mature protein causes the modification and replacement in the loop AB near position 22, which leads to the elimination of hydrogen bonds.

In figures 1A and 1B shows that the loop AB is not laid and serving the loop.

On the original IFNα-17 wild-type residue G22 is very close to the residue R144. It is known that this residue R144 is involved in the binding of interferon α-2 (IFNα-2) with its receptor. Patterns IFNα-2 and IFNα-17 are very similar, and, in all likelihood, the rest R144 in IFNα-17 participate in binding with its receptor.

Thus, G22R-mutated protein has a three-dimensional conformation that is different from the natural conformation of the protein IFNα-17 wild-type.

Computer molecular modeling also allows us to predict that the presence of the amino acid arginine in position 22 leads to a significant change in the structure and function of a natural protein wild tee is and IFNα -17, namely at the level of binding of IFNα-17 with its receptor.

Genotyping of polynucleotides of the present invention can be implemented so as to determine the frequency of alleles of these polynucleotides in this population.

Determination of functional properties of polypeptides of the present invention can be similarly implemented using test their biological activity.

This can be measured, for example, signal transduction, maturation of dendritic cells, release of cytokines by T-lymphocytes, cytokine release by monocytes, in vitro or in vivo antiviral activity of the polypeptides of the present invention, cellular antiproliferative activity of polypeptides of the present invention with respect to cell lines of Daudi Burkitt, cellular antiproliferative activity of polypeptides of the present invention relative to the cell line TF-1 and comparison with IFNα-17 wild-type or IFNα-2 wild type, selected as representative of the commercial product Intron A.

The present invention also relates to the application of polynucleotides and polypeptides of the present invention, as well as therapeutic molecules, obtained and/or identified on the basis of these polynucleotides and polypeptides, and in particular about what ractice and treatment of certain disorders and/or diseases.

Brief description of the graphical material

Figure 1A shows a model of the encoded protein of the present invention comprising the SNP G45R, and protein IFNα-17 wild type. In figure 1B, on a larger scale, shows a model of the lower part of each of the proteins presented on figure 1A.

In figures 1A and 1B black "tape" represent the structure of a protein IFNα-17 wild-type and white "ribbons" are patterns G22R-mutated protein IFNα-17.

The figure 2 presents the test results for the evaluation of antiproliferative action G45R-mutated IFNα-17 on the cell line TF-1. This figure is on x-axis the concentration of IFNα (ng/ml), and the ordinate axis pending the degree of inhibition of cell proliferation (%). Antiproliferative effect G45R-mutated IFNα-17 (black diamonds) compared with the antiproliferative action of IFNα-2 wild-type (white squares).

The figure 3 presents the test results for the evaluation of antiproliferative action G45R-mutated IFNα-17 cell line Daudi Burkitt. This figure is on x-axis the concentration of IFNα (ng/ml), and the ordinate axis pending the degree of inhibition of cell proliferation (%). Antiproliferative effect G45R-mutated IFNα-17 (black diamonds) compared with the antiproliferative action of IFNα-2 wild-type (white to agrati).

The figure 4 presents estimates of survival of mice previously infected with the virus, VSV and processed G45R-mutated protein IFNα-17, compared with mice treated with IFNα-2 wild-type, or compared with untreated mice. In this figure, the abscissa axis represents time survival (days), and the ordinate axis delayed relative survival VSV-infected mice. Black diamonds refer to data for VSV-infected mice treated G45R-mutated IFNα-17, black squares refer to data for VSV-infected mice treated with IFNα-2 wild type, and where there's no shading triangles refer to data obtained for raw VSV-infected mice.

Detailed description of the invention

Definition

The expression "nucleotide sequence of the original gene wild type" should be understood as "the nucleotide sequence of SEQ ID NO:1 human gene IFNα-17".

This sequence is available in Genbank under the registration number V00532 and described in the works:

Olopade et al.: "Mapping of the shortest region of overlap of deletions of the short arm of chromosome 9 associated with human neoplasia"; Genomics; 14: 437-443; 1992.

Lawn, R. M. et al.: "DNA sequence of two closely linked human leukocyte interferon genes"; Science 212 (4499), 1159-1162 (1981).

The terms "natural protein IFNα-17 wild-type or IFN proteinα-17 wild-type" refers to a Mature white is, encoded by the nucleotide sequence of the original gene IFNα-17 wild type. Natural immature protein IFNα-17 wild type corresponds to the peptide sequence represented in SEQ ID NO:2.

The term "polynucleotide" means polyribonucleotide or polyethoxylated, which may be a modified or unmodified DNA or RNA.

The term "polynucleotide" means, for example, single-stranded or double-stranded DNA; DNA, consisting of a mixture of one or more single-stranded regions or one or more double-stranded regions, single-stranded or double-stranded RNA, and RNA, consisting of a mixture of one or more single-stranded regions and one or more double-stranded regions. The term "polynucleotide" can also mean RNA and/or DNA that includes one or more of triplex areas. The term "polynucleotide" can also mean DNA and RNA containing one or more bases, modified so that they had the skeleton, modified for purposes of stabilization or for other purposes. The term "modified base" means, for example, unusual bases such as inosine.

The term "polypeptide" means a peptide, Oligopeptide, an oligomer or a protein containing at least two amino acids attached to each other, the normal is Oh or a modified peptide bond, such as, for example, in cases isothermic peptides.

The polypeptide may comprise amino acids that are not included in the 20 amino acids specified by the genetic code. The polypeptide may consist of amino acids, modified in the course of natural processes, such as posttranslational processes of ripening, or during chemical processes, well known to specialists. Such modifications are described in detail in the literature. These modifications can occur in any part of the polypeptide: a peptide skeleton, in the amino acid chain, or even the carboxy - or amino-ends.

The polypeptide can be done after extensive processing ubication, or it may be cyclic, with or without branching branching. This type of modification can be the result of natural or artificial post-translational processes, well known to experts.

For example, the term "polypeptide modifications include acetylation, acylation, ADP-ribosylating, amidation, covalent binding of flavin, covalent binding with gem, covalent binding to a nucleotide or nucleotide derivative, covalent binding of a lipid or lipid derivative, covalent binding to phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, education disul innych links, demethylation, formation of cysteine, formation of Pyroglutamate, formirovanie, gamma-carboxylation, glycosylation, including the introduction of PEG, the formation of GPI-anchors, hydroxylation, iodination, methylation, monitorowanie, oxidation, proteolytic processes, phosphorylation, prenisolone, racemization, sunilolivera, sulfation, joining amino acids, such as originin, or accession ubicacin. Such modifications are described in detail in the literature: PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, New York, 1993, POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983, Seifter et al. "Analysis for protein modifications and nonprotein cofactors", Meth. Enzymol. (1990) 182: 626-646 and Rattan et al. "Protein Synthesis: Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663: 48-62 per.

The terms "isolated polynucleotide" or "isolated polypeptide" mean respectively polynucleotide or polypeptide, such as polynucleotide or polypeptide, as defined previously, and separate from the human body or produced by any other technical means.

The term "identity" refers to the degree of identity of nucleotide or polypeptide sequences.

The term "identity" is well known in the art and well described in the literature. COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., Ed., Oxford University Press, New York, 1998; BIOCOMPUTING INFORMATICS AND GENOME PROJECT, Smith, D.W., Ed., Academic Press, New York, 1993; COMPUER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin H.G., Ed, Humana Press, New Jersey, 1994; and SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987.

Methods commonly used to determine identity and similarity between two sequences, is also well described in the literature. Cm. GUIDE TO HUGE COMPUTER, Martin J. Bishop, Ed, Academic Press, San Diego, 1994, and Carillo, H. &Lipton, D., Siam J. Applied Math (1988) 48:1073.

Polynucleotide, which, for example, 95% identical to the nucleotide sequence of SEQ ID NO:1 represents polynucleotide containing up to 5 point mutations per 100 nucleotides, compared with the specified sequence.

Such point mutations can be one (or several) substitutions, additions and/or deletions in one (or more) nucleotide.

Similarly, a polypeptide which is, for example, 95% identical to the amino acid sequence of SEQ ID NO:2 is a polypeptide containing up to 5 point mutations per 100 amino acids, compared with the specified sequence.

Such point mutations can be one (or several) substitutions, additions and/or deletions in one (or more) amino acid.

Polynucleotide and polypeptides of the present invention, which are not completely identical, respectively:

- the nucleotide sequence of SEQ ID NO:1, containing at least one of the following SNP: DS and 808Ins(a)

- amino acid in which sledovatelnot SEQ ID NO:2, containing SNP G45R,

- amino acid sequence of SEQ ID NO:3 containing SNP G45R,

considered as variants of these sequences.

Usually polynucleotide the present invention has a biological activity that is similar or almost similar activity nucleotide sequence of SEQ ID NO:1, containing at least one SNP: DS and 808Ins(a).

Similarly, the polypeptide of the present invention usually has a biological activity that is similar or almost similar activity:

- amino acid sequence of SEQ ID NO:2, containing the SNP G45R, and/or

- amino acid sequence of SEQ ID NO:3, possibly containing SNP G45R.

A variant of the present invention can be obtained, for example, by site-directed mutagenesis or direct synthesis.

The term "SNP" means any natural change in the basis of the nucleotide sequence. This SNP at nucleotide sequence can be coded, silent and non-coding.

"Coding SNP is a polymorphism contained in the coding sequence of the nucleotide sequence that results in an amino acid modification in the amino acid sequence encoded by the specified nucleotide sequence. In this case, in a broader sense, the term "SNP" is also meant a mutation in the amino acid sequence.

"Silent SNP is a polymorphism contained in the coding sequence of the nucleotide sequence, which does not entail amino acid modification in the amino acid sequence encoded by the specified nucleotide sequence.

"Non-coding" the SNP is a polymorphism contained in the non-coding sequence of the nucleotide sequence. This polymorphism may in particular be present in the intron, splicing, promoter transcription or enhancer sequence.

The term "functional SNP" means the SNP defined above, which is present in the nucleotide sequence or amino acid sequence with functionality.

The term "functionality" means the biological activity of the polypeptide or polynucleotide.

The functionality of the polypeptide or polynucleotide of the present invention may consist in preserving, enhancing, reducing or suppressing the biological activity of the polypeptide encoded by the nucleotide sequence of the original wild-type gene or the sequence specified polynucleotide.

The functionality of the polypeptide or polynucleotide of the present invention may also be to change the nature of the biological asset is the spine of the polypeptide, encoded by the nucleotide sequence of the original wild-type gene or the sequence specified polynucleotide.

Biological activity can be, in particular, is associated with the affinity or lack of affinity of the polypeptide of the present invention in relation to the receptor.

Polynucleotide

The first object of the present invention is selected polynucleotide, including:

a) a nucleotide sequence that is at least 90% identical, preferably at least 95% identical, and more preferably at least 99% identical to the sequence of SEQ ID NO:1 or its coding sequence (from nucleotide 639 to nucleotide 1208), with the specified nucleotide sequence includes at least one of the following coding SNPS: DS and 808Ins(a), or

b) a nucleotide sequence complementary to the nucleotide sequence (a).

In accordance with the present invention the nucleotide sequence (a) may contain SNP DS or 808Ins(a)or SNP DS and 808Ins(a).

It should be noted that in the context of the present invention this numbering coincides with the provisions of the SNP in the nucleotide sequence of SEQ ID NO:1.

The present invention also relates to the selected polynucleotide containing:

a) nucleotide the sequence of SEQ ID NO:1 or its coding sequence, each of these sequences contains at least one of the following coding SNPS: DS and 808Ins(a) or

b) a nucleotide sequence complementary to the nucleotide sequence (a).

Polynucleotide of the present invention, preferably, consists of a sequence of SEQ ID NO:1 or its coding sequence, and each of these sequences comprises at least one of the following coding SNPS: DS and 808Ins(a).

In accordance with the present invention polynucleotide defined above, includes one coding SNP selected from the group consisting of DC and 808Ins(a).

The present invention also relates to the selected polynucleotide, consists of:

a) the nucleotide sequence of SEQ ID NO:1 or its coding sequence, where each of these sequences comprises at least one of the following SNP: DS and 808Ins(a), or

b) a nucleotide sequence complementary to the nucleotide sequence (a)

when this is specified, the selected polynucleotide consists of at least 10 nucleotides.

Preferably the selected polynucleotide defined above, consists of 10-40 nucleotides.

The present invention also relates to the selected polynucleotide, codereuse polypeptide, including:

a) aminokislotna the sequence of SEQ ID NO:2 or

b) amino acid sequence containing amino acid situated between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2,

where each of the amino acid sequences (a) and (b) contains the following coding SNPS: G45R.

It should be noted that in the context of the present invention this numbering coincides with the position of the SNP G45R in the amino acid sequence of SEQ ID NO:2.

Another object of the present invention is selected polynucleotide encoding the polypeptide, including:

(a) amino acid sequence of SEQ ID NO:3 or

b) amino acid sequence containing amino acid situated between positions 24 and 57 amino acid sequence of SEQ ID NO:3.

Each of the amino acid sequences of a) and b) defined above polypeptide can also contain SNP G45R.

In accordance with a preferred object of the present invention defined above, the polypeptide contains one coding SNP, such as SNP defined above.

More preferably selected polynucleotide of the present invention encodes a polypeptide that contains the entire amino acid sequence of SEQ ID NO:2 or a part thereof and having a coding SNP G45R.

More preferably selected polynucleotide of the present invention encodes a polypeptide that contains su the amino acid sequence of SEQ ID NO:3 or its part and, you may also have the coding SNP G45R.

Polynucleotide of the present invention preferably consists of a DNA or RNA molecule.

Polynucleotide of the present invention can be obtained by standard methods of synthesis of DNA or RNA.

Polynucleotide of the present invention containing the SNP DS can also be obtained using site-directed mutagenesis of this nucleotide sequence of a gene IFNα-17 by modifying the nucleotide sequence of the wild-type by means of substitution of guanine (g) cytosine (C) at position 771 nucleotide sequence of SEQ ID NO:1.

Polynucleotide of the present invention containing the SNP 808Ins(a)can also be obtained using site-directed mutagenesis of the nucleotide sequence of a gene IFNα-17 by adding adenine (a) at position 808 of the nucleotide sequence of SEQ ID NO:1.

Site-directed mutagenesis, which can be realized in such a way, well known to specialists. In this connection may be mentioned the publication T.A. Kunkel, 1985, "Proc.Natl. Acad. Sci., USA, 82:488.

Selected polynucleotide may also include, for example, the nucleotide sequence encoding the amino acid sequence of pre-, Pro - or pre-Pro-protein or marker amino acid sequence, such as a hexa-his-tag peptide.

Polynuclear the Ted of the present invention may also be associated with nucleotide sequences, encoding other proteins or protein fragments, in order to obtain hybrid proteins or other products for cleaning.

Polynucleotide of the present invention may also include a nucleotide sequence such as 5'- and/or 3'-non-coding sequences, such as transcribed or retranscribing sequence, translated or untranslated sequence, a sequence of signal splicing, polyadenylation sequence, a sequence that communicates with the ribosome, or even sequences that stabilize mRNA.

Nucleotide sequence complementary to a nucleotide or polynucleotide sequence, means that it can gibridizatsiya with this nucleotide sequence in harsh environments.

The terms "stringent hybridization conditions" means, primarily, but not necessarily, chemical conditions, allowing to be hybridization, in case the said nucleotide sequences have an identity of at least 80%, preferably greater than or equal to 90%, even more preferably greater than or equal to 95%, and most preferably greater than or equal to 97%.

Stringent conditions can be obtained by methods well known in the art, for example, by inkberrow the deposits polynucleotides at 42° C in a solution containing 50% formamide, 5 x SSC (150 mm NaCl, 15 mm triacrylate), 50 mm sodium phosphate (pH of 7.6), 5 x denhardt's solution, 10% dextran sulfate, and 20 μg denatured sperm DNA, salmon, followed by washing the filters in 0.1 x SSC at 65°C.

In accordance with the scope of the present invention, if the hard conditions can be carried out hybridization of nucleotide sequences having an identity of 100%, it is considered that this nucleotide sequence is strictly complementary to the nucleotide sequence described in (a).

It should be noted that in the context of the present invention the nucleotide sequence complementary to any nucleotide sequence that contains at least one antisense SNP of the present invention.

For example, if this nucleotide sequence contains the SNP DS, its complementary nucleotide sequence contains the nucleotide g in equivalent position 771.

Identification, hybridization and/or amplification of polynucleotide containing SNP

The present invention also relates to the use of all or part of:

a) polynucleotide that 80-100% (preferably at least 90%, more preferably 95%, and particularly preferably 100%identical nucleate the Noah sequence SEQ ID NO:1, and/or

b) polynucleotide of the present invention containing at least one SNP,

for the purposes of identification, hybridization and/or amplification of all or part of polynucleotide that 80-100% (preferably at least 90%, more preferably 95%, and particularly preferably 100%) identical to the nucleotide sequence of SEQ ID NO:1, or if necessary its coding sequence (from nucleotide 639 to nucleotide 1208), where each of these sequences comprises at least one of the following SNP: DS, 808Ins(a).

Genotyping and incidence of SNP

The present invention also relates to the use of all or part of:

a) polynucleotide that 80-100% (preferably at least 90%, more preferably 95%, and particularly preferably 100%) identical to the nucleotide sequence of SEQ ID NO:1, and/or

b) polynucleotide of the present invention containing at least one SNP,

for genotyping of all or part of polynucleotide, which is 80-100% (preferably at least 90%, more preferably 95%, and particularly preferably 100%) identical to the nucleotide sequence of SEQ ID NO:1, or if necessary its coding sequence (from nucleotide 639 to nucleotide 1208), where each of these sequences included the em at least one of the following SNP: DS, 808Ins(a).

In accordance with the present invention genotyping can be carried out at the individual or population of individuals.

In the context of the present invention, the term "genotyping" refers to the process genotyping in one individual or population of individuals. The genotype consists of the alleles present in one or more specific loci.

The term "population of individuals" means a group of individuals, selected at random or involuntary way. Such individuals can be people, animals, microorganisms or plants.

Usually a group of individuals includes at least 10 individuals, and preferably from 100 to 300 individuals. These individuals can be selected based on their ethnicity or their phenotype, and in particular can be chosen such individuals who suffer from such disorders and/or diseases such as carcinoma, melanoma, lymphoma, leukemia, and cancer of the liver, neck, head and kidneys, cardiovascular disease; metabolic disease that is not associated with the immune system, such as obesity, infectious diseases such as hepatitis b and C and AIDS; pneumonia, ulcerative colitis, diseases of the Central nervous system such as Alzheimer's disease, schizophrenia and depression; Otto the provision of transplant tissue or organs; wound healing; anemia in patients subjected to dialysis; allergies; asthma; multiple sclerosis; osteoporosis; psoriasis; rheumatoid arthritis; Crohn's disease, autoimmune diseases and disorders; disorders of the gastrointestinal tract or even disorders associated with treatment with chemotherapy.

Preferably in this group of individuals was genotyped functional SNP of the present invention.

For genotyping SNP there are many technologies (see, for example, Kwok Pharmacogenomics, 2000, vol.1, pp.95-100. "High-throughput genotyping assay approaches"). These technologies are based on one of the four following principles: hybridization of allele-specific oligonucleotides; the elongation of oligonucleotides by dideoxynucleotide, optionally in the presence of deoxynucleotides; legirovanii allele-specific oligonucleotides or splitting of allele-specific oligonucleotides. Each of these technologies can be associated with the detection system, such as direct measurement or polarized fluorescence or mass spectrometry.

Genotyping can be, in particular, is carried out with minisequencing using "hot" ddNTP (2 different ddNTP labeled with different fluorophores) and "cold" ddNTP (its 2 different ddNTP) in combination with the use of polarization-fluorescent scan of the as. The Protocol minisequencing with the reading of the polarized fluorescence (FP-TDI Technology or Fluorescence Polarization Template-direct Dye-Terminator Incorporation).

This can be carried out on the product obtained after amplification with polymerase chain reaction (PCR) DNA from each individual. This PCR product is chosen so that it includes the gene region of polynucleotide containing the analyzed SNP. After the last stage in PCR-thermoacetica the tablet is placed on the polarization-fluorescent scanner to read the labeled bases using filters fluorophore-specific excitation and emission. The intensities of labeled bases are shown in the graph.

For PCR amplification, in the case of a single SNP of the present invention, the sense and antisense primers, respectively, can be easily selected by any person in accordance with the position of the SNP of the present invention.

So, for example, as primers for PCR amplificatio can serve the following sense and antisense nucleotide sequence, respectively:

SEQ ID NO:4: Sense primer: TTCAAGGTTACCCATCTCAASEQ ID NO:5: Antisense primer TTAGTCAATCAGGATCATTGC.

Nucleotide sequences allow you to amplify a fragment having a length of 655 nucleotides from nucleotide 591 and to nucleotide 1245 in nucleotide sequence SEQ ID NO:1.

Then for a group of individuals carry out statistical analysis of the frequency of occurrence of each allele (allelic frequency), encoded by the gene containing the SNP, where the specified analysis allows to determine the importance and distribution of these alleles in different subgroups, and in particular, if necessary, in different ethnic groups that make up this population of individuals.

Data obtained by genotyping, analyzed to assess the frequency distribution of different alleles observed in the studied populations. Determining the frequency of occurrence of alleles can be implemented using a computer program such as SAS-suite® (SAS or SPLUS® (MathSoft). Comparison of allelic distribution of SNP of the present invention in different ethnic groups of a population of individuals can be performed using the computer program ARLEQUIN® and SAS-suite®.

SNP of the present invention used as genetic markers

Because SNP, modifying the functional sequence of genes (e.g., promoter, splicing sites, the coding region), probably directly associated with susceptibility or resistance to disease, all SNP (functional or nonfunctional) can serve as valuable markers for the identification of one or more of the ENES, responsible for these pathological conditions, and therefore, they can be indirectly associated with the specified pathological conditions (see Cargill et al. (1999). Nature Genetics 22:231-238; Riley et al. (2000). Pharmacogenomics 1:39-47; Roberts L. (2000) Science 287:1898-1899).

Thus, the present invention also relates to a data Bank, which includes at least one of the following SNP: DS, 808Ins(a) polynucleotide gene IFNα-17.

While it is clear that the SNP are numbered in accordance with the nucleotide sequence of SEQ ID NO:1.

The specified data Bank can be analyzed in order to determine statistically relevant associations between:

(i) at least one of the following SNP: DS, 808Ins(a) polynucleotide gene IFNα-17,

(ii) the disease or resistance to disease.

More preferably the present invention relates to a method of determining statistically relevant associations between at least one SNP selected from the group consisting of DC, 808Ins(a) polynucleotide gene IFNα-17, and disease or resistance to disease, including:

a) genotyping a group of individuals;

b) determination of the distribution of the specified disease or resistance to the disease in this group of individuals:

(C) comparison of genotyping data with the data distribution shown what about the diseases or resistance to a specific disease; and

d) analysis of this comparison to identify statistically relevant associations.

The present invention also relates to the use of at least one of the following SNP: DS, 808Ins(a) polynucleotide gene IFNα-17 in order to obtain diagnostic/prognostic kits for the detection or resistance to disease.

SNP of the present invention, such as SNP defined above, may be directly or indirectly associated with the disease or resistance to disease.

Preferably these diseases can be diseases mentioned above.

Expressing the vector and cell-hosts

The present invention also relates to a recombinant vector comprising at least one polynucleotide of the present invention.

This can be used with many expressing systems, including, but not limited to, chromosome, episome and derived from viruses. More specifically, used recombinant vectors can occur from bacterial plasmids, transposons, yeast Epsom, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papillomaviruses, such as SV40, a virus cowpox virus, adenoviruses, Fox poxviruses, pseudorabies and retroviruses.

Such recombi is based vectors can also be cosignee or famiglie derivatives. The nucleotide sequence may be incorporated into recombinant expressing vector by methods well known in the art, such as methods described in Molecular Cloning: A Laboratory manual, Sambrook et al. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

The recombinant vector can include nucleotide sequences that regulate the expression of polynucleotides, as well as nucleotide sequences that provide for the expression and transcription of polynucleotide of the present invention and translation of the polypeptide of the present invention, where these sequences are chosen in accordance with the used cells of the host.

For example, in the recombinant vector may be integrated with an appropriate secretion signal, so that the polypeptide encoded by polynucleotides of the present invention, heading directly into the lumen of the endoplasmic reticulum, periplasmatic on the membrane or in the extracellular space.

The present invention also relates to the cell host comprising the recombinant vector of the present invention.

Introduction of the recombinant vector into the cell host may be carried out by methods well known in the art, such as methods described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., 2nd ed., McGraw-Hill Prfessional Publishing, 1995 and MOLECULAR CLONING: A LABORATORY MANUAL, supra, for example, transfection by calcium phosphate, transfection of DEAE-dextran, transfection, microinjection, transfection with cationic lipids, electroporation, transduction or infection.

Such cells-owners can be, for example, bacterial cells, such as cells of streptococci, staphylococci, E. coli or Bacillus subtilis; the cells of fungi, such as yeast cells and Aspergillus cells, Streptomyces, insect cells such as cells of Drosophila S2 and Spodoptera Sf9, animal cells such as cells of Cho, COS, HeLa, S, KSS, SOME 293 and human cells subjected to treatment, or even plant cells.

Cell-hosts can be used, for example, for expression of the polypeptide of the present invention or as an active product in the pharmaceutical compositions described below.

Polypeptide

The object of the present invention is also allocated to the polypeptide comprising the amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99% identical to all or part of:

(a) amino acid sequence of SEQ ID NO:2 or

b) amino acid sequence containing amino acid situated between positions 24 and 189 of the amino acid sequence is SEQ ID NO:2,

each of these amino acid sequences (a) and (b) contains the following coding SNPS: G45R.

The polypeptide of the present invention may also include all or part of:

(a) amino acid sequence of SEQ ID NO:2 or

b) amino acid sequence containing amino acid situated between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2,

each of these amino acid sequences (a) and (b) contains the following coding SNPS: G45R.

More specifically, the polypeptide of the present invention may consist of all or part of:

(a) amino acid sequence of SEQ ID NO:2 or

b) amino acid sequence containing amino acid situated between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2,

each of these amino acid sequences (a) and (b) contains the following coding SNPS: G45R.

The object of the present invention is also allocated to the polypeptide comprising the amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95%and even more preferably at least 99% identical to all or part of:

(a) amino acid sequence of SEQ ID NO:3 or

b) amino acid serial is a major, containing amino acids located between positions 24 and 57 amino acid sequence of SEQ ID NO:3,

each of these amino acid sequences (a) and (b) contains the following coding SNPS: "57Q frame 57".

The polypeptide of the present invention may also include all or part of:

(a) amino acid sequence of SEQ ID NO:3 or

b) amino acid sequence containing amino acid situated between positions 24 and 57 amino acid sequence of SEQ ID NO:3.

More specifically, the polypeptide of the present invention may consist of all or part of:

(a) amino acid sequence of SEQ ID NO:3 or

b) amino acid sequence containing amino acid situated between positions 24 and 57 amino acid sequence of SEQ ID NO:3.

Each of these amino acid sequences (a) and (b) the specified polypeptide can also contain SNP: G45R.

Preferably the polypeptide of the present invention contains one coding SNP selected from the group consisting of G45R, "57Q frame 57".

More preferably the polypeptide of the present invention contains amino acids 24-189 amino acid sequence of SEQ ID NO:2 and has a coding SNP G45R.

More preferably the polypeptide of the present invention contains amino acids 24-57 the amino acid is based sequence SEQ ID NO:3.

The present invention also relates to a method for obtaining the above-mentioned polypeptide, which opened earlier cell host is cultivated in a culture medium, and the specified polypeptide isolated from the culture medium.

The polypeptide can be purified from culture medium host cells by methods well known in the art, such as deposition autroprime agents such as salts, in particular ammonium sulfate, ethanol, acetone or triperoxonane acid; acid extraction; ion exchange chromatography; chromatography on phosphocellulose; hydrophobic chromatography; affinity chromatography; chromatography on hydroxyappatite or size-exclusion chromatography.

The term "cultural environment" means the environment from which emit or purified polypeptide of the present invention. This environment can consist of the extracellular environment and/or cell lysate. If the specified conformation of the polypeptide has been modified in the process of separation or purification of the active conformation of the specified polypeptide may be recovered by methods well known to specialists.

Antibodies

The present invention also relates to a method for immunospecific antibodies.

The term "antibody" means a monoclonal, polyclonal, chimeric, single-chain, "humanitariannet" antibody, and that the same Fab-fragments, including products library expressed Fab or immunoglobulins.

Immunospecific antibody can be obtained by immunization of an animal with the polypeptide of the present invention.

The present invention also relates to immunospecificity the antibody against the polypeptide of the present invention defined above.

The polypeptide of the present invention or its fragments, similar, one of its variants, or a cell expressing the polypeptide, can also be used for producing immunospecificity antibodies.

The term "immunospecificity" refers to the antibody with a higher affinity to the polypeptide of the present invention than in relation to other polypeptides known to specialists.

Immunospecificity antibodies can be obtained by introducing the polypeptide of the present invention, one of its fragments, its equivalent, or epitope fragment, or cells expressing this polynucleotide, to a mammal, preferably non-human, by methods well known to specialists.

To obtain monoclonal antibodies can be used standard methods of producing antibodies from cell lines, such as the hybridoma technique (Kohler et al., Nature (1975) 256:495-497), Triana technology, hybridoma technology with COI is whether the human b-cells (Kozbor et al., Immunology Today (1983) 4:72) and hybridoma technique using EBV (Cole et al., "The EBV-hybridoma technique and its application to human lung cancer", Monoclonal Antibodies and Cancer Therapy (Vol. 27, UCLA Symposia on Molecular and Cellular Biology, New Series)(eds. R.A. Reisfeld and S.Sell), pp.77-96, Alan R.Liss, Inc. N.Y. 1985, pp.77-96).

Can also be used in methods of producing single-chain antibodies, such as, for example, the antibody described in U.S. patent No. 4946778.

For the production of " humanized" antibodies can also be used transgenic animals, such as a mouse.

Agents that interact with a polypeptide of the present invention

The present invention also relates to a method of identification of the agent that activates or inhibits the polypeptide of the present invention, including:

a) obtaining a recombinant vector comprising polynucleotide of the present invention containing at least one coding SNP,

b) obtaining host cells comprising the recombinant vector (a),

(C) contacting host cells (b) with a test agent and

d) determining an activating or inhibitory effect generated by the test agent.

The polypeptide of the present invention can also be used in the method of screening compounds that interact with them.

These compounds may represent an activating agents (agonists) or inhibiting agent is (antagonists) in relation to the activity, characteristic of the polypeptide of the present invention. Such compounds may also be ligands or substrates of the polypeptide of the present invention. Cm. Coligan et al., Current Protocols in Immunology 1(2), Chapter 5 (1991).

In General, for the implementation of this method first, it is desirable to produce the appropriate cell hosts expressing the polypeptide of the present invention. Such cells can be, for example, mammalian cells, yeast, insects such as Drosophila, or bacteria, such as E.coli.

Then these cells or membrane extracts of cells incubated in the presence of test compounds.

Can then be conducted to assess the ability of test compounds to contact the polypeptides of the present invention, as well as to inhibit or activate the functional response.

Stage (d) of the method described above can be performed using the test agent, marked direct or indirect way. This stage may also include the test in a competitive binding using labeled or unlabeled agent and labeled competing agent.

You can also define, produces whether the test agent is an alarm activation or inhibition in cells expressing the polypeptide of the present invention, using appropriate means for detecting selected in the under detektivami signal.

Such activating or any abscopal agents can be polynucleotide, and in some cases, oligonucleotides or polypeptides, such as, for example, proteins or antibodies.

The present invention also relates to a method of identification of the agent, activated or inhibiting the polypeptide of the present invention, including:

a) obtaining a recombinant vector comprising polynucleotide of the present invention containing at least one coding SNP,

b) obtaining host cells comprising the recombinant vector (a),

(C) adherence to host cells (b) in the presence of the test agent and

d) determining an activating or inhibitory activity of this polypeptide with a test agent.

The agent is activated or inhibiting the polypeptide of the present invention is an agent that produces a response accordingly, when the activation or inhibition in the presence of the specified polypeptide.

Agents, activated or directly or indirectly inhibiting the polypeptide of the present invention, can consist of polypeptides, such as, for example, membrane or nuclear receptors, kinases, and more preferably tyrosine kinase, transcription factors or polynucleotide.

Detection

The present invention also relates the I for method of analysis of the biological properties of polynucleotide of the present invention and/or of the polypeptide of the present invention the individual, including the implementation of at least one of the following procedures:

a) determining the presence or absence of polynucleotide of the present invention in the genome of the individual;

b) determining the level of expression of polynucleotide of the present invention the individual;

(C) determining the presence or absence of the polypeptide of the present invention the individual;

d) determining the concentration of the polypeptide of the present invention the individual and/or

e) determine the functionality of the polypeptide of the present invention the individual.

These biological properties can be analyzed in an individual or in a sample taken from the individual.

These biological properties allow genetic diagnosis and to determine whether the individual has the disease or is it also affected by the risk of the emergence of this disease, or Vice versa, if a particular individual partial resistance to the development of this disease, ailments or disorders associated with the presence of polynucleotide of the present invention and/or of the polypeptide of the present invention.

These diseases can be disorders and/or diseases, such as cancer and tumors, infectious diseases, venereal diseases, immuno-associated diseases the project and/or autoimmune diseases and disorders, cardiovascular diseases, metabolic diseases, diseases of the Central nervous system and disorders associated with the use of chemotherapy.

Specified cancers and tumors are carcinomas, including metastatic carcinoma of the kidney, melanoma, lymphomas, including follicular lymphoma and T-cell skin lymphoma, leukemia, including leukemia retikulez, chronic lymphocytic leukemia and chronic myeloid leukemia, liver cancer, neck, head and kidneys, multiple myeloma, carcinoid tumors and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in AIDS cases.

These infectious diseases are viral infections, including chronic hepatitis b and C and HIV/AIDS, infectious pneumonia and sexually transmitted diseases such as genital warts.

These immune and autoimmune diseases can be graft rejection of tissue or organs; allergies; asthma; psoriasis; rheumatoid arthritis; multiple sclerosis; Crohn's disease and ulcerative colitis.

Specified diseases of metabolism can be non-immune disease, such as obesity.

Specified diseases of the Central nervous system can be Alzheimer's disease, Parkinson's disease, schizophrenia and depression.

These diseases the disorders can also be disease, associated with wound healing, anemia in patients subjected to dialysis, and osteoporosis.

This method also allows you to establish a genetic diagnosis of the disease or resistance to disease associated with the presence of the individual mutant allele encoded by a SNP of the present invention.

Preferably, in stage (a) can be detected by the presence or absence of polynucleotide containing at least one coding SNP defined above.

Detection of polynucleotide can be carried out in biological samples from the subject individual, such as cells, blood, urine, saliva, or in samples taken at biopsy or autopsy of a particular individual. For direct detection or detection after PCR amplification may be, for example, used the genomic DNA. In a similar manner can also be used RNA or cDNA.

Can then be compared to the nucleotide sequence of polynucleotide of the present invention with a nucleotide sequence found in the genome of the individual.

Comparison of the nucleotide sequences can be carried out by sequencing; methods DNA hybridization; the difference in the mobility of DNA fragments in gel electrophoresis carried out with or without using denaturing what their agents; or the difference of the melting temperatures (see Myers et al., Science (1985) 230:1242). Such modifications in the specific dot patterns of nucleotide sequences can be detected using tests for protection from the action of nucleases, such as Mcasa or nuclease S1, by enzymatic hydrolysis and chemical splitting agents. Cm. Cotton et al., Proc.Natl. Acad. Sci., USA (1985) 85:4397-4401. Oligonucleotide probes containing a fragment of polynucleotide the present invention can also be used for screening.

To determine the expression of polynucleotide of the present invention and to identify the genetic variability of the specified polynucleotide can be used many methods well known in the art (see, Chee et al., Science (1996), Vol.274, pp.610-613).

In stage (b) the expression level of polynucleotide can be measured through the quantification of the level of RNA encoded by the specified polynucleotide (and coding for the polypeptide), by methods well known in the art, such as PCR, CRL, method of protection from RNase, using Northern blot analysis and other hybridization methods.

In stages C) and d) the presence or absence and concentration of the polypeptide of the present invention in an individual or in a sample taken from the individual, can be determined by methods well known special is the plates, for example, using radioimmunoassay, tests on competitive binding, Western blot analysis and ELISA.

Then in stage d) the specific concentration of the polypeptide of the present invention can be compared with the concentration of the wild-type protein, normally present in an individual.

A specialist may be determined that the threshold above or below which there is a sensitivity or, on the contrary, resistance to disease, ailment or disorder listed above publications, known from the prior art, or by using traditional tests or analyses, such as those mentioned above.

In stage e) determining the functionality of the polypeptide of the present invention can be carried out by methods well known in the art, for example, using in vitro tests, such as tests, mentioned above, or using cells of the hosts expressing the specified polypeptide.

Therapeutic compounds and treatment of diseases

The present invention also relates to therapeutic compound containing the polypeptide of the present invention as the active agent.

The present invention also relates to the use of the polypeptide of the present invention for the production of therapeutic compounds for the prevention or treatment of various disorders and/or ill the deposits of the person. These diseases can be disorders and/or diseases, such as cancer and tumors, infectious diseases, venereal diseases, immuno-associated diseases and/or autoimmune diseases and disorders, cardiovascular diseases, metabolic diseases, diseases of the Central nervous system and disorders associated with the use of chemotherapy.

Specified cancers and tumors are carcinomas, including metastatic carcinoma of the kidney, melanoma, lymphomas, including follicular lymphoma and T-cell skin lymphoma, leukemia, including leukemia retikulez, chronic lymphocytic leukemia and chronic myeloid leukemia, liver cancer, neck, head and kidneys, multiple myeloma, carcinoid tumors and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in AIDS cases.

These infectious diseases are viral infections, including chronic hepatitis b and C and HIV/AIDS, infectious pneumonia and sexually transmitted diseases such as genital warts.

These immune and autoimmune diseases can be graft rejection of tissue or organs, allergies, asthma, psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease and ulcerative colitis.

These bolesna and metabolism can be non-immune disease, such as obesity.

Specified diseases of the Central nervous system can be Alzheimer's disease, Parkinson's disease, schizophrenia and depression.

These diseases and disorders may also be the healing of wounds, anemia in patients subjected to dialysis, and osteoporosis.

Preferably the polypeptide of the present invention can also be used for the production of therapeutic compounds for the prevention or treatment of various disorders and/or diseases in humans, such as some viral infections, such as chronic hepatitis b and C, leukemia, including leukemia retikulez and chronic myeloid leukemia, multiple myeloma, follicular lymphoma, carcinoid tumor, malignant myeloma, metastatic carcinoma, kidney disease, Alzheimer's disease, Parkinson's disease, and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in the case of AIDS, genital warts and/or sexually transmitted diseases.

Some compounds, allowing to obtain the polypeptide of the present invention, and the compound obtained or identified using the specified polypeptide or on the basis of this polypeptide, can also be used for therapeutic treatment of the human body, i.e. they can be used in the us as a therapeutic connection.

Therefore, the present invention also relates to a drug containing as an active agent polynucleotide of the present invention, comprising at least one above-defined coding SNP, the above-defined recombinant vector as defined above cell host and/or defined above antibody.

The present invention also relates to the application of polynucleotide of the present invention containing at least one above-defined coding SNP, the above-defined recombinant vector as defined above cell host and/or defined above antibody for the manufacture of a medicinal product for the prevention and/or treatment of various disorders and/or diseases. These diseases can be disorders and/or diseases, such as cancer and tumors, infectious diseases, venereal diseases, immuno-associated diseases and/or autoimmune diseases and disorders, cardiovascular diseases, metabolic diseases, diseases of the Central nervous system and disorders associated with the use of chemotherapy.

These cancer diseases and tumors are carcinomas, including metastatic carcinoma of the kidney, melanoma, lymphomas, including follicular lymphoma and T-cell lymphoma is expected, leukemia, including leukemia retikulez, chronic lymphocytic leukemia and chronic myeloid leukemia, liver cancer, neck, head and kidneys, multiple myeloma, carcinoid tumors and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in AIDS cases.

These infectious diseases are viral infections, including chronic hepatitis b and C and HIV/AIDS, infectious pneumonia and sexually transmitted diseases such as genital warts.

These immune and autoimmune diseases can be graft rejection of tissue or organs, allergies, asthma, psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease and ulcerative colitis.

Specified diseases of metabolism can be non-immune disease, such as obesity.

Specified diseases of the Central nervous system can be Alzheimer's disease, Parkinson's disease, schizophrenia and depression.

These diseases and disorders may also be the healing of wounds, anemia in patients subjected to dialysis, and osteoporosis.

Preferably the present invention also relates to the use of polynucleotide of the present invention containing at least one above-defined coding SNP, the above-defined recombinant vector, opredelennoye cell host and/or defined above antibody, for the manufacture of a medicinal product for the prevention and/or treatment of various disorders and/or diseases in humans, such as some viral infections, such as chronic hepatitis b and C, leukemia, including leukemia retikulez and chronic myeloid leukemia, multiple myeloma, follicular lymphoma, carcinoid tumor, malignant melanoma, metastatic carcinoma of the kidney, Alzheimer's disease, Parkinson's disease, and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in the case of AIDS, genital warts or venereal disease.

The dose of the polypeptide and other compounds of the present invention, used as the active agent, depends on the choice of connection, therapeutic indications, route of administration, the nature of the composition, of a particular individual and doctor's appointments.

When used as an active agent, the polypeptide of the present invention is usually administered in a dose comprising from 1 to 15 IU (international units). The recommended dose can be administered either subcutaneously or intramuscularly 1 to 5 times a week for 1-12 months.

The present invention also relates to pharmaceutical compositions which contain as active agent, at least one of the above-mentioned connection, such as polypeptide of the present invention, polynucleotide of the present invention containing at least one above-defined SNP, the above-defined recombinant vector as defined above cell host and/or defined above antibody, and pharmaceutically acceptable carrier.

In these pharmaceutical compositions the active agent is mainly present in a physiologically effective dose.

These pharmaceutical compositions may consist, for example, solid or liquid and may be present in the pharmaceutical form used in modern medicine, such as, for example, conventional tablets or tablet coatings, gel capsules, granules, caramel, suppositories, and preferably the preparations and powders for injection. These pharmaceutical forms can be obtained by standard methods.

The active agent(s) may be entered in the fillers commonly used in pharmaceutical compositions, such as talc, Arabic gum, lactose, starch, dextrose, glycerol, ethanol, magnesium stearate, cocoa butter, aqueous or anhydrous media, fats of animal or vegetable origin, paraffin derivatives, glycols, various wetting agents, dispersing or emulsifying agents and preservatives.

The active agent(s) of the present invention may be introduced by the Department is Ino or in combination with other compounds, such as therapeutic compounds, such as other cytokines, such as interleukins or interferons.

Various formulations of pharmaceutical compositions adapted in accordance with the method of administration.

The pharmaceutical compositions can be introduced in various ways known to the specialists.

The present invention also relates to diagnostic compositions, which, as the active agent contains at least one of the above-mentioned connection, such as a polypeptide of the present invention, the entire polynucleotide of the present invention or part thereof, the above-defined recombinant vector as defined above cell host and/or defined above antibody and a suitable pharmaceutically acceptable carrier.

This diagnostic composition may contain, for example, a suitable filler, such as a filler commonly used in diagnostic compositions, such as buffers and preservatives.

The present invention also relates to the application:

a) a therapeutically effective amount of the polypeptide of the present invention and/or

b) polynucleotide of the present invention and/or

(C) defined above, the host cell is taken from the individual being treated, and

to obtain a therapeutic compound intended DL is increasing the level of expression or activity of the polypeptide of the present invention the individual.

So, for example, treatment of an individual in need of raising the level of expression or activity of the polypeptide of the present invention can be made by several methods.

The individual may be entered therapeutically effective amount of the polypeptide of the present invention together with a pharmaceutically acceptable carrier.

Can also be enhanced endogenous production of the polypeptide of the present invention by introducing the individual polynucleotide of the present invention. For example, the specified polynucleotide can be embedded in expressing retroviral vector. Such a vector can be isolated from cells infected with the retroviral plasmid vector containing RNA encoding a polypeptide of the present invention, so that the transduced cells were produced infectious viral particles containing the desired gene. Cm. Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, Chapter 20, Human Molecular Genetics, Strachan & Read, BIOS Scientifics Publishers Ltd (1996).

In accordance with the present invention, it is preferable to polynucleotide containing at least one coding SNP, for example SNP defined above.

The individual may also be introduced owned cell owners who were previously taken from the individual and modified so that they expressed the polyp is Ted of the present invention, as explained above.

The present invention also relates to the application:

a) a therapeutically effective amount of a previously defined immunospecific antibodies and/or

b) polynucleotide that inhibits expression of polynucleotide of the present invention

to obtain a therapeutic compound intended to reduce the level of expression or activity of the polypeptide of the present invention the individual.

Thus, the individual may be entered therapeutically effective amount of the inhibiting agent and/or antibodies, such as antibody, as defined above, or it can be introduced in combination with a pharmaceutically acceptable carrier.

Can also be reduced endogenous production of the polypeptide of the present invention by introducing a specified individual complementary polynucleotide of the present invention that inhibits the expression of polynucleotide of the present invention.

Preferably can be used complementary polynucleotide containing at least one coding SNP, such as SNP defined above.

The present invention also relates to the use of protein IFNα-17 to obtain drugs for prevention or treatment of a patient suffering from a disorder or disease caused by vari is Tom IFNα -17 and associated with the presence in the genome of the specified patient nucleotide sequence that is at least 95%, preferably 97%, more preferably 99%, and particularly preferably 100%) identical to the nucleotide sequence of SEQ ID NO:1, provided that said nucleotide sequence comprises one of the following SNP: DS, 808Ins(a).

The specified medication, preferably used for the prevention or treatment of one of the diseases selected from the group consisting of cancers and tumors, infectious diseases, venereal diseases, immune-related diseases and/or autoimmune diseases and disorders, cardiovascular diseases, metabolic diseases, Central nervous system diseases and disorders associated with the use of chemotherapy.

These cancer diseases and tumors are carcinomas, including metastatic carcinoma of the kidney, melanoma, lymphomas, including follicular lymphoma and T-cell skin lymphoma, leukemia, including leukemia retikulez, chronic lymphocytic leukemia and chronic myeloid leukemia, liver cancer, neck, head and kidneys, multiple myeloma, carcinoid tumors and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in AIDS cases.

The decree is nymi infectious diseases are viral infections, including chronic hepatitis b and C and HIV/AIDS, infectious pneumonia and sexually transmitted diseases such as genital warts.

These immune and autoimmune diseases can be graft rejection of tissue or organs, allergies, asthma, psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease and ulcerative colitis.

Specified diseases of metabolism can be non-immune disease, such as obesity.

Specified diseases of the Central nervous system can be Alzheimer's disease, Parkinson's disease, schizophrenia and depression.

These diseases and disorders may also be the healing of wounds, anemia in patients subjected to dialysis, and osteoporosis.

Connection-mimetic polypeptide IFNα-17 containing SNP DS of the present invention

The present invention also relates to new compounds having biological activity, mostly similar to the activity of the polypeptide with:

a) the amino acid sequence SEQ ID NO:2 or

b) amino acid sequence containing amino acid situated between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2;

provided that the above amino acid sequence (a) and (b) contain SNP G45R.

Specified biological activity can be measured,for example, by assessing the maturation of dendritic cells, release of cytokines CD4+- or CD8+T-lymphocytes release cytokines by monocytes, in vitro or in vivo antiviral activity, cellular antiproliferative activity against cell lines TF-1 or cellular antiproliferative activity against cell lines of Daudi Burkitt, as described in the experimental part.

As mentioned in the experimental part, in comparison with IFNα-2 wild type, G45R-mutated IFNα-17 has:

- higher ability to stimulate the release of IFN-gamma in CD4+T-lymphocytes

- higher ability to stimulate the release of IFN-gamma and IL-10 CD8+T-lymphocytes

- lower the ability to stimulate the release of IL-10 and IL-12 by monocytes,

a similar antiproliferative effect on cells TF-1,

- higher antiproliferative effect on cells of the cell line Daudi Burkitt,

- higher antiviral activity in vitro in cell culture infected VS,

- higher antiviral activity in vivo in a murine EMCV-model.

As was also mentioned in the experimental part, G45R-mutated IFNα-17, compared with IFNα-17 wild-type, has a lower antiproliferative effect on the cell lines On the UDI Burkitt.

The new compound of the present invention, such as compound defined above, may have biological activity, in General, similar activity G45R-mutated IFNα-17.

The specified connection may also have biological activity, such as stimulation of the release of IFN-gamma in CD4+- or CD8+T-lymphocytes and the release of IL-10 CD8+T-lymphocytes, antiviral activity in vitro, or antiviral activity in vivo, which is even higher than the activity of G45R-mutated IFNα-17.

The specified connection may also have biological activity, such as stimulation of the release of IL-10 and IL-12 by monocytes, which is even lower than the activity of G45R-mutated IFNα-17.

The specified connection can be biochemical compound, such as, for example, polypeptide or peptide, or organic molecule, such as, for example, a synthetic peptide-mimetic.

The present invention also relates to the use of the polypeptide of the present invention containing the SNP G45R, to identify the connection, such as connection specified above.

The present invention also relates to a method of identifying compounds of the present invention, comprising the following stages:

a) determining the biological asset the spine of the tested compounds such as, for example, maturation of dendritic cells, release of cytokines CD4+- or CD8+T-lymphocytes, cytokine release by monocytes, cellular antiproliferative activity against cell lines TF-1 or cellular antiproliferative activity against cell lines of Daudi Burkitt, in vitro or in vivo antiviral activity;

(b) comparison:

(i) the activity of the test compounds identified in stage a), with

(ii) the activity of the polypeptide with the amino acid sequence SEQ ID NO:2 or the amino acid sequence containing amino acids between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2, provided that the above amino acid sequence containing the SNP G45R and

C) determining, on the basis of the comparison carried out in stage (b)whether the test compound, in General, similar, or lower, or a higher activity compared with the activity of the polypeptide with the amino acid sequence SEQ ID NO:2 or the amino acid sequence containing amino acids between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2, provided that the above amino acid sequence containing the SNP G45R.

Preferably, the test compound may be previously the preliminary identified from combinatorial libraries of synthetic peptide high-throughput, or it can be constructed by computer simulation of the medicinal product, so that it had the same three-dimensional structure as a polypeptide with the amino acid sequence SEQ ID NO:2, or amino acid sequence containing amino acids between positions 24 and 189 of the amino acid sequence of SEQ ID NO:2, provided that the above amino acid sequence containing the SNP G45R.

Methods of identification and design of compounds well-known to experts.

These methods can be found, for example, in the publication:

- R.B. Silverman (1992). "Organic Chemistry of Drug Design and Drug Action". Academic Press, 1st edition (January 15, 1992).

Anderson S. and J. Chiplin (2002). "Structural genomics; shaping the future of drug design" Drug Discov. Today. 7(2): 105-107.

- Selick H.E., Beresford A.P., Tarbit M.H. (2002). "The merging importance of predictive ADME simulation in drug discovery". Drug Discov. Today. 7(2): 109-116.

- R. Burbidge, M. Trotter, B. Buxton, Holden S. (2001). Drug design by machine learning: support vector machines for pharmaceutical data analysis". Comput. Chem. 26(1): 5-14.

Kauvar L.M. (1996). "Peptide mimetic drugs: a comment on progress and prospects". 14(6): 709.

Compounds of the present invention can be used to produce a medicinal product intended for the prevention or treatment of one of the diseases selected from the group consisting of cancers and tumors, infectious diseases, venereal diseases, immuno-associated illness is evani and/or autoimmune diseases and disorders, cardiovascular diseases, metabolic diseases, Central nervous system diseases and disorders associated with the use of chemotherapy.

These cancer diseases and tumors are carcinomas, including metastatic carcinoma of the kidney, melanoma, lymphomas, including follicular lymphoma and T-cell skin lymphoma, leukemia, including leukemia retikulez, chronic lymphocytic leukemia and chronic myeloid leukemia, liver cancer, neck, head and kidneys, multiple myeloma, carcinoid tumors and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in AIDS cases.

These infectious diseases are viral infections, including chronic hepatitis b and C and HIV/AIDS, infectious pneumonia and sexually transmitted diseases such as genital warts.

These immune and autoimmune diseases can be graft rejection of tissue or organs, allergies, asthma, psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease and ulcerative colitis.

Specified diseases of metabolism can be non-immune disease, such as obesity.

Specified diseases of the Central nervous system can be Alzheimer's disease, Parkinson's disease, schizophrenia and depression.

These diseases and R is straitway can also be healing, anemia in patients subjected to dialysis, and osteoporosis.

Preferably the compounds of the present invention can be used to produce a medicinal product intended for the prevention and/or treatment of one of the diseases selected from the group consisting of some viral infections, such as chronic hepatitis b and C, leukemia, including leukemia retikulez and chronic myeloid leukemia, multiple myeloma, follicular lymphoma, carcinoid tumor, malignant melanoma, metastatic carcinoma of the kidney, Alzheimer's disease, Parkinson's disease, and tumors that arise due to immunodeficiency, such as Kaposi's sarcoma in the case of AIDS, genital warts and/or sexually transmitted diseases.

Experimental part

Example 1: Simulation of a protein encoded by polynucleotide nucleotide sequence containing the SNP G45R, and the protein encoded by the nucleotide sequence of the original gene of wild-type

In the first stage, the designed three-dimensional structure of IFNα-17 based on patterns IFNα-2, which is available in the PDB (code access 1ITF), using computer software Modeler (MSI, San Diego, CA).

Then a fragment of the Mature polypeptide modified so that it could reproduce the mutation G45R.

This m is adapted fragment spent a thousand stages molecular minimization using AMBER and DISCOVER (MSI: Molecular Simulations Inc.).

Then using the same programs and the same force fields conducted two series of calculations of molecular dynamics parameters.

In each case conducted 50000 stages of calculations at 300 K, and then spent 300 stages trim.

The results of this simulation are visualized in figures 1A and 1B.

Example 2. The expression of natural IFNα-17 wild-type and G45R-matirovannogo IFNα-17 in yeast

a) Cloning of natural IFNα-17 wild-type and matirovannogo IFNα-17 in eukaryotic expressing vector pPicZα-topo

The nucleotide sequence encoding the Mature part of the natural IFNα-17 wild-type and G45R-mutated IFNα-17, amplified by PCR using as template genomic DNA from the individual who is heterozygous for the SNP.

For amplification can be used the following PCR primers:

SEQ ID NO:6: sense primer: TGTGATCTGCCTCAGACCCAC.

SEQ ID NO:7: antisense primer: TCAATCCTTCCTCCTTAATATTTTTTGC.

PCR products were built in eukaryotic expressing vector pPicZα-topo under the control of the hybrid promoter AOH induced by methanol (TOROTM-cloning; Invitrogen Corp.).

This vector provides heterologous expression of eukaryotic proteins in the yeast Pichia pastoris.

After checking nucleotide the th sequence of the region of the vector, which encodes the recombinant proteins, this vector was linearizable using restricteduser enzyme PmeI and strain P. pastoris (Invitrogen) transformed these recombinant expressing vectors.

b) Heterologous expression in P. pastoris and purification of proteins IFNα-17 wild-type and G45R-mutated IFNα-17

Two saturated pre-culture in 50 ml of BMGY medium (2% peptone, 1% yeast extract, of 1.34% YNB, 1% glycerol, 100 mm potassium phosphate, 0.4 mg/l Biotin, pH 6.0)containing clone that encodes a protein IFNα-17 wild-type or clone encoding G45R-mutated IFNα-17, were grown in 24-48 hours at 30°C, stirring at 200 revolutions per minute (rpm).

When the cell density in the culture has reached saturation (which corresponded to an optical density 12, measured at a wavelength of 600 nm), this culture was used for inoculation, at 5 OD/ml, 250 ml BMMY medium (2% peptone, 1% yeast extract, of 1.34% YNB, 0.5% methanol, 100 mm potassium phosphate, 0.4 mg/l Biotin, pH 6.0).

Then protein expression was induced with methanol at a final concentration of 1% over 24 hours at 30°and with stirring in a flask for culturing at 180 Rev/min

Due to the presence of a signal peptide sequence "alpha factor"above coding sequences, proteins secretarials yeast in the cultural panorama of the remote control. The alpha factor is usually cleaved during processing.

The suspension was centrifuged and the protein was isolated from the resulting supernatant by HPLC.

In the preliminary stage, carried out by ultrafiltration (Labscale, the cut-off of 5000 Da, Millipore) followed by dialysis, achieved a tenfold concentration of yeast supernatant in buffer 50 mm Tris-HCl, pH of 9.0, 25 mm NaCl.

In the first chromatographic stage was performed protein purification by affinity column with separate (Blue Sepharose, Amersham Pharmacia). The presence of protein in the collected fractions was confirmed, on the one hand, by electrophoresis in LTO-SDS page, and on the other hand, by immunodetection using specific antibodies directed against the protein IFNα-17. In this stage, the purity of the desired protein was more than 75%.

In the second stage of purification gel filtration was allowed to replace the buffer in the collected fractions corresponding to proteins IFNα-17, instead of 50 mm Tris pH of 9.0, 25 mm NaCl.

The last stage of purification consisted in the separation of proteins on ion-exchange chromatographic column.

Fractions containing recombinant protein were injected with on anion-exchange column (ResourceQ of 6.0 ml, Pharmacia), previously equilibrated in Tris buffer 50 mm, pH 9, NaCl, 25 mm. Elution of proteins was performed using gradient within 0.25 and 1 M NaCl in Tris buffer,50 mm, pH 9.

The purity of the desired protein was assessed by electrophoresis in LTOs/PAG-gel, and the protein concentration was measured using densitometry (Quantity one, Biorad) and ICA-analysis (bicinchoninic acid - copper sulfate, Sigma).

Finally, the purified protein IFNα-17 wild-type and G45R-mutated protein IFNα-17 obtained in accordance with the described Protocol on a larger scale to produce large quantities of protein, used for functional tests described below.

Example 3. Evaluation of immunomodulatory activity of natural IFNα-17 wild-type and G45R-mutated IFNα-17

IFN type I (IFN-alpha and IFN-beta) is able to modulate some immune system function. It was demonstrated that they accelerate the maturation of dendritic cells (DC): that is, enhance the expression of MHC-class I molecules (HLA-ABC) and II (HLA-DR), increased expression of molecules involved in co-stimulation of T-lymphocytes, CD80-, CD86 and CD83 molecules, and increase the incentive function of T lymphocytes.

a) Action G45R-mutated IFNα-17 on the maturation of dendritic cells

First explored immunomoduliruushim activity G45R-mutated IFNα-17 in relation to the maturation of dendritic cells and compared it with the activity of IFNα-2 wild type, selected as representative of the commercial product Intron A.

To do this, first come and dendritic cells from monocytes in the peripheral blood of an adult individual, cultivated in the presence of the cytokines GM-CSF and IL-4. After cleaning using the cleaning kit CD14+-these cells dendritic cells maintained in the presence of 100 ng/ml IFNα-2 wild-type or G45R-mutated IFNα-17 and their phenotype was determined using FACS analysis by assessing the expression of molecules of the MHC molecules of class I and II and markers CD40, CD80, CD86, CD83 and CD1. The stage of maturation of these dendritic cells were also compared with the stages of maturation of dendritic cells derived without IFNα-processing, i.e. unstimulated dendritic cells, which was used as a control.

Average values of fluorescence intensity for each marker and for the three experimental conditions, expressed in arbitrary units, are presented in the following table:

HLA

ABC
HLA

DR
CD40CD80CD86CD83CD1a
No. IFNα- IFNα no14215524640242165
G45R-mutated IFNα-1716518931650321949
IFNα-2 wild-type 17927649187572651

The results of this test demonstrated that G45R-mutated protein IFNα-17 able to stimulate the maturation of dendritic cells.

b) Action G45R-mutated IFNα-17 on the release of cytokines by T-lymphocytes

Together with immunomodulating activity G45R-mutated IFNα-17 also investigated by assessing the release of cytokines by T-lymphocytes in the presence of the mutated protein IFNα-17 and in the presence or in the absence of a strong antigen (SEB), is used to simulate the immune response against cytotoxic reactions. This test is also carried out in the presence of IFNα-2 wild type, used as a control and selected as representative of the commercial product Intron A.

To do this, a healthy donor were isolated mononuclear cells of peripheral blood (MCPC) and stimulated for 16 hours in the appropriate medium containing anti-CD3 and anti-CD28 antibodies or SEB. Each culture was added to 4 μg/ml IFNα-2 wild-type or G45R-mutated IFNα-17. After stimulation, T cells were labeled extracellular anti-CD3, anti-CD4 and anti-CD69 antibodies or anti-CD3, anti-CD8 and anti-CD69 antibodies were labeled intracellular specific antibodies directed against cytokines Th1-type (IF-gamma) or against cytokines of the Th2-type (IL-10). Fluorescent cells were analyzed using FACScalibur and CellQuest.

The results showed that G45R-mutated IFNα-17 and IFNα-2 wild type did not stimulate the release of IL-10 and IFN-gamma, and therefore not activated T-lymphocytes in the absence of SEB.

In contrast, G45R-mutated IFNα-17 and IFNα-2 wild-type stimulated release of cytokines (IL-10 and IFN-gamma) SEB-activated T-lymphocytes, as shown in the table below. This table demonstrated the release of cytokines by T-lymphocytes in the presence of SEB, expressed as the percentage of CD4+-, CD69+cells or CD8+,CD69+cells for CD4+T-lymphocytes and CD8+T-lymphocytes, respectively, and specify the percentage of CD69+cells from the total number of cells.

T-lymphocyteIFN-gammaIL-10CD69+cells/total
CD4+CD69+Negative control11,97,51,26
G45R-IFNα-1737,27grade of 20.062,94
IFNα-2 wild-type19,624,682,7
CD8+CD69+Negative control8,73 0,654,69
G45R-IFNα-1736,77,029,54
IFNα-17 wild-type16,374.26 deaths10,02

These results clearly demonstrated that G45R-mutated IFNα-17 strongly stimulates the release of cytokines (IFN-gamma) CD4+T-lymphocytes and CD8+T-lymphocytes, pre-activated antigen SEB. In this test, the level of production of interferon gamma in CD4+T-lymphocytes in the presence of G45R-mutated IFNα-17 was higher than in the presence of IFNα-2 wild type and level of production of interferon gamma and IL-10 CD8+T-lymphocytes in the presence of G45R-mutated IFNα-17 was higher than in the presence of IFNα-2 wild-type.

(C) Action G45R-mutated IFNα-17 on the release of cytokines by monocytes

And finally, together with immunomodulating activity G45R-mutated IFNα-17 investigated by measuring the release rate of the cytokines by monocytes in the absence or in the presence of bacterial toxic agent (LPS). This test is also carried out in the presence of IFNα-2 wild type, used as a control and selected as representative of the commercial product Intron A.

To do this, a healthy donor were isolated mononuclear cells of the peripheral cu is VI (MCPC) and tested for their phenotype to determine the relative amount of CD64+CD4dim cells (CD64 and CD4dim are markers of monocytes in the blood). After culturing overnight, these MCPC incubated in culture medium alone (estimulando cells) or in the presence of LPS stimulated cells). Each culture was added to 4 μg/ml IFNα-2 wild-type or G45R-mutated IFNα-17. After cultivation, the cells were labeled extracellular anti-CD64 and anti-CD4dim antibodies and intracellular specific antibodies directed against cytokines Th1-type (TNF-alpha), IL-12 and IL-10.

Fluorescent cells were analyzed using FACScalibur and CellQuest.

The results showed that G45R-mutated IFNα-17 and IFNα-2 wild type did not stimulate the release of cytokines (IL-10, IL-12 and TNF-alpha) in the absence of LPS.

In contrast, monocytes released cytokines in the presence of LPS. The release of IL-10 and IL-12 by monocytes in the presence of LPS further increased in the presence of IFNα-2 wild type, but not in the presence of G45R-mutated IFNα-17, as shown in the table below. This table demonstrated the release of cytokines by monocytes in the presence of LPS, expressed as the percentage of CD64+, CD4dim-cells, and percentages CD4dim, CD64+cells from the total number of cells.

IL-10IL-12TNF-αCD4dimCD64+cells/everything about
IFNα no16,21charged 8.5213,883,1
G45R-IFNα-1717,478,340,922,86
IFNα-2 wild-type49,3434,4850,872,71

Thus, the absence of a synergistic effect observed in the presence of G45R-mutated IFNα-17 and LPS, suggesting that G45R-mutated IFNα-17 may have immunosuppressive action.

Example 4. Evaluation of in vitro antiproliferative action G45R-mutated IFNα-17 cell line erythroleucus TF-1

Action G45R-mutated IFNα-17 also assessed in relation to cell lines of erythroleucus TF-1. This test is also carried out in the presence of IFNα-2 wild type, used as a control and selected as representative of the commercial product Intron A.

For this, cells TF-1 was subjected to contact with increasing concentrations of mutant IFNα-21 or IFNα-2 wild-type (0.001 to 1000 ng/ml) and measured cell proliferation.

The results presented in figure 2, showed that G45R-mutated IFNα-17 has a weak antiproliferative effect on cells TF-1, and this action is similar to action of IFNα-2 wild-type, which gives the OS the Finance to assume, what hematologica toxicity G45R-mutated IFNα-17 does not exceed the toxicity of IFNα-2 wild-type.

Example 5. Assessment of the ability of G45R-mutated IFNα-17 to induce antiproliferative activity in human cell lines lymphoma, Daudi Burkitt

These tests were carried out on two different proteins IFNα-17, namely G45R-mutated protein IFNα-17 and natural protein IFNα-17 wild type. Cells (human cell lines lymphoma, Daudi Burkitt, hereafter referred to as "cells, Daudi"), pre-cultured in medium RPMI 1640 (which has been added 10% fetal calf serum and 2 mm L-glutamine), then inoculable in 96-well tablets when cell density of 4 x 104cells/well.

In each well, cells Daudi was subjected to contact with increasing concentrations of proteins or natural IFNα-17 wild-type or G45R-mutated IFNα-17.

Concentrations of the studied IFNα-17 (natural protein wild-type or mutated protein) ranged from 0.003 pmol up to 600 nmol (final concentration in the wells).

Then cells Daudi were incubated for 66 h at 37aboutWith 5% CO2then in these cultures was added to the reagent Uptiblue (Uptima). After additional incubation for 4 hours, the degree of cell proliferation was quantitatively assessed by measuring fluores is enzie, emitted at a wavelength of 590 nmol (excitation 560 nm).

Antiproliferative activity of natural IFNα-17 wild-type or G45R-mutated IFNα-17 measured as IC50corresponding to the concentration of protein IFNα-17, in picomoles (pmol), which inhibits 50% of cell growth.

Carried out four similar experiments, each of which was repeated four times. The value of the IC50for each experiment is presented in the following table:

ExperimentIC50(pmol)Ratio
IFNα-17 wild-typeG45R-IFNα-17
10,321,515
20,607,2012
30,221,537
40,8110,9914

The average IC50 values measured for IFNα-17 wild type was 0.49 pmol, and the average IC50 values measured for G45R-mutated IFNα-17, was 5,31 PM. Thus, the average ratio of IC50 values for the mutated IFNα-17 to the value for natural IFNα-17 wild type reached 10,80 (with a standard deviation of 3.46)

This test showed that in the case of G45R-mutated IFNα-17 antiproliferative activity is strongly inhibited (i.e. approximately 5 to 15 times less) compared with IFNα-17 wild-type.

Similar independent experiments were performed using IFNα-2 wild type for comparison of antiproliferative action G45R-mutated IFNα-17 on cell proliferation, Daudi with the action of IFNα-2 wild type. For this, cells Daudi were cultured in the presence of concentrations of G45R-mutated IFNα-17 or IFNα-2 wild type comprising in the range from 0.001 to 10 ng/ml.

Conducted three similar experiments. The results of one representative experiment are given in figure 3.

These results demonstrated that the antiproliferative effect G45R-mutated IFNα-17 on the proliferation of cell lines Daudi Burkitt higher than the effect of IFNα-2 wild-type.

Example 6. Evaluation of antiviral activity G45R-mutated IFNα-17

IFN plays an important role in antiviral defense. The antiviral activity of IFN partly due to IFN-induced enzyme systems, such as:

- 2'5'-oligoadenylate synthetase, the enzyme that catalyzes the synthesis of adenosine oligomer. These oligomers activate RNase L, endoribonuclease, which, after activation, R will sreset viral RNA;

- MX proteins (GTP), which inhibit the synthesis and/or maturation of viral transcripts. This activity mainly affects the influenza virus;

- PKR protein (or kinase R), which is activated by double-stranded RNA. Activated PKR inhibits protein synthesis.

The antiviral activity of IFN is also induced by other mechanisms, such as, in the case of retroviruses, the inhibition of penetration of viral particles in cells, replication, binding and release of the particles, as well as the infectivity of viral particles.

And finally, IFN has mediated antiviral effect through the modulation of some functions of the immune system, and in particular through the stimulation of the response to cell-mediated reactions (including an increase in molecules MHC class I and II, increasing the production of IL-12 and IFN-gamma, increased CTL activity and so on).

Antiviral activity G45R-mutated IFNα-17 also assessed in vitro in cell culture and in vivo in murine models. Both tests were conducted in parallel with IFNα-2 wild type, used as a control and selected as representative of the commercial product Intron A.

a) Antiviral activity in vitro in cell culture

This analysis allows to evaluate the antiviral activity of G45R-mutated IFNα-17 and IFNα-2 wild-type cell of the cult of the re using vesicular stomatitis virus (VSV).

For this human epithelial WISH cells were cultured for 24 hours in the presence of decreasing concentrations of G45R-mutated IFNα-17 or IFNα-2 wild type. Then these cells were infected with vesicular stomatitis virus (VSV) for another 24-48 hours and were evaluated by lysis of the cells.

The antiviral effect of different tested IFNα determined by comparing the value of the IC50corresponding concentrations of IFN, inhibiting 50% lysis of the cells, induced by virus VSV.

A similar experiment was performed twice, and the value of the IC50measured in one representative experiment are presented in the following table:

IFNα-2 wild-typeG45R-mutated IFNα-17
IC50(ng/ml)42

Thus, G45R-mutated IFNα-17 possessed antiviral activity in cell culture infected with VSV, and this antiviral activity was higher than the activity of IFNα-2 wild-type.

(b) Antiviral activity in vivo in a murine model

This test was performed in vivo in EMCV-mouse models (mice, infected with the virus encephalomyocarditis).

Human IFN was found dose-dependent antiviral activity in mice, and this is aktivnosti was, basically, 100-1000 times less than the activity of a similar amount of mouse IFN (Meister et al., (1986). J. Gen. Virol. 67, 1633-1644).

Intraperitoneal injection of virus encephalomyocarditis mice (EMCV) has led to the rapid progression of the fatal disease characterized by lesions of the Central nervous system and encephalitis (Finter NB (1973). Front.Biol. 2:295-360). It was found that mouse and human interferon alpha are effective to protect mice from lethal EMCV infection (Tovey & Maury (1999). J. interferon Cytokine Res. 19:145-155).

Groups of 20 six-week Swiss mice were infected intraperitoneally (I.P. Pavlova.) 100 x LD50EMCV, and the same dose was injected with another one hour, and then daily for 3 days was administered 2 mg drugs G45R-mutated IFNα-17 and IFNα-2 wild type. As a control group served as the animals treated only filler. For the survival of these animals were daily observed for 21 days.

The results presented in figure 4, indicate that the relative survival rate of mice treated G45R-mutated IFNα-17, was much higher than the survival rate of untreated mice, and even higher than the survival rate of mice treated with IFNα-2 wild type. These data demonstrated strong antiviral activity G45R-mutated IFNα-17 in murine models in vivo.

All obtained results which you demonstrated what G45R-mutated IFNα-17 has unique biological properties.

1. Selected polynucleotide that encodes a polypeptide with antiviral, antiproliferative and/or immunomodulatory activity, where polynucleotide essentially corresponds to the

a) a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:1 or its coding sequence, provided that said nucleotide sequence has a single nucleotide polymorphism SNP g771c, based on the numbering of SEQ ID NO:1; or

b) a nucleotide sequence complementary to the nucleotide sequence (a).

2. Selected polynucleotide according to claim 1, characterized in that the nucleotide sequence specified polynucleotide at least 99%identical to the nucleotide sequence of SEQ ID NO:1 or its complementary sequence.

3. Selected polynucleotide according to claim 1 or 2, characterized in that it encodes a polypeptide containing the amino acid sequence of SEQ ID NO:2 and having SNP G45R, based on the numbering of SEQ ID NO:2.

4. Selected polynucleotide that encodes a polypeptide with antiviral, antiproliferative and/or immunomodulatory activity, where polynucleotide essentially corresponds to the

a) part or all after the outermost SEQ ID NO:1, or its coding sequence, provided that this nucleotide sequence contains the SNP g771c, based on the numbering of SEQ ID NO:1, or

b) a nucleotide sequence complementary to the nucleotide sequence (a), with the specified polynucleotide consists of at least 40 nucleotides.

5. Selected polynucleotide according to claim 4, characterized in that it encodes a polypeptide having the SNP G45R, based on the numbering of SEQ ID NO:2.

6. The selected polypeptide with antiviral, antiproliferative and/or immunomodulatory activity, essentially corresponding to the amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2, provided that the amino acid sequence of the specified polypeptide contains SNP G45R, based on the numbering of SEQ ID NO:2.

7. The selected polypeptide with antiviral, antiproliferative and/or immunomodulatory activity, essentially corresponding to the amino acid sequence that is at least 95% identical to amino acids 24-189 amino acid sequence of SEQ ID NO:2, provided that the amino acid sequence of the specified polypeptide contains SNP G45R, based on the numbering of SEQ ID NO:2.

8. The selected polypeptide according to claim 6 or 7, characterized in that the specified aminoxy the pilot sequence at least 99% identical to the amino acid sequence of SEQ ID NO:2.

9. The selected polypeptide according to any one of p-8, characterized in that it has the same or substantially the same biological activity as the interferon alpha 17.

10. The use of selected polynucleotide according to claims 1 to 5 comprising the expression vector to enhance the endogenous production of the polypeptide by PP-9.

11. The use of a selected polypeptide according PP-9 as a therapeutic agent with antiviral, antiproliferative and/or immunomodulatory activity.

12. The use of a selected polypeptide according PP-9 to obtain drugs for prevention or treatment of a disease selected from the group consisting of various types of tumors and malignant tumors, infectious diseases, immunologic and autoimmune diseases, cardiovascular diseases, metabolic diseases, diseases of the Central nervous system, disorders associated with chemotherapy, wound healing, anemia in patients undergoing dialysis, and/or osteoporosis.

13. The application indicated in paragraph 12, where these infectious diseases include viral infections, including chronic hepatitis b and C, HIV/AIDS, infectious pneumonia and sexually transmitted diseases such as genital warts.



 

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83 cl, 32 dwg, 12 tbl, 16 ex

FIELD: organic chemistry, genetics.

SUBSTANCE: invention refers to genetic engineering. Recombinant plasmid DNA pYAll-39 contains: - Pstl-Pstl-DNA vector fragment pUC19 sized 2700 nucleotide pairs, - Pstl- Pstl - alphoid DNA fragment of human lymphocytes high-specific for centromeric areas of 4th and 9th human chromosomes sized 680 nucleotide pairs. The latter is produced from Pstl restriction fragments of alphoid DNA total fraction as chromosomes hybridization in situ under special conditions of high stiffness with standard saline solution with 55% phormamide. Genetic marker is -Ampr - gen of ampicillin-resistance. Invention enables to reliably identify 4th and 9th human chromosomes in norm and pathology, including preimplantation, prenatal and postnatal diagnostics of chromosome anomaly including aneuploidy at various cancer types, as well as for cytogenetic human genome mapping.

EFFECT: identification of 4th and 9th human chromosomes in norm and pathology.

1 cl, 4 ex

FIELD: technological processess.

SUBSTANCE: oligomeric polypeptide dimmers include ligand-binding leptyne domains. Domains are connected by means of flexible polypeptide linker molecules. Linker molecules might possibly include sites of sensitivity to proteases.

EFFECT: releases biologically active cytokines when injecting to human being or animal.

31 cl, 13 dwg

FIELD: technological processes.

SUBSTANCE: protein variants are suggested that possess lysozyme activity. Protein-based antibacterial substance is described. DNA molecule is discovered that codes the specified protein, and also procariotic cell is discovered that contained the specified DNA molecule. Method is described to prepare protein with the help of procariotic cell.

EFFECT: simplifies preparation of antibacterial substance in commercially significant volumes.

13 cl, 3 ex

FIELD: medicine, molecular biology, antibodies.

SUBSTANCE: invention relates to an antibody raised against CCR5 and comprising: (i) two light chains wherein each light chain comprises product of plasmid expression and designated as pVK:HuPRO140-VK (ATCC - PTA-4097), and (ii) two heavy chains wherein each heavy chain comprises product of plasmid expression and designated as pVg4:HuPRO140 HG2-VH (ATCC - PTA-4098), or plasmid designated as pVg4:HuPRO140 (mut B+D+I)-VH (ATCC - PTA-4099), or fragment of such antibody binding with CCR5 on a human cell surface. Invention relates to nucleic acid encoding light and heavy chains of antibody, expression vector, cell-host transformed with at least one vector, and a method for preparing antibody. Antibody is used as an active component in composition used for inhibition of infection of cells CD4 + HIV-1, and to a pharmaceutical composition used in treatment of a patient with HIV-1 infection. Also, invention relates to antibody conjugate against CCR5 and its using. Use of antibodies provides enhancing effectiveness of prophylaxis and treatment of HIV-1 infection.

EFFECT: valuable medicinal properties of antibody.

31 cl, 23 dwg, 3 ex

FIELD: medicine, immunology, chemistry of peptides.

SUBSTANCE: invention discloses peptides GNA33 representing mimetics of epitopes of microorganisms Neisseria meningitides of the serogroup B showing definite amino acid sequences given in the description and able to induce production of antibodies eliciting complement-mediated bactericidal activity and/or opsonic activity against indicated microorganisms in a mammalian subject. Indicated peptides are used as components of a composition used in a method for production of the immune response reaction against indicated microorganisms in a mammalian subject. Also invention concerns a polynucleotide encoding indicated peptide and recombinant expression vector comprising indicated polynucleotide. Also invention discloses a method for preparing peptide GNA33 by culturing the cell-host comprising recombinant expression vector and assay for the presence of antibodies raised against meningococci B in biological sample using GNA33 peptide. Using the invention provides effectiveness of vaccine raised against MenB and safety for its using for a patient.

EFFECT: valuable biological and medicinal properties of peptides.

11 cl, 6 tbl, 8 dwg, 6 ex

The invention relates to the field of the biotechnology industry, in particular the production of virus-inducer for the production of human leukocyte interferon
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