Vns-met-histones

FIELD: biotechnologies.

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

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

17 cl, 3 dwg, 6 tbl, 7 ex

 

The present invention provides a nucleic acid molecule which encodes a polypeptide consisting of the two methionine residues in the first and second N-terminal amino acid residues, connected by peptide bonds Mature the eukaryotic histone. The present invention also relates to a vector containing the specified nucleic acid molecule, a host transformed by the specified vector, the polypeptide encoded by the nucleic acid molecule, and pharmaceutical and diagnostic compositions. The present invention also relates to the use of nucleic acid molecules, vectors, hosts, and polypeptide of the present invention for the preparation of compositions for the treatment of diseases. In addition, the present invention relates to a method for checking the presence of the nucleic acid molecule or polypeptide in the sample and to the set.

Throughout this description contains many documents. Revealing the essence of the content of these documents, including manufacturers ' instructions, fully incorporated here by reference.

Currently there is a huge economic interest in the production of high levels of recombinant proteins like histones. Production of large quantities of recombinant proteins is of interest not only for what the devices of sufficient protein for studies of their properties and functions, but also to ensure that large quantities of protein for therapeutic use.

A huge number of parameters must be taken into account for a successful production at a high level, and purification of recombinant proteins. Important parameters include the terms in the expression, regulation of translation and mRNA stability, targeted delivery and degradation of the protein (Makrides, S., Microbiological Reviews, 1996: 512).

One approach to improve the production, detection, and purification of recombinant proteins is the use of a broad selection of partners merge (Makrides, S., Microbiological Reviews, 1996: 512). We developed detailed methods to include affinity tags for purification and detection of recombinant proteins. Such affinity tags United advantageous ability to make possible more effective cleaning and at the same time to allow easy detection of the recombinant protein on the basis of the label. However, in many cases adding a fairly large affinity tag may be disadvantageous due to adverse effects on translation, arrangement and activity of the protein. Especially when used for therapeutic applications it is often necessary subsequent removal of affinity tags, which thereby weakens some positive effects (for example, light detection), which affinity tag attached protein (Gellissen, G. “Production of Recombinant Proteins”, 2005, WILEY-VCH Vrlag GmbH& Co., KgaA, Weinheim).

The inclusion of a methionine residue at the N end of each polypeptide in the process of creation is part of the universal signal for the initiation of translation used by prokaryotes and eukaryotes. TheE. colithe removal of this N-terminal methionine residue succeeds cytoplasmic enzyme methionine-aminopeptidase (map) (Hirel et al., Biochemistry, 1998, 86: 8247).

It was found that efficient cleavage of N-terminal methionine residue of recombinant eukaryotic proteins produced in prokaryotes, such asE. colidepends on connecting with the amino acid methionine. Although there are conflicting data for some amino acids seem to agree that the probability of removal is greatest for small and uncharged amino acid residues Ala, Gly, Pro, Ser, Val, Cys, and Thr. Apparently, large side chains unfavorable for removal of methionine (Hirel et al., Biochemistry, 1989, 86: 8247; Frottin et al., Mol. & Cell Proteomics, 2006, 12: 2336; Gellissen, G. “Production of recombinant Protein”, 2005, WILEY-VCH Verlag GmbH&Co., KgaA, Weinheim).

I believe that the removal of N-terminal methionine plays an important role in the stability of the protein (Giglione et al., EMBO J., 2003, 1: 13), and in the proper functioning of the protein, as determined, for example, MEF-2C, human hemoglobin, interleukin-2, homologues of RNase A or ribonuclease frog (Meierhans and Allemann, J. Biol. Chem. 1998, 273: 26052; Adachi, K. et al., Protein xpr. Purif., 2000, 20; 37; Endo, S. et al., Biochemistry, 2001, 40: 914; Boix, E. et al., J. Mol. Biol., 1996, 257: 992; Liao, Y.D. et al., Nucleic Acids Res., 2003, 31: 5247; Varshavsky, A., Proc. Natl. Acad. Sci., 1996, 93: 12142). Additional doctrine concerning why nature has kept such a specialized enzyme system to remove the residue of methionine, is to reuse the cellular pool methionine to conserve this essential amino acids (Hirel et al., Biochemistry. 1989, 86: 8247).

In ER describes the recombinant production of proteins histones inE. coli. Such recombinant production of human proteins is considered to be advantageous for therapeutic applications, as well as more efficient and cost-effective in comparison with products from the thymus of a person or a calf. In addition, the recombinant production of proteins makes possible a better quality control during the production process.

Pyo and others (Pyo, S.H. et al., Protein Expr. Purif., 2001, 1: 38) describe the production of recombinant histone N inE. coliusing strongly basic properties of histone to develop effective way large-scale purification of recombinant protein.

Although the prior art has been demonstrated products at a high level of recombinant proteins, there is, however, a real need in finding appropriate ways to identify the result R is combinatori protein. As discussed above, the use of affinity tags such as His-tag, is widely used in the art, but may be problematic in the production of proteins for therapeutic applications.

Thus, underlying the present invention technical problem was the provision of improved recombinant eukaryotic polypeptides which, for example, simplify the production and detection.

The solution to this technical problem is achieved by the embodiments characterized in the claims.

Accordingly, in the first embodiment, the present invention relates to a nucleic acid molecule that (a) encodes a polypeptide consisting of (AA) the two methionine residues in the first and second N-terminal amino acid residues, connected by peptide bond (ab) Mature the eukaryotic histone; (b) encodes a polypeptide consisting of (ba) of the two methionine residues in the first and second N-terminal amino acid residues, connected by peptide bond (bb) Mature eukaryotic a polypeptide sequence which is at least 80% identical the sequence of the Mature eukaryotic histone (a) and which substantially retains its biological activity; or (C) hybridizes in the rigid conditions with a complementary chain of a molecule of nucleic acid, encoding the polypeptide (a) or (b)with the specified nucleic acid molecule encodes a polypeptide that has at least two N-terminal methionine residue and in which substantially retains the biological activity of the polypeptide (a) or (b).

In accordance with the present invention, the molecules of nucleic acids include DNA, for example, cDNA or genomic DNA, RNA (e.g. mRNA), in synthetic or semi-synthetic form, further synthetic or semi-synthetic derivatives of DNA or RNA (e.g., NCP or phosphorothioate) and copolymers, as sense and antisense chain. They may contain additional non-natural or derivatives of the nucleotide bases, as it will be quickly obvious skilled in the art specialists. In a preferred embodiment, the nucleic acid molecule is DNA, including genomic DNA.

For the purposes of the present invention, peptide-nucleic acid (NCP) is a polyamide type analogue of DNA, and are commercially available Monomeric units for derivatives of adenine, guanine, thymine and cytosine (Perceptive Biosystems). Some of the components of DNA, such as phosphorus trioxide phosphorus or derivative desoxyribose, not present in the NCP. As described by Nielsen and others (Science 254: 1497 (1991)) and Egholm and other Nature 365: 666 (1993)), RNA binding which are specifically and tightly to complementary DNA strands and are not subject to degradation by nucleases. In fact, the NCP is associated stronger with DNA than DNA itself. This is probably due to the fact that there is no electrostatic repulsion between the two circuits, as well as the fact that the backbone of the polyamide is more flexible. Because of this, duplexes PNK/DNA are formed in a wider range of conditions hardness than duplexes, DNA/DNA, which makes the performance of multiplex hybridization easier. Due to the strong binding can be used probes smaller than with DNA. In addition, more likely, you can define a mating defect on one basis when using hybridization PNK/DNA, because the defect pairing for a single reason, a 15-Mer PNK/DNA lowers the melting point (T.sub.m) 8-20° PNK/DNA in comparison with 4-16° for 15-dimensional duplex DNA/DNA. The absence of charged groups in the NCP indicates that hybridization can be performed at low ionic strength and to reduce the possible interfering effects of the salt during analysis.

Used here, the term "polypeptide" is a group of molecules which comprise more than 30 amino acids. In accordance with the present invention, the group of polypeptides include proteins". The polypeptides may further form dimers, trimers and oligomers of higher order, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such di is a career, trimers, etc. may be identical or non-identical. In the corresponding structures of the highest order is called Homo - or heterodimers, Homo - or heterotrimeric etc. Homo - and heterodimer etc. also fall under the definition of "protein". The polypeptides may also be fused proteins, and partner in the merger attached to the C-end of the polypeptide of the present invention. These components are specified fused proteins, which are not sequences of histones or fragments or variants described here above, include amino acid sequences that impart desired properties, such as modified/increased stability, modified/enhanced solubility and/or the ability for targeted delivery to one or more specific cell types, or would be able to report excellent biological activity. For example, provide proteins, merged with antibodies specific for cell surface markers, or recognize antigen fragments of such antibodies. In addition, the present invention also covered peptidomimetics of such polypeptides in which amino acid(s) and/or peptide bond(s) replaced by functional equivalents. Such functional equivalents include all known amino acids other than the 20 codere the s genes of amino acids, such as selenocysteine. The terms "polypeptide" and "protein" also refer to natural and modified polypeptides/proteins, the modification is carried out, for example, when glycosylation, acetylation, phosphorylation, etc. Such modifications are widely known in this technical field.

The term "methionine", in accordance with the present invention, are well known to the skilled in the art specialist. Methionine is an essential amino acid encoded by the codon AUG in accordance with the standard genetic code. Specified methionine, because it is found in eukaryotes, contributes in the preferred implementation of the present invention. N-formylmethionine prokaryotes also included in the meaning of the term "methionine" and contributes to an alternative implementation of the present invention.

Used herein, the terms "first and second N-terminal amino acid residues" refers to amino acid residues found at positions 1 and 2 of the polypeptide of the present invention. These residues are also referred to in the art as the last and penultimate residues at N-end. In other words, the first methionine residue located in the N-terminal position of the original product broadcast polypeptide, which itself contains a methionine at its N end.

Use is used here, the term "peptide bond" is well known to the skilled in the art specialist and refers to a chemical bond, formed between two molecules of the amino acid and the carboxyl group of one amino acid reacts with the amino group of another amino acid.

The term "Mature eukaryotic histone", in accordance with the present invention belongs to the histone, deprived of its initial N-terminal methionine. As is well known to the skilled in the art specialist, broadcast polypeptides used the universal signal of translation initiation, which leads to the inclusion of methionine as the starting amino acid residue at the N end of the translated polypeptide. In eukaryotes, and partly also in prokaryotes, this N-terminal methionine is cleaved at the "Mature" polypeptide.

In accordance with the present invention, the term "histone" refers to a group of proteins, including the core histones H2A (no access in Swiss-Prot for H2A man - R), H2B (no access in Swiss-Prot for H2B human R), H3 (no access in Swiss-Prot for N man - R) and H4 (no access in Swiss-Prot for H4 man - R), and the family of the linker histone H1 (see below No. of access to Swiss-Prot). Classically histones known as structural components of the cell nucleus, where they act as "coils", which is wound DNA, and play a key role in gene regulation. However, histones exhibit a wide pair is here (Reichhard, R. et al., Proc. Natl. Acad. Sci. 1985, 82: 4871; Reichhard, R. et al., FEBS 1985, 188: 63). Installed, for example, that the histones are systematically as hormones and regulatory factors, as well as have an important protective function.

Thanks to its versatility, the histones were important in a number of therapeutic approaches. For example, it was found that the histones H1, H2A and H2B as well as stimulate healthy peripheral lymphocytes (Cebecauer, L. et al. Rheumatologia 1991, 5: 107). It is established that histone H1 enhances the regeneration of muscle tissue by stimulating the proliferation of cultured myoblasts (Henriquez, J.P. et al., J. Cell Sci. 2002, 115: 2041), changing the picture of diseases characterized amyloidogenic fibrils (Duce, J.A. et al. J. Mol. Biol. 2006, 361: 493), and stimulates stem cells (Semina et al. Radiation Biology and Oncology, 1994, 34: 544). Histone H1 is also used for diagnosis, prevention and treatment of ulcerative colitis and its clinical subtypes (Braun, J. et al., U.S. patent No. 6074835). In addition, it was found that histone H1 and histone cores capable of carrying biologically active substances through the blood-brain barrier (Pardridgem W.M. et al., J. Pharmacol. Exp. Ther. 1989, 251: 821). In addition, in the European patent 0392315 demonstrates hormonal or hormone-like activity of histone H1 and its subtypes. Role of histone in autoimmune diseases, including, for example, systemic lupus erythematosus (SLE), is installed, for example, in Heb the European patent 0532979 or patent application WO 03/044054. It is established that the function of histones, in addition, include antibiotic functions (U.S. patent No. 6565854 and 6884423) and antiviral functions (WO 2005/112975). In addition, it is shown the application of histones to prevent platelet aggregation (WO 02/067907) and for the treatment of thrombocytopenia (WO 2006/119912).

Found that histones play a major role in the treatment of cancer. Vani and others (Vani, G. et al., Chemotherapy 2003, 49: 252) demonstrate, for example, that the histone H1 enhances the immune status and immune response in animals, supporting experimental breast cancer. Also found that antioxidant status with cancer individuals is amplified using histone H1 (Vani, G. et al., Chemotherapy 2005, 51: 57). Determined that the processing is sensitive to estrogen in human breast cancer cells by histone H1 reduces the number of estrogen receptors (Vani, G. and Devi, C.S., Mol. Cell Biochem. 2005, 272: 151). In U.S. patent No. 5812257 demonstrates treatment induced by irradiation of leukemia or carcinoma histones H1 or H2A:H2B as well as. Histones can also be potentially useful for the treatment of cancer by capturing pathogenic extracellular DNA and circulation throughout the body pathogenic nucleus (Le Lann-Terrisse et al. (1997) Cancer Immunol. Immunother., 43: 337).

In accordance with the present invention the nucleic acid molecule can encode a polypeptide consisting of the two methionine residues as the first and second N-terminal amino acid residues, connected via a peptide bond with Mature eukaryotic a polypeptide sequence which is identical to at least 80%, more preferably 85%, more preferably 90% sequence Mature eukaryotic histone (a) and which substantially retains its biological activity. Even more preferably, the nucleic acid molecule can encode a polypeptide consisting of the two methionine residues in the first and second N-terminal amino acid residues, connected by peptide bonds Mature the eukaryotic polypeptide sequence which is identical to at least 95% and most preferably 98% sequence Mature eukaryotic histone (a) and which substantially retains its biological function.

In accordance with the present invention, the term "sequence identity in percent" refers to the number of matches ("hits") are identical nucleotides/amino acids of two or more aligned sequences of nucleic acids or amino acid sequences in comparison with the number of nucleotides or amino acid residues that comprise the full length sequences of nucleic acids or amino acid sequences (or comparable frequently is). In other words, using the combination, with two or more sequences or subsequences percentage of amino acid residues or nucleotides that are the same (for example, the identity of 80% or 85%)can be identified by comparing and combining the (sub)sequences for maximum compatibility across the comparison window or around the target area, as determined using the algorithm for comparison of sequences known in the art, or after combining manual and visual inspection. This definition also applies to the complement of the test sequence. Preferred nucleic acid molecules and/or polypeptides in accordance with the present invention are molecules of nucleic acids/polypeptides, which is characterized by the identity exists throughout the area, whose length is at least about 15-25 amino acids or nucleotides, more preferably throughout the area, whose length is at least about 50-100 amino acids or nucleotides. Skilled in the art experts know how to determine the sequence identity percentage between sequences using, for example, algorithms such as algorithms based what's on the computer program CLUSTALW (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 1988, 85: 2444), which are known in the art.

Although the FASTDB algorithm usually does not take into account internal mismatched deletions or additions in the sequence, i.e. omissions in the calculation, this can be corrected manually in order to avoid overestimation of identical sequences in %. However, CLUSTALW takes into account the gaps in the sequences in the calculations of their identity. Meet the requirements of skilled in the art specialists are algorithms BLAST and BLAST 2.0 (Altschul, Nucl. Acids Res., 1977, 25: 3389). The program BLASTN for nucleic acids sequences is the default word length (W)component 11, an expectation (E) of 10, M=5, N=4 and the comparison of both circuits. For amino acid sequences, the BLASTP program uses the default word length (W), component 3, and expectation (E) - 10. In the matrix of quantitative estimates BLOSUM62 (Henikoff, Proc. Natl. Acad. Sci., 1989, 89: of 10,915) used a combination of (B)of 50, expectation (E) of 10, M=5, N=4 and the comparison of both circuits. All these programs can be used for the purposes of the present invention. All the above programs can be used in accordance with the present invention.

In accordance with the present invention the activity substantially preserved, if you get at least 20% of the biological the banking activity of the corresponding Mature eukaryotic histone, referred to in paragraph (a)above. Preferably, when the stored at least 50%, such as at least 60%, at least 75% or at least 80% activity. More preferred is saving at least 90%, e.g. at least 95%, even more preferred at least 98%, such as 99%, of the biological activity. Most preferred is complete, that is 100%, the preservation of biological activity. Also in accordance with the present invention are polypeptides having increased biological activity compared to the corresponding Mature the eukaryotic histone referred to in paragraph (a), i.e. more than 100% of the enzymatic activity of the reference histone. Methods of assessment of biological activity of the (poly)peptide are well known to the skilled in the art specialist and include, without limitation, measurement of the enzymatic activity, cytotoxicity, release of cytokines, hemolysis, or the expression of biomarkers. In particular, the cytotoxicity assays are assays that use in vitro or in vivo cell culture, which is treated, for example, poly(peptide), such as histones, and which is determined using detection methods cells the rate of change of cell death after treatment. Biological activity is ü can also be determined using ELISA assays, especially in the case of antibodies.

Used herein, the term "hybridizes/hybridization" refers to the pairing of the nucleic acid molecule with (partially) complementary chain of this molecule nucleic acid, thereby forming a hybrid.

In the art knows how to perform experiments on hybridization of nucleic acid molecules. Accordingly, the skilled in the art specialist knows what conditions are hybridization he must use in order to make possible a successful hybridization. In relation to creating suitable conditions for hybridization cope in standard books-texts such as Sambrook and Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989) or Higgins and Hames (Eds.) “Nucleic acid hybridization, a practical approach,” IRL Press Oxford, Washington DC (1985). In one preferred embodiment, hybridization is carried out in harsh conditions.

"Stringent hybridization conditions" refer to conditions that include, for example, incubation overnight at 65°C. in 4 × SSC (600 mm NaCl, 60 mm sodium citrate) followed by washing at 65°C in 0.1 × SSC for one hour. Alternatively, the hybridization conditions can include incubation overnight at 42°C in a solution containing 50% formamide, 5 × SSC (750 mm NaCl, 75 mm is the atrium citrate), 50 mm sodium phosphate (pH of 7.6), 5 × denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, degraded as a result of hydrodynamic shear DNA from salmon ROE, followed by washing the filters in 0.1 × SSC at approximately 65°C. the abovementioned hybridization conditions are also known to the skilled in the art specialist as "highly stringent hybridization conditions". There are molecules of nucleic acids which hybridize with nucleic acid molecules of the present invention under conditions of lower stringency for hybridization ("low stringency for hybridization"). Change the rigidity of hybridization and signal detection is carried out, mainly through the manipulation of the concentration of formamide (lower percentages of formamide result in less stiffness), salt conditions, or temperature. For example, conditions of lower stringency include incubation over night at 50°C. in 4 × SSC or incubation over night at 37°C in a solution containing 6 × SSPE (20 × SSPE=3M NaCl; 0,2M NaH2PO4; 0,02M EDTA, pH 7,4), 0,5% SDS, 30% formamide, 100 μg/ml of blocking DNA from salmon ROE, followed by washes at 50°C in 1 × SSPE, of 0.1 × SDS. In addition, to achieve even lower stringency washing is performed after the hard hybridization, it is possible to conduct the ri higher concentrations of salt (e.g., 5 × SSC). Note that changing the above-mentioned conditions can be performed through the inclusion and/or replacement reagents options to block, used to attenuate background in experiments on hybridization. Typical reagents for blocking include denhardt's reagent, BLOTTO, heparin, denatured DNA from salmon ROE and a commercially available patented composition. When including specific reagents for blocking may require modification of the above-described hybridization conditions due to compatibility issues. Such modifications can usually be carried out by a qualified technician immediately. Hybridization complex may be formed in solution (e.g., analysis of the Cot or Rot) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid substrate (e.g., membranes, filters, chips, beamed conclusions or slides, on which, for example, were recorded cells). Here above the option exercise preferably refers to highly stringent conditions and in the alternative case to the conditions of lower stringency.

In addition to the above, the term "nucleic acid molecule, hybridization in harsh environments complementares chain molecules of nucleic acid, encoding the polypeptide (a) or (b)referred to in paragraph (C), preferably refers to sequences which display a sequence identity constituting at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 97%nucleotide sequence as described above in paragraphs (a) or (b).

As indicated here above, preferred in accordance with the present invention are molecules of nucleic acids which are capable of hybridizing with the nucleic acid molecules of the present invention or their parts in (highly) stringent conditions of hybridization, i.e. which do not cross-hybridize with nucleic acid molecules, an unrelated nucleotide sequences. In accordance with paragraph (C), above, the present invention also embraces nucleic acid molecule, related but not identical to the nucleic acid molecules of (a) or (b) for each sequence. In addition, the present invention includes, in accordance with paragraph (C) fragments of the nucleic acid molecules (a) or (b). For all embodiments falling within paragraph (C), it is important in accordance with this embodiment, to kodiruemyi this molecule, nucleic acid, polypeptide had at least two N-terminal methionine residue and retain or essentially retain the biological activity of the histone (a) or (b).

In addition, in the preferred embodiment, the present invention also relates to a nucleic acid molecule, the sequence of which is degenerate in comparison with the sequence described above, the nucleic acid molecule of paragraph (a) or (b). When used in accordance with the present invention the term "non degenerate due to the genetic code" means that because of the redundancy of the genetic code of different nucleotide sequences encode the same amino acid.

Although there are a number of well-known in the art of affinity tags, which merge with the polypeptides in order to make possible more light production and detection, these labels often need to be removed when used in therapeutic applications. In contrast, affinity labels, the authors present invention was surprised to find bis-Met-polypeptides that exhibit biological properties similar to the biological properties of their natural counterparts, and, therefore, the polypeptides of the present invention can be used for therapeutic purposes. Because functionality is peptides of the present invention is clearly not changed, at least when using the tests applied by the authors of the present invention, the removal of residues of methionine is not necessary. In addition, the removal of residues of methionine does not occur during production. As outlined above, the separation of N-terminal methionine residue largely depends on the size of the second amino acid residue. Because the polypeptides of the present invention contain as the second amino acid residue of an additional methionine, one of the observed effects is that only a low percentage, i.e. in the range of approximately 20%, the two N-terminal residues of methionine is cleaved inE. coli. The remaining approximately 80% of cases the two N-terminal methionine residue is not hatshepsuts. During the production of the polypeptide of the present invention in prokaryotes, such asE. colithe last N-terminal methionine may also be formirovanie. However, the authors of the present invention have not received any formirovanii products, as verified by mass spectrometry. Also could not observe cleavage of only one methionine residue. Not wanting to be limited by any theory, suggest that cleavage of the first N-terminal residue of methionine leads to rapid removal of the second N-terminal methionine, resulting in Tsaplina both methionine residues.

Consequently, bis-Met-histones present invention provide the advantage of easy detection in the presence of endogenous histones. For example, although bis-Met-histones can not be separated from their endogenous counterparts through various methods HPLC (RPC; SEC; IEX) or electrophoresis (SDS-PAGE; CE), bis-Met-histones can be easily distinguished using mass spectrometry (or tandem mass spectrometry with ionization by elektrorazpredelenie (ESI-MS), for example, the same fraction HPLC with reversed phase (see examples). This allows you to control the pharmacokinetics of therapeutic histones during clinical trials without the use of isotope labelling or specific antibodies against the investigational medicinal product.

Furthermore, surprisingly, it was found that the histones that contains two methionine residue as the first and second N-terminal amino acid residues, exhibit advantageous properties when recombinant products. Thus, the authors of the present invention found that a significantly higher level of histone can be obtained after the introduction of two methionine residues. Although production in the bacterial cell after fermentation bis-Met-histone was not significantly higher with surprisingly it was found significantly different behavior bis-Met-histone first key stages the processing in the main direction. While bis-Met-histone was eluted at an estimated concentration of salt, recombinant histone devoid of additional methionine residues could not be eluted from the column MacroPrep High S, except for very high concentrations of salt, and it was impossible to continue to clean effectively. Therefore, bis-Met-histone behaves very well on column MacroPrep High S that enables efficient and high yield purification process.

Accordingly, the present invention is based on the new fact that the presence of two methionine residues at the N end of histones provides bis-Met-histones, which allow easy detection in the presence of endogenous histones and make possible the efficient production of recombinant proteins.

In a preferred embodiment, the histone selected from the group consisting of histone N, N, N, N, N, N and H1t egg.

Access numbers in Swiss-Prot for subtypes of histone H1 is N - R; N - Q02539, H1.2 - P16403, H1.3 - P16402, H1.4 - P10412, H1.5 - Q14529 and H1t-R. The sequence of the nucleic acid and amino acid sequence of histone N person shown in SEQ ID NO: 6 and 7. The sequence of the nucleic acid and amino acid sequence of histone N person shown in SEQ ID NO: 8 and 9. The sequence of the nucleic acid and amino acid follow etelnost of histone N person shown in SEQ ID NO: 10 and 11. The sequence of the nucleic acid and amino acid sequence of histone N person shown in SEQ ID NO: 12 and 13.

In another embodiment, the present invention provides a nucleic acid molecule that is complementary to the nucleic acid molecule of the present invention.

Molecules of nucleic acids are "complementary"if they are natural contact with each other in contributing to salt and temperature conditions by using mating grounds. For example, the sequence "A-G-T binds to a complementary sequence T-C-A". "Complementarity" in accordance with the present invention is related to a complete base pairing of nucleotides along the length of the nucleic acid molecules of the present invention. Thus, tagged with a detectable label molecule of nucleic acid, is not exactly complementary to the nucleic acid molecule of the present invention, will not give a detectable signal, if selected corresponding to the conditions of hybridization and washing. Such complementary nucleic acid molecule can, for example, be used as probes in Northern or southern blotting drugs RNA or DNA.

In another aspect the present invention provides antisense oligo - is whether polynucleotide the nucleic acid molecules of the present invention, moreover, the oligonucleotide includes a nucleotide complementary triplets of nucleotides coding for the first two N-terminal methionine residue of the histone of the present invention, and has a minimum length, which is 10 nucleotides.

These antisense oligonucleotides can for example be used as primers for sequencing analyses or as probes in Northern or southern blotting drugs RNA or DNA. The antisense oligonucleotides of the present invention preferably include at least 10, preferably at least 15, for example, at least 25 consecutive nucleotides. More preferably, the antisense oligonucleotides of the present invention include at least 100, more preferably at least 200, and most preferably at least 500 nucleotides in length. Such a nucleic acid molecule can also be used, for example, as a probe in the analysis method of protection from the action of RNase or as antisense probe for inhibiting the expression of histones present invention. Skilled in the art specialist knows how to prepare and use such probes (see, for example, Sambrook and Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory N.Y. (2001)).

In another alternative in which the version of the implementation of the present invention provides a vector comprising the nucleic acid molecule of the present invention. Preferably the vector is a plasmid, cosmid, virus, bacteriophage or another vector used, for example, usually when genetic engineering. In a further embodiment, the present invention provides a vector comprising the complementary nucleic acid molecule or antisense oligonucleotide of the present invention.

The nucleic acid molecule of the present invention can be embedded in a separate commercially available vectors. Non-limiting examples include prokaryotic plasmid vectors, such as the number of pUC, pBluescript (Stratagene), a number of expression vectors pET, including vectors pETduet (Novagen) or pCRTOPO (Invitrogen), and the vectors that are compatible with expression in mammalian cells, such as pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples of plasmid vectors suitable forPichia pastorisinclude , for example, plasmid pAO815, pPIC9K and pPIC3.5K (all from Invitrogen).

The above nucleic acid molecule of the present invention can also be embedded into a vector of terms of creating a translational fusion with another molecule of nucleic acid. Another nucleic acid molecule can to the activated protein which may, for example, to increase the solubility and/or to facilitate cleaning of the fused protein. Non-limiting examples include pET32, pET41, pET43. The vectors may also contain additional expressing a nucleic acid molecule encoding one or more chaperones that facilitate proper stacking of proteins. Suitable for the expression of bacterial hosts include, for example, strains originating from BL21 (e.g., BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosetta®.

In relation to methods of modifying vectors, see Sambrook and others, above. Typically, the vectors may contain one or more started replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, such as resistance to antibiotics, and one or more expression cassettes. Suitable beginning of replication (ori) include, for example, start replication Col E1, SV40 virus and M13.

Embedded in the vector coding sequences can, for example, be synthesized using standard methods or to isolate from natural sources. Ligation of coding sequences with regulatory transcription elements and/or other encoding amino acid sequences can be performed using well known methods. Regulatory transcription elements (part of CAS expression is Yety), ensuring expression in prokaryotes or eukaryotic cells, a well-known skilled in the art specialists. These elements include regulatory sequences that allow initiation of transcription (e.g., codon of translation initiation, the promoters, enhancers and/or insulators), the portions of the inner landing ribosomes (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include enhancers of transcription and translation and/or natural associated or heterologous promoter region. Preferably the nucleic acid molecule of the present invention is functionally connected with the controlling expression of a sequence, which makes possible the expression in prokaryotes or eukaryotic cells. The vector can also include a nucleotide sequence encoding a signal for secretion as additional regulatory elements. Such sequences are well known to the skilled in the art specialist. In addition, depending on the expression system to the coding sequence of the nucleic acid molecules of the present invention can add earnie sequence, able to direct the expressed polypeptide in the cell compartment. Such leader sequences are known in the art.

Possible examples of regulatory elements ensuring initiation of transcription, include the promoter of cytomegalovirus (CMV), SV40 promoter, the RSV promoter (rous sarcoma virus), the lacZ promoter, the promoter gai10, the promoter of the elongation factor 1α human CMV enhancer, promoter CaM-kinase, polyhedral promoter of the virus multiple nuclear polyhedrosis Autographa californica (AcMNPV) or the SV40 enhancer. For expression in prokaryotes have been described many promoters, including, for example, tac-lac-promoter, the lacUV5 promoter, or trp. Examples of further regulatory elements in prokaryotes and in eukaryotic cells include signals termination of transcription, such as the site of the SV40-poly-a or site tk-poly-a and signals polyhedral polyadenylation SV40, lacZ and AcMNPV, 3' from the nucleic acid molecule.

In addition, it is preferable that the vector of the present invention include breeding marker. Examples of breeding markers include resistance to neomycin, ampicillin, hygromycin, kanamycin, etc. Specially designed vectors allow you to move the Shuttle to the principle of DNA between different hosts, such as bacteria cells of fungi or bacteria - animal cells (for example, the system Gataway®, available from Invitrogen).

Expression vector in accordance with the present invention is able to control the replication and expression of molecules of the nucleic acid and encoded polypeptide of this invention. In this area known in the art suitable expression vectors comprising the described regulatory elements, such as vectors for the expression of cDNA Okayama-Berg pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen, used, among others, in the accompanying examples), pSPORT1 (GIBCO BRL) or pGEMHE (Promega) or prokaryotic expression vectors such as lambda gt11, pJOE, the number of pBBR1-MCS, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1 or preferably the vector pET (Novagen).

Described here above, the nucleic acid molecule of the present invention can be designed for direct introduction or introduction via liposomes, the phage or viral vectors (e.g. adenoviral, retroviral) vectors into the cell. In addition, the baculovirus system or systems based on cowpox virus or virus Semliki forest can be used as eukaryotic expression systems for nucleic acid molecules of the present invention.

Typical vectors for expression in mammalian cells include the promoter element, which mediates the initiation of transcription of mRNA, protein-coding sequence, and signals required for the termination of transcri the tion and polyadenylation of the transcript. In addition, can also be included such items as the start of replication, the gene of resistance to the drug, the regulators (as part of an inducible promoter). The lac promoter is typical of the inducible promoter used for prokaryotic cells, which can be induced using an analog of lactose isopropylthio-β-D-galactoside ("IPTG"). For recombinant expression and secretion of interest of the nucleic acid molecule can be ligitamate between, for example, leader of the PelB signal, which directs the recombinant protein in periplasm, and gene III in pagemode called pHEN4 (described Ghahroudi and others in 1997, FEBS Letters 414: 521-526). Additional elements could include enhancers, Kozak sequence and introns, flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved when used early and late promoters from SV40, the long terminal repeats (LTRS) from retroviruses, such as RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, you can also use cellular elements (e.g., the actin promoter of the person). Suitable for use to practice the present invention expression vectors include, for example, such vectors as pSVL and pMSC (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr ATCC 37146) and pBC12MI (ATCC 67109). Hosts are mammalian cells, which could be used include human cells Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127, Cos 1, Cos 7 and CV1 cells, quail QC1-3, mouse L-cells and cells of the Chinese hamster ovary (CHO). In the alternative case, the recombinant polypeptide can be Express in stable cell lines that contain the gene construct integrated into the chromosome. Cotransfected breeding marker such as dhfr, gpt, resistance to neomycin, hygromycin, makes possible the identification and selection of transfected cells. Transfected nucleic acid can also amplify the expression of large amounts of the encoded polypeptide. Marker DHFR (dihydrotetrazolo useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful breeding marker is the enzyme glutaminase (GS) (Murphy et al. 1991, Biochem J. 227: 277-279; Bebbington et al. 1992, Bio/Technology 10: 169-175). Using these markers, mammalian cells grown in a selective culture medium and select the cells with the highest stability. As indicated above, expression vectors will preferably include at least one breeding marker. Such markers include dihydrotetrazolo, resistance to G418 or neomycin to the cultural eukaryotic cells and genes of resistance to tetracycline, kanamycin or ampicillin for cultivationE. coliand other bacteria.

In addition, the present invention relates to a host, methods of genetic engineering using the nucleic acid molecules of the present invention or the vector of the present invention. The specified host can be obtained by introducing into the host specified nucleic acid molecule or vector(s), who in his presence mediate the expression of the polypeptide of the present invention.

The host may be any prokaryotic or eukaryotic cell. Suitable prokaryotes/bacteria are those that are typically used for cloning, for example,E. coli(for example, strains BL21(DE3), HB101, DH5α, XL1 Blue, Y1090 and JM101E. coli),Salmonella typhimurium,Serratia marcescens,Pseudomonas putida,Pseudomonas fluorescens,Streptomyces lividans,Lactococcus lactis,Mycobacterium smegmatisorBacillus subtilis. Suitable eukaryotic host may be an animal cell, such as Cho, COS, 293 cells and melanoma Bowes, an amphibian cell, the cell fish, insect cell, such as cells of Drosophila S2 and Spodoptera Sf9, cell fungus, plant cell, transgenic, non-people, animals or transgenic plants.

In a preferred embodiment of the present invention the host is a bacterium, yeast cell, insect cell, the years of the fungus, the cell of a mammal or plant cell. This technology is widely known suitable environment and conditions for culturing the above-described host cells. In a preferred embodiment, the master of the developed methods of genetic engineering using a nucleic acid molecule or vector of the present invention, isE. colifor example, strains originating from BL21 (e.g., BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosetta®.

In a further embodiment, the present invention also relates to a method for obtaining able to Express the polypeptide of the present invention bacteria or eukaryotic cells, including the genetic engineering of bacteria or eukaryotic cells using the vector of the present invention. The term "genetic engineering" refers to a method of introducing into the cell's genetic information or modification of the genetic information of the cell. This is usually performed by travelrobe or transforming a host cell with the nucleic acid molecule. The introduction of design in the cell-master can be performed using transfection using calcium phosphate-mediated DEAE-dextran transfection mediated by cationic lipid transfection, electroporation, transduction, infection or other methods. what these methods are described in many standard laboratory manuals, such as Sambrook and others, cited in the above location. This nucleic acid molecule introduced into a cell of the host, includes an open reading frame encoding a polypeptide of the present invention.

In an additional embodiment, the present invention relates to a method for producing the polypeptide of the present invention, comprising culturing the host of the present invention in suitable conditions, and the allocation of the produced polypeptide of the present invention from the specified host or culture.

In the art there are a large number of suitable methods for producing polypeptides in a suitable host. If the owner is a single-celled organism, such as prokaryotes, the cell is mammalian or insect skilled in the art specialist can refer to a range of cultivation conditions. Suitably, if the produced protein is obtained from the environment for cultivation, lysates subjected to cultivation of organisms, or selected (biological) membranes using well known methods. In the case of a multicellular organism the host may be a cell that is part of or is part of the body, for example, specified a host cell may be suitable for harvesting the plant part. Preferably the second method involves recombinant production of the protein in the host, as specified above. For example, nucleic acid sequences, including nucleic acid molecule in accordance with the present invention, it is possible to synthesize by PCR and embed in the expression vector. Subsequently, the suitable host can be transformed expression vector. After that, the host is subjected to cultivation for the production of the desired polypeptide, which is isolated and purified. Such methods are widely known in the art (see, for example, Sambrook, etc. above).

An alternative method of producing the polypeptide of the present invention is in vitro translation of mRNA. Suitable for use in accordance with the present invention is a cell-free expression systems include rabbit reticulocytosis lysate, an extract from wheat germ, canine pancreatic microsomal membranes, the S30 extract ofE. coliand related systems of transcription/translation, such as TNT system (Promega). These systems allow you to Express recombinant polypeptides adding vectors for cloning, DNA fragments or RNA sequences containing coding region and the corresponding promoter elements.

In addition to recombinant production, polypeptide (protein), protein fragments or protein of the present invention can producera the AMB synthetically, for example, by direct peptide synthesis using solid-phase techniques (cf with Stewart et al. (1969) Solid Phase Peptide Synthesis; Freeman Co., San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154).

Synthesis of synthetic proteins can be performed using the methods manually or with automation. Automated synthesis can be accomplished, for example, using peptide synthesizers Applied Biosystems 431A (Perkin Elmer, Foster City, CA) according to instructions provided by the manufacturer. Various fragments can be chemically synthesized separately and combined using chemical methods to obtain the full-length molecule. As described above, can be used in chemical synthesis, such as solid-phase method described Houghton (Proc. Natl. Acad. Sci., 1985, 82: 5131). In addition, the polypeptide (protein), protein fragments or protein of the present invention it is possible to produce semi-synthetic, for example, by combining recombinant and synthetic products. All polypeptides (proteins), which has two methionine residue as the first and second N-terminal amino acid residues, connected by peptide bond (s) with Mature the eukaryotic histone; (b) with Mature eukaryotic a polypeptide sequence which is identical to at least 80% sequence Mature eukaryotic histone and in which significant about the time retains its biological activity; or (C) with another polypeptide of the present invention described above, as well as fragments of such a polypeptide (protein) and fused proteins, are within the scope of the present invention, regardless of the used for their production method products. This is because the amino acid sequences of all these proteins (also) is encoded by the nucleic acid molecule of the present invention.

Isolation and purification of proteins can be accomplished using any of several known methods, for example and without limitation, ion exchange chromatography, gel filtration, affinity chromatography, liquid chromatography, high pressure (HPLC), HPLC with reversed phase chromatography based on hydrophobic interactions and preparative disc electrophoresis. For methods for the isolation/purification of proteins may require modification of the polypeptides of the present invention using conventional methods. For example, the protein can optionally add his-tag tag to allow purification on a column of Nickel. Other modifications can cause increase or decrease of the activity, allow the production of proteins at higher levels or to simplify purification of the protein.

In an alternative embodiment, the present invention provides a polypeptide encoded by what alcolol nucleic acid of the present invention or produced through the method of the present invention.

The present invention also provides a composition comprising the nucleic acid molecule or a vector or host, or polypeptide of the present invention. In these compositions also, optionally, contains an antibody, aptamer or phage of the present invention, described further below.

Used in accordance with the present invention, the term "composition" refers to a composition that contains at least one of the compounds. Optionally it may contain additional molecules that can modify the properties of the compounds of the present invention, thus, for example, inhibiting, stabilizing, inhibiting, modulating and/or activating their function. The composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of powder(s), tablets (pills), solution(s) or aerosol(s).

In a preferred embodiment, the composition of the present invention, in addition, contains a number of Mature eukaryotic histone.

Preferably, such a composition comprises a polypeptide of the present invention (bis-Met-histone) in a mixture with Mature the eukaryotic histone. Consequently, the composition may contain a mixture of histones that contains two methionine residue at the N end, and histone deprived of both methionine residues. Preferably the mixture is located a ratio of 90% of the polypeptide of the present invention to 10% of Mature eukaryotic histone. More preferably the mixture is in the ratio of 80% to 20%, more preferably 70% to 30%. Even more preferred mix ratio is 50% to 50%, 30% to 70% or 20% to 80%. Most preferably, when the mixture is in the ratio of 10% of the polypeptide of the present invention to 90% of Mature eukaryotic histone. This mixture may be due to partial removal of methionine from bis-Met-histone due to insufficient activity of methionine-amino peptidases of the host organism. Alternatively, to obtain the above-mentioned mixture of Mature eukaryotic histone can be added to the bis-Met-histone of the present invention. Preferably the histones in a mixture of histones are of the same type, such as H1 or H2A.

In another preferred embodiment, the composition is a pharmaceutical composition, optionally additionally contains a pharmaceutically acceptable carrier and/or diluent.

In accordance with the present invention, the term "pharmaceutical composition" refers to compositions for administration to the patient, which is preferably a human patient. The pharmaceutical composition of the present invention contains compounds mentioned above. The pharmaceutical composition of the present invention may optionally and additionally contain a pharmaceutically acceptable carrier. Under "Pharma is efticiency acceptable carrier" is meant a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or excipient compositions of any type. Examples of suitable pharmaceutical carriers are well known in the art and include solutions of sodium chloride, phosphate buffered solutions of sodium chloride, water, such emulsion as the emulsion of the type oil-in-water, various types of wetting agents, sterile solutions, organic solvents, including DMSO and the like, Preferably the carrier is a carrier for parenteral administration, more preferably a solution that is isotonic with the blood of the recipient. Media appropriately contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to the recipient in all used doses and concentrations, and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts; antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) polypeptides, e.g polyalanine or tripeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, g is caminova acid, aspartic acid, or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; hepatoblastoma agents, such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium; and/or nonionic surfactants such as Polysorbate, poloxamer or PEG.

Used herein, the term "parenteral" refers to introduction methods, which include intravenous, intramuscular, intraperitoneal, epigastric, subcutaneous and intraarticular injection or infusion.

Compositions containing such media can be prepared using well known conventional methods. Typically, the composition is prepared by bringing the components of the pharmaceutical composition in constant and direct contact with liquid carriers or finely powdered solid carriers, or both. Then, if necessary, the product shape of the desired composition.

These pharmaceutical compositions can enter the subject in a suitable dose. Scheme doses will be determined by a staff physician and clinical factors. As is well known in the field of medicine, the dose for any one patient depends on many factors, including the size of the patient, the surface area, age, the particular link is, which should be administered, sex, time and route of administration, General health and other drugs administered simultaneously. A therapeutically effective amount in this situation, it can be easily determined using conventional experimentation and known to the ordinary practitioner or doctor and evaluated them. As a rule, the scheme in the form of a regular injection of the pharmaceutical composition should be in the range of doses from 1 mg to 20 g per day. However, a more preferred dosage would be in the range of 0.01 mg to 100 mg, even more preferably from 0.01 mg to 50 mg, and most preferably from 0.01 mg to 10 mg per day.

The components of the pharmaceutical composition used for therapeutic administration, must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., membranes with pore size 0.2 microns).

The components of the pharmaceutical compositions will usually be stored in containers for single or multiple doses, for example sealed ampoules or vials, as an aqueous solution or as a freeze-dried composition for recreation. As an example, lyophilized compositions, 10-ml vials filled with 5 ml of sterile filtered 1% (weight to volume) aqueous solution and resultyou the second mixture is subjected to lyophilization. The infusion solution is prepared by recreating dried connection(s)using bacteriostatic water for injection.

Can also contain preservatives and other additives, such as, for example, antimicrobial agents, antioxidants, hepatoblastoma agents, inert gases, etc. in Addition, the pharmaceutical composition may contain additional agents depending on the intended use of the pharmaceutical composition.

The pharmaceutical composition can in particular be used for the treatment of diseases, preferably disorders, selected from those described here above diseases, including, for example, cancer, thrombocytopenia, infections such as bacterial, viral or fungal infections, autoimmune diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis, ulcerative colitis, or diseases characterized by amyloidogenic fibrils, such as Alzheimer's disease (AD) and Parkinson's disease (PD), leishmaniasis, some forms of myopathy or cardiovascular disorders associated with thrombosis.

Cancer, in accordance with the present invention, refers to a class of diseases or disorders characterized by uncontrolled division of cells and their ability to spread, or by direct germination in the adjacent fabric cerasinus, or by implantation in remote locations with metastasis (in which cancer cells are transported through the bloodstream or lymphatic system).

Thrombocytopenia, in accordance with the present invention, refers to the presence of a relatively small number of platelets in the blood, while the normal number of platelets, typically is in the range from 140,000 to 400,000 in mm3.

Infection, in accordance with the present invention, is detrimental colonization of a host organism alien species. During infection with an infectious organism tries to use the resources of the host for reproduction (usually at the expense of the owner). The reaction of the host to infection is inflammation.

Bacterial infectious diseases, in accordance with the present invention include, but without limitation, bacterial meningitis, cholera, diphtheria, Listeria, pertussis, pneumococcal pneumonia, salmonellosis, tetanus, typhoid, tuberculosis or infection of the urinary tract.

Viral infectious diseases, in accordance with the present invention include, but without limitation, mononucleosis, AIDS, chickenpox, common cold, cytomegalovirus infection, dengue fever, hemorrhagic fever, Ebola, viral disease of the mouth and extremities, hepatitis, influenza, mumps, polio, is Edinstvo, smallpox, viral encephalitis, viral gastroenteritis, viral encephalitis, viral meningitis, viral pneumonia, or yellow fever.

Fungal infectious diseases, in accordance with the present invention include, but are not limited to, aspergillosis, blastomycosis, candidiasis, Coccidioides, cryptococcosis, histoplasmosis or dermopathy foot.

Autoimmune diseases, in accordance with the present invention relate to diseases that are the result of an overactive immune response of the body to substances and tissues normally present in the body. Autoimmune diseases are well known to the skilled in the art specialist and shall include, but without limitation, lupus erythematosus, acute disseminated encephalomyelitis, aplastic anemia, autoimmune hepatitis, diabetes, multiple sclerosis, retro-bulbar neuritis or rheumatoid arthritis.

Lupus erythematosus, in accordance with the present invention, refers to a chronic (long-lasting) autoimmune disease in which the immune system becomes overactive and has a corrosive effect on normal tissue. This aggressive action leads to inflammation and causes symptoms. Lupus is not specific to a particular body type autoimmune sabol is found.

Rheumatoid arthritis, in accordance with the present invention, is an autoimmune disease because the immune system has a corrosive effect on bone joints.

Ulcerative colitis, in accordance with the present invention, is a form of inflammatory bowel disease (IBD). Ulcerative colitis is a form of colitis, a disease of the intestines, particularly the colon, that includes characteristic ulcers, or open sores in the colon. The main symptom of active disease is usually diarrhea with blood, with a gradual beginning. Ulcerative colitis, however, is a systemic disease that affects many parts of the body outside of the intestine.

Diseases characterized by amyloidogenic fibrils, in accordance with the present invention, are diseases that share as a common symptom that usually soluble peptide amyloid-beta or protein alpha synuclein aggregates in ordered fibrillar structures, usually leading to increased oxidative damage, excitotoxicity, and changes in the cell cycle. Diseases characterized by amyloidogenic fibrils include, but without limitation, Alzheimer's disease (AD) and Parkinson's disease (PD).

Alzheimer's disease pre who is a neurodegenerative disease, characterized by progressive deterioration of cognitive process together with reduced activities of daily living and neuropsychiatric symptoms or behavioral changes. It is the most common type of dementia.

Parkinson's is a degenerative disease of the Central nervous system that often impairs motor skills and speech of the patient.

Leishmaniasis is trypanosome disease caused originally Leishmania, the organism is a parasite class of simple Tripanosoma. Leishmaniasis is transmitted through certain types of mosquitoes, and its symptoms are skin wounds, fever, lesions of the spleen and liver, and anemia.

Myopathies are neuromuscular diseases in which muscle fibers do not function, resulting in muscle weakness. Several classes of myopathy known and include, but without limitation, for example, muscular dystrophy, congenital myopathy, chromosomal or inflammatory myopathy myopathy.

Cardio-vascular disorders associated with thrombosis, in accordance with the present invention relate to violations, including, but not limited to, deep vein thrombosis or myocardial infarction. Especially preferred are cardio-vascular disorders associated with thrombosis, mediated by the γ-thrombin./p>

In another preferred embodiment, the composition of the present invention is a diagnostic composition.

In accordance with the present invention, the term "diagnostic composition" refers to compositions for diagnosis in individual patients, their potential reactions to pharmaceutical compositions of the present invention or the curability. The diagnostic composition of the present invention contains the above-mentioned compounds. The diagnostic composition may further contain an appropriate buffer(s) and enzymes such as reverse transcriptase, thermostable polymerase, etc. Diagnostic compositions can be packaged in a container or multiple containers.

The present invention also provides a method of treatment and/or prevention of a disease selected from cancer, thrombocytopenia, infections such as bacterial, viral or fungal infections, autoimmune diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis, ulcerative colitis, or diseases characterized by amyloidogenic fibrils, such as Alzheimer's disease (AD) and Parkinson's disease (PD), myopathy or cardiovascular disorders associated with thrombosis involving the introduction of a pharmaceutical composition of this izaberete the Oia needy in this subject.

The present invention also provides the use of nucleic acid molecules or vectors, or non-human host, or of the polypeptide of the present invention for the preparation of compositions for therapeutic and/or diagnostic purposes.

In a preferred embodiment, therapeutic goal is the treatment of cancer, thrombocytopenia, infections such as bacterial, viral or fungal infections, autoimmune diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis, ulcerative colitis, or diseases characterized by amyloidogenic fibrils, such as Alzheimer's disease (AD) and Parkinson's disease (PD), myopathy or cardiovascular disorders associated with thrombosis.

In a further embodiment, the present invention provides an antibody or aptamer or phage that specifically bind with the nucleic acid molecule or polypeptide of the present invention, but is not associated with the corresponding Mature the eukaryotic histone.

The specified antibody can be monoclonal or polyclonal antibody.

The term "antibody" includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies or fragments thereof that specifically bind the specified peptide or polypeptide, and shall also include bespecifically antibodies synthetic antibodies, antibody fragments, such as Fab, F(ab2)'-, Fv or scFv fragments etc, or a chemically modified derivative of any of them. Monoclonal antibodies can be prepared, for example, using methods originally described by Kohler and Milstein in Nature 256 (1975), 495, and Galfre in Meth. Enzymol. 73 (1981), 3, which include the fusion of mouse myeloma cells with spleen cells derived from immunized mammals, with modifications developed in the art. Furthermore, antibodies or fragments thereof against the above-mentioned peptides can be obtained using the methods described, for example, Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. When derivatives of these antibodies are obtained by the phage display technique, surface plasmon resonance, used in the BIAcore system can be used to increase the efficiency of antibodies in phage that bind to the epitope of the peptide or polypeptide of the present invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Production of chimeric antibodies is described, for example, in WO 89/09622. Further sources of antibodies used in accordance with the present invention are the so-called xenogenic antibodies. The General principle of production of xenogenic antibodies, such as human antibodies in mice, described, n is the sample, in WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. Antibodies used in accordance with the present invention, or their respective immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, using a deletion(s), insertion(s), substitution(s), add(I) amino acids and/or recombination(s) and/or any other modification(s) amino acids known in the art, or separately, or in combination. Ways of introducing such modifications in the DNA sequence that determines the amino acid sequence of the chain of immunoglobulin, well known to the skilled in the art specialist, see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

The term "monoclonal or polyclonal antibody" (see, Harlow and Lane (1988), quoted in place) also refers to derivatives of these antibodies have maintained or substantially maintained their binding specificity. While particularly preferred variants of these derivatives are further defined here below, in other preferred derivatives of such antibodies are chimeric antibodies, including, for example, murine or rat variable region and a constant region of a human.

The term "scFv-fragment (single-chain Fv-f is agment) rather construed in the art, and this fragment is preferred due to its small size and ability of the recombinant production of such fragments.

Preferably the antibody, aptamer, fragment or derivative in accordance with the present invention is specifically associated with the target protein, polypeptide or its fragment or epitope, the presence or absence of control.

The term "specifically binds", as used in accordance with the present invention means that the antibody, etc. does not cross react or essentially does not cross react with polypeptides with similar structures or Mature a eukaryotic polypeptide, having two N-terminal residues of methionine. The cross-reactivity of the investigated panel of antibodies, etc. can be checked, for example, by assessing binding of the specified panel of antibodies, etc. under normal conditions (see, for example, Harlow and Lane (1988), quoted in place) with interest the polypeptide, as well as with a number of more or less (structurally and/or functionally) closely related polypeptides. Only those antibodies that are bound to interest the polypeptide/protein, but not bound, or essentially no contact with any of the other polypeptides, which are preferably expressed in the same tissue that represents what the overall interest of the polypeptide, are specific to the interest of the polypeptide/protein and selected for further studies in accordance with the method of the present invention.

In a particularly preferred embodiment of the method of the present invention, the specified antibody or antigennegative part are or come from human antibodies or gumanitarnogo antibodies. The term "humanitariannet antibody" means, in accordance with the present invention, an antibody of nonhuman origin, in which at least one complementarity determining section (CDR) in the variable regions, such as CDR3, and preferably all 6 CDRs, was replaced by a CDR of an antibody of human origin, having the desired specificity. Optional inhuman constant region of an antibody is replaced by(s) constant region(s) human antibodies. Methods of producing humanized antibodies are described, for example, in EP-A1 0239400 and WO 90/07861.

Aptamers are DNA or RNA, which have been selected from random pools based on their ability to bind with other molecules. Were selected aptamers that bind nucleic acid, proteins, small organic compounds, and even entire organisms. Database aptamer contains at http;//aptamer.icmb.utexas.edu/.

To cretae, aptamers can be classified as aptamers in the form of DNA or RNA or peptide aptamers. While the first of these two aptamers consist of a (usually short) chains of oligonucleotides, the latter aptamers consist of a short variable peptide domain, attached at both ends to a protein skeleton.

Aptamers in the form of nucleic acids are varieties of nucleic acids, which were developed through repeated rounds of in vitro selection or equivalently SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.

Peptide aptamers are proteins that develop to make noise in the interaction of other proteins inside the cells. They consist of a variable peptide loop attached at both ends to a protein skeleton. This double structural relationship significantly increases the binding affinity of the peptide aptamer to levels comparable to the binding affinity of antibodies (nanomolar range). The length of the variable loops usually 10-20 amino acids, and the frame can be any protein that has the ability to good solubility. Currently most the used frame protein is a bacterial protein thioredoxin, when this variable loop insert into regenerating the active site, which is a loop-Cys-Gly-Pro-Cys - protein wild type, with cross-circuit two cysteines capable of forming a disulfide bridge. Selection of peptide aptamers can be performed using various systems, but the most used system is currently the yeast dvuhserijnaya system.

Aptamers are used for biotechnological and therapeutic applications, as they provide the ability to molecular recognition, competing with abilities usually used biomolecules, in particular antibodies. In addition to their ability to distinguish between recognition, aptamers have the advantage over antibodies, because they can be constructed completely in a test tube, it is easy to produce by chemical synthesis, they possess desirable storage stability and do not have and have minor imaginosity in therapeutic applications.

Unmodified aptamers are rapidly eliminated from the bloodstream, with the period of existence is minutes, hours, mainly due to degradation by nucleases and clearance from the body by the kidneys, the result of significantly lower molecular weight aptamer. Currently, the use of unmodified aptamers focus naliczenie transient States, such as blood clotting, or treatment of such organs as the eye, where possible local delivery. This rapid clearance may be an advantage in applications such as in vivo diagnostic imaging. Scientists have several modifications such as 2'-fluoro-substituted pyrimidines, the binding of polyethylene glycol (PEG), with which the period of existence of aptamers can easily be expanded to a time scale, constituting a day or even a week.

Phages in accordance with the present invention relate to recombinant phages and widely known in the art and are described, for example, Griffiths and others in EMBO J. 1994, 13: 3245. Phages can carry fragments or derivatives of antibodies with the desired binding specificity to a polypeptide of the present invention in the form of a fused protein on its surface, and the merger partner is a surface molecule of the phage.

In a preferred embodiment of the method of the present invention, the specified antibody or aptamer or phage distinguishable mark. While aptamers preferably radioactively mark using3N or32R or a fluorescent marker, such as described above marker, phage or antibody can, for example, to mark appropriately (using131I as a pre is respectful of radioactive label) or mark, such mark, as His-tag, FLAG-tag or myc-tag.

In an alternative embodiment, the present invention provides a diagnostic composition containing a specified antibody, aptamer and/or phage. This composition may further contain an appropriate buffer(s) and enzymes such as reverse transcriptase, thermostable polymerase, etc.

Specified diagnostic composition can be used to verify the presence of the polypeptide of the present invention, for example, in the immunoassay using the antibody of the present invention. Used herein, the term "immunoassay" includes means, such, for example, thus, the Western blot turns, ELISA, RIA (radioimmunoassay), indirect immunofluorescent methods, etc. Such methods are widely known in the art and are described, for example, Harlow and Lane, above.

In an alternative embodiment, the present invention provides a method for checking the presence of the nucleic acid molecule or polypeptide of the present invention, including the analysis of the sample obtained from the subject, the presence of a specified nucleic acid molecule or polypeptide.

Methods of test sample for the presence of the nucleic acid molecules of the present invention include, but without limitation, the analyses using amplifica the AI, sequencing and hybridization of nucleic acids.

Examples of analyses using nucleic acid amplification and means for their implementation include, but without limitation, PCR (including "nested" PCR, RT-PCR, analysis extension using PCR techniques, amplification-based nucleic acid sequences (first NASBA), PCR for analysis of conformational polymorphism single-stranded DNA (SSCP)), amplification system to identify mutations (ARMSTM) and analyses of linear elongation using amplification system to identify mutations (ALEXTM). The details of such methods can be found in engineering, see, e.g., Newton et al., Nucleic Acids Res. 17 (1989) 2503-2516; Agrawal (Ed.), “Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Methods in Molecular Biology, 20)”, Humana Press, 1993; Haque et al., Diagn. Mol. Pathol. 7 (1998) 248-252; Innis et al. (Ed.), “PCR Applications: Protocols for Functional Genomics, Academic Press, 1999; Chen and Janes (Ed.), “PCR Cloning Protocols: From Molecular Cloning to Genetic”, 2ndEdition, Humana Press, 2002; Pissardet et al., Clin. Chem. 48 (2002) 769-772; Steemers et al., Nature Meth. 3 (2006) 31-33: Kakavas et al., J. Clin. Anal. 20 (2006) 1-7.

Examples of sequencing analyses include, without limitation, approaches to sequence analysis by direct sequencing, fluorescent SSCP in an automated DNA sequencing machine, persecutione. These techniques are conventional in the art, see, for example, Adams et al. (Ed.), “Automated DNA Sequencing and Analysis”, Acadmic Press, 1994; Alphey, “DNA Sequencing: From Experimental Methods to Bioinformatics”, Springer Verlag Publishing, 1997; Ramon et al., J. Transl. Med. 1 (2003) 9; Meng et al., J. Clin. Endocrinol. Metab. 90 (2005) 3419-3422.

Examples of analyses of hybridization include, without limitation, Northern and southern blotting, heteroduplex analysis, detection of mutations using hybridization using specific sequences of oligonucleotides, hybridization using allele-specific oligonucleotides on a DNA chip analysis, based on technology Illumina''s®, analyses based BeadArray®, see, for example, Barnes et al., Nucleic Acids Res. 33 (2005) 5914-5923; Fan et al., Biotechniques 39 (2005) 583-588; Shen et al., Mutat. Res.-Fund. Mol. M. 573 (2005) 70-82; Steemers and Gunderson, Pharmacogenomics, 6 (2005) 777-782.

Examples of assays based on detection of proteins include, without limitation, stage methods, such as ion exchange chromatography, gel filtration, affinity chromatography, chromatography based on hydrophobic interactions, HPLC with reversed phase, disc electrophoresis, capillary electrophoresis, Western blotting, immunoprecipitation, amino acid sequencing, spectroscopic methods (UV, circular dichroism, IR, fluorescence) and mass spectrometry (e.g., MS-QTOF), see, for example, Soejima and Koda, Transfusion 45 (2005) 1934-1939; Yeh et al., Anesth. Play mode display. 101 (2005) 1401-1406; Chou et al., Am. J. Clin. Pathol. 124 (2005) 330-338.

The above-described assays known in the art, such as the er, from standard books-texts, such as Lottspeich, Engel “Bioanalytik” Spektrum Akademischer Verlag (2006); Sambrook and Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989); Higgins and Hames (Eds.) “Nucleic acid hybridization, a practical approach,” IRL Press Oxford, Washington DC (1985); Nollau et al., Clin. Chem. 43 (1997), 1114-1128. Some of the analyses described in the accompanying examples.

In another preferred embodiment of the method of the present invention the specified sample is blood, serum, plasma, fetal tissue, saliva, urine, tissue mucosa, mucus, tissue, vaginal tissue of the fruit obtained from the vagina, skin, hair, hair follicle, or other human tissue. Preferably the sample is blood, serum, plasma, saliva, urine, tissue mucosa, mucus.

The present invention also relates to a kit comprising the nucleic acid molecule, vector, not a human host, the polypeptide or antibody, aptamer and/or phage of the present invention in one or more containers.

The figures are shown:

Figure 1. Schematic representation of the purification procedure.

Figure 2. QTOF Mass spectrum B1, clinical experimental series M-H1A-P02.

Figure 3. QTOF Mass spectrum B2, clinical experimental series M-H1A-pool.

The present invention now b the children described by reference to the following examples, which are only illustrative and should not be construed as limiting the scope of the present invention.

Example 1: Cloning of constructs hH1.3

Construction of plasmid vector pEGT1-rH1.3S1

As shown in SEQ ID NO: 1, histones demonstrate a strong positive charge, which is a consequence of a very high content of lysine residues. Because the use of codons for lysine differs greatly betweenEscherichia coliand man, was optimised codons to adapt the sequence of histone N person to use codons inE. coli.

Received synthetic gene, the sequence of which is provided as SEQ ID NO: 2. Artificial sequence was flanked by two restriction sites, namely for BspH1 and BamH1, in order to make possible the subsequent introduction of the expression vector pEGT1. Codon of translation initiation ATG was included in the Nco1 restriction site CCATGG. Initial ATG was doubled, which provides a site for BspH1 TCATGA sticky end CATG which is compatible with Nco1. Thus, the second residue of methionine was included after the first methionine residue. An additional site for BamH1 GGATCC was introduced after the termination codon TAA. Amino acid sequence encoded by this artificial gene provided in SEQ ID NO: 3.

Optimized the Yong cut out from the containing plasmid by cleavage BspH1 and BamH1 and have been built into the expression vector pEGT1, linearized using Nco1 and BamH1, in accordance with standard protocols to obtain plasmids pEGT1-rH1.3S1.

Subjected to legirovanie vector pEGT1-rH1.3S1 was introduced in electrocompetent strain BL21[DE3]E. coliusing electroporation, using a standard Protocol, and transformed cells were selected on plates with LB supplemented with kanamycin. He was selected one clone, and sequence encodes a histone insert was tested for its exact accordance with SEQ ID NO: 2.

Construction of plasmid vector pEGT1-rH1.3S2

To prevent insertion of the second methionine in the design rH1.3S1 used the second synthetic gene. The second source codon TCC encoding serine was replaced with AGC, also encodes a serine, to ensure compatibility with the site for BspH1. The DNA sequence provided as SEQ ID NO: 4, and the recombinant protein encoded by this artificial gene provided as SEQ ID NO: 5. Used the strategy of cloning, with the same cloning strategy outlined above for pEGT1-rH1.3S1, since synthetic gene of SEQ ID NO: 4 was flanked by a restriction site for BspH1 TCATGA codon-initiated and restriction site for BamH1 CCATGG through one pair of bases after codon-terminator.

The optimized gene was cut out from the containing plasmid by cleavage BspH1 and BamH1, and was built in expression the th vector pEGT1, linearized using Nco1 and BamH1, in accordance with standard protocols to obtain plasmids pEGT1-rH1.3S2.

Subjected to legirovanie vector pEGT1-rH1.3S2 was introduced in electrocompetent strain BL21[DE3]E. coliusing electroporation, using a standard Protocol, and transformed cells were selected on plates with LB supplemented with kanamycin. He was selected one clone, and sequence encodes a histone insert was tested for its exact accordance with SEQ ID NO: 4.

Example 2: Recombinant production of histone N

Strain

Used for cooking rH1.3S the bacterium is a recombinant strain BL21 (DE3)/pEGT1/H1.3SEscherichia coli. Constructs used for transformation of strain BL21 (DE3)E. coli. Three clones were selected for conducting the selection expression, and one clone was selected for preparation preparation of inoculum (Pre-MS-05L23-H1B).

Basic seed (MS-06D05-H1B) was obtained using Pre-MS-05L23-H1B), and working seed (WS-06D06-H1B) was obtained using the basic seed.

Cultivation seed

Each of the two shake flasks for 2 litres, each containing 500 ml of medium YES (30 g/l yeast extract, 5 g/l NaCl), seeded with 100 µl of the working seed (WS-06D06-H1B). The culture is incubated at 37°C with shaking at 270 rpm out within 5 hours (+/-0,5 h) to achieve an OD (600 nm), component of more than 1.5.

Fermentation

The fermentor 100 liters are made using 100 liters of medium NRJ18. The fermentor is sterilized for 30 minutes at 123°C. After sterilization and before inoculation aseptically add 50 ml SAG471 (antifoam). The fermentor inoculated with culture inoculum with achievement of theoretical initial optical density at 600 nm component is 8.75 × 10-7.

The calculated amount of inoculum added to the bottle to migrate containing 500 ml of medium YES.

The fermentation is carried out overnight at 37°C. During the fermentation process, the pH is maintained at pH of 7.0±0.2 with the help of the periodic addition of 4 M NaOH and 2.24 M HNO3. Dissolved oxygen on the basis of feedback regulate with shaking at 30%.

When the culture reaches OP600between 15 and 20, the culture induce a solution of 1 mm IPTG (23,8 g dissolved in 500 ml of highly purified water).

After induction for 1 h 30 min OP600is more than 24, and the fermenter is cooled below 16°C. the pH Regulation remain at the level of pH 7.0 ą 0.2. During the cooling keep constant the other parameters, except for pressure, which is reduced to 400 mbar, and the shaking of 200 rpm

When the ambient temperature falls below 16°C, determine the volume of the culture. The whole culture is centrifuged to IP is the use of 2 Beckman-centrifuge JA10, equipped with a rotor JLA 8.1000 (±6 l/centrifuge): 5200 rpm - 4°C - 20 minutes.

During the stage of centrifugation of the cellular precipitation receive and store then at -20°C.

The destruction of cells in the homogenizer using high pressure

The day before the destruction of the cells concentrated cells corresponding to 100 liters of culture was thawed at room temperature.

On the day of the destruction of the cellular precipitation was diluted to 250 g/l in 20 mm Na2HPO4.12H2O, pH 7.0, and the temperature of the suspension is increased to 30°C.

The suspension is then homogenized propeller stirrer Heidolph R2R100. Cells then are lysed in the homogenizer PONY with the use of high pressure (800 bar). Cell suspension twice treated through the cell homogenizer.

Example 3: Purification of proteins

All stages of the purification is carried out using the total volume of the fermenter (i.e. 100 l).

1. Precipitation of 2.5% perchloric acid and extraction 8M urea

To the resulting cell mass is added 1/7 volume of 20% HClO4(final concentration: 2.5 percent). The suspension is homogenized before the third cycle in the homogenizer PONY at 250 bar. Solution save when lightly shaken for one hour at room temperature. Then the suspension is centrifuged for 15 minutes (12200 g - 7000 rpm, 4°C). The supernatant is collected, bring rndo 4,0±0,1 using 10M NaOH and filtered through a membrane of 0.45/0.22 μm Sartopore 2 (2000 cm 2in sterile soft tank.

Add urea to obtain 8 M concentration in double the target volume, while the volume was adjusted with 20 mm buffer Na2HPO4, pH 7.0. The solution remain overnight with mild shaking at room temperature. Then the pH was adjusted to 4.0±0.1 s using 37% HCl or 10 M NaOH.

2. Anion-exchange chromatography on Q-Sepharose for fast flow (QSFF) - type with a minus sign

The purpose of this phase is to reduce the content of endotoxins and DNA. Anion exchange chromatography is performed using a Q-Sepharose for fast flow (Amersham Biosciences, catalog No. 17-0510-05), which is filled with a column Moduline 350/500 (Millipore BioProcess Division, catalogue No. 86351211).

Column fill in high purity water with a flow rate of eluent 120 cm/h (115,4 l/h). The dimensions of the compacted layer columns are: diameter = 25 cm, the cross-sectional area = 961 cm2layer = 18 cm, filled volume = 17,314±0,962 HP Column disinfected using 1.5-2.5 column volumes (CV) of 1M NaOH + 2M NaCl with the duration of the contact, component 2 hours, at a flow rate of 96.2 l/h (100 cm/h -1603 ml/h).

All chromatographic stage performing with a linear flow rate of 100 cm/h (± 96,2 l/h). pH stabilize 1-2 CV of 50 mm ammonium acetate + 1 M NaCl, pH of 4.0. The column then balance 3,5-5 CV of 50 mm acetate, ammo the Oia + 8 M urea, pH of 4.0.

The solution from the extraction of urea (see section 1) was diluted to approximately 1.5 times 50 mm ammonium acetate + 8 M urea, pH 4,0, to obtain a conductivity less than 10 MS/see Only 8 CV solution from the extraction of urea (before breeding) load at the same time. Protein N1 get in the duct, when the trim - elution performed with a 1.5-2.5 CV of 50 mm ammonium acetate + 8 M urea, pH of 4.0.

After elution of the column purified using 1.5 to 2.5 CV of 50 mm ammonium acetate + 1 M NaCl, pH of 4.0. This elution in 1 M NaCl eliminates DNA and endotoxins. Then the column disinfected using 1.5 to 2.5 CV of 1M NaOH + 2M NaCl (2 h) and stored at room temperature in 20 mm NaOH.

3. Cation-exchange chromatography on MacroPrep High S (MHS-E) - type plus

Cation exchange chromatography is performed using MacroPrep High S (Bio-Rad Laboratories, catalogue number 156-0033), which is filled with a column Vantage 180/500 (Millipore BioProcess Division, catalogue No. 87018001). Column fill in high purity water with a flow rate of eluent 260 cm/h (66,1 l/h). The dimensions of the compacted layer columns are: diameter = 18 cm, the cross-sectional area = 254,4 cm2layer = 36 cm, filled volume = 9,16±0,25 L.

Column disinfected using 1.5 to 2.5 CV of 1M NaOH + 2M NaCl with the duration of the contact, component 2 hours, at speeds on the Oka 40 l/h (157 cm/h). The maximum flow rate may be 250 cm/h pH stabilize 1-2 CV of 50 mm ammonium acetate + 2 M NaCl, pH of 4.0. The column then balance 4-5,5 CV of 50 mm ammonium acetate, pH of 2.0.

the pH of the fraction QSFF-FT (see section 2) was adjusted to 2.0 using 37% HCl. This solution load without prior dilution with a velocity component 125 cm/h (±31.8 l/h). Binding capacity of the gel is 5-15 mg/ml matrix. After loading the column balance 2-3 CV of 50 mm ammonium acetate, pH of 2.0, a flow rate of 157 cm/h (40 l/h). The maximum flow rate of 200 cm/h

The elution is performed using a linear gradient of conductivity in 10 CV between 25% (0.5 M NaCl) and 75% (1.5 M NaCl) using 50 mm ammonium acetate, pH of 2.0, and 50 mm ammonium acetate + 2 M NaCl, pH of 2.0. The elution is conducted with the velocity component 157 cm/h (40 l/h). The maximum flow rate of 200 cm/h Peak elution get a 2-liter fractions are analyzed by electrophoresis in SDS-page prior to the merger. After the unification of the pool MHS-E stored at -20°C until the next stage of treatment or at 2-8°C when used within 24 hours.

Then the column disinfected using 1.5 to 2.5 CV of 1M NaOH + 2M NaCl (2 h) and stored at room temperature in 20 mm NaOH.

4. Concentration - diafiltrate

Concentration is carried out with the use of two cassettes Sartocon (0,6m 2, cut-off 5 kDa) membranes from hydrosart (Hydrosart) Sartorius (Sartopore, catalogue No. 3021442906 E-SC). Membrane fixed in the holder, which is connected with the system Proflux M13 (Millipore Bioprocess Division. The membrane is washed with water for injection (WFI). Disinfection is carried out by continuous recirculation of 0.5 M NaOH for 60 minutes. Then the membrane was washed with 20 mm Na2HPO4. 12 H2O until the pH of filtrate = 7,0±0,1. Then the membrane balance PBS, pH 7,4 (8 g/l NaCl, 0,19 g/l KH2PO4, of 2.38 g/l Na2HPO4) until the pH of filtrate = 7,4±0,1.

The inlet pressure and the outlet pressure set at 1.5±0.1 bar and 1.2±0.1 bar, respectively.

Several eluates with MacroPrep High S unite together and, in accordance with the total amount of protein, concentrated to a volume appropriate for theoretical concentration of 30 mg/ml After concentration, the concentrate is subjected to diafiltration against 10 volumes of PBS, pH 7,4 (8 g/l NaCl, 0,19 g/l KH2PO4, of 2.38 g/l Na2HPO4). Concentrate collect and spend 7 leaching of the membrane, each washing using 150 ml of PBS, pH 7,4 (8 g/l NaCl, 0,19 g/l KH2PO4, of 2.38 g/l Na2HPO4within 3 minutes, using the same process parameters. The series concentrates close during leaching.

Analysis of the protein using BSA carried out on the concentrate and each of the individual fractions of leaching. The concentrate is combined with about obrannye leaching fractions to obtain the total concentration exceeding 12 mg/ml, greater than 90%, if possible.

The membrane was washed with WFI. Disinfection is carried out by continuous recirculation of 0.5 M NaOH for 60 minutes. The membrane is then stored in 0.1 M NaOH.

5. Sterilization by filtration

Sterilization by filtration of concentrate + selected fractions leaching perform on the filter 1000 cm20,45/0,22 Sartopore 2 (Sartorius, catalogue No. 544-1307-H8-00) at room temperature. The membrane was washed with about 500 ml of PBS, pH 7,4 (8 g/l NaCl, 0,19 g/l KH2PO4, of 2.38 g/l Na2HPO4).

Filtering is done using a peristaltic pump with a flow rate component of approximately 100 ml/min, and the filtrate is collected in a sterile and free from pyrogens bottle for a single application of 5 l or 10 l

In accordance with the analysis using the BCA performed on the filtered mass concentration set at 10 mg/ml using PBS, pH 7,4 (8 g/l NaCl, 0,19 g/l KH2PO4, of 2.38 g/l Na2HPO4)added during filtration. After sampling sterile mass distribute the aliquot in PETG 2000-ml Nalgene-bottle (±1500 ml 1700 ml/bottle). Sterile mass stored at -20°C. a Schematic summary of the stages of obtaining provided in figure 1.

Example 4: purification Efficiency hH1.3 and bis-Met-hH1.3

Cultivation BL21[DE3]-bis-Met-rH1.3 50-l fermenter was led as a result to the output at the time of collection, forming at least 600 mg/l of culture, as determined by analysis by electrophoresis in SDS-PAG serial dilution subjected to lysis of cells in General. The final output after a complete cleaning process exceeded 500 mg/l of purified bis-Met-rH1.3.

Cultivation BL21[DE3]-hH1.3 50-l fermenter has resulted in the release at the time of collection, which shall be at least 600 mg/l of culture, as determined by analysis by electrophoresis in SDS-PAG serial dilution subjected to lysis of cells in General. Cells were subjected to treatment by means of homogenization and precipitation using HClO4in accordance with a standard Protocol. The results were consistent with the expectation. Download on MacroPrep High S also carried out as usual, but protein rhH1.3 could not be eluted from the column using a linear gradient of conductivity in 10 CV between 30% (0.6 M NaCl) and 75% (1.5 M NaCl) using a 10 mm NaCH3COO, pH of 2.0, and 10 mm NaCH3COO + 2 M NaCl, pH of 2.0. Although protein rhH1.3 was elyuirovaniya 2 M NaCl, this stage it was impossible to efficiently purify the protein, and the protein could not be subjected to further processing. Thus, treatment should be considered as failed. This failure was confirmed in two independent experiments cleanup, which began with two independent fermentati.

Example 5: Effect of bis-Met-histone H1.3 in vitro

Analysis of the area in which euromania

To measure the effect of recombinant histone as an antimicrobial and antiviral agent conducted an analysis of the zone of inhibition in accordance with standard methods. In addition, they tested the effect of recombinant histone as an antifungal agent. Bacteria and fungi were grown in the presence of bis-Met-histone H1.3, obtained in accordance with the methods outlined above and have determined the average diameter of the zone (see table 1).

Both gram-positive and gram-negative bacteria and fungi were effectively destroyed, as shown in table 1.

9,8
Table 1
Analysis of zone of inhibition
Proven bodyThe drug concentration [ág/ál]Average zone diameter [mm]
B. megaterium Bacillus511,4
2,5010,5
1,25the 9.7
of 0.6258,6
0,317,9
the positive control LL-37
Escherichia coli D2120,007,4
10,006,3
5,004,8
2,500
1,250
the positive control LL-375
Candida albicans20,0011,8
5,008,1
2,506,1
1,254,4
positive controls:
Nystatin20,9

Analysis of cytotoxicity

This cell analysis is the identification of toxic effects of histone-sensitive histone line leukemic cells (e.g., U-937). Cell viability of cancer - leukemia after incubation with various concentrations of histone controlled through research AlamarBlue, based on measurement of the fluorescence of the indicator of oxidation in which Stanovlenie, which varies in response to the viability of the cells. Anticancer activity of histone determined by IC50, which correspond to the concentration of histone, which is determined by the 50% viability of cancer cells. Experimental series used for the analysis of cytotoxicity, as well as clinical trials, are summarized in table 2. As shown in table 1, all of the tested samples show similar high cytotoxicity against lines of tumor cells U-937, regardless of different contents N and bis-Met (compare with table 3).

Table 2
Cytotoxicity of experimental series used in a clinical trial
Samples (drug product)IC50 [μm]
experimental series 1: M-NA-P023,2±0,5
experimental series 2: M-NA-Pol3,1±0,5
experimental series 3: M-NA-Pol3,1±0,5
experimental series 4: M-NA-Pol2,2±0,5

In addition to bacteria and fungi shown in table 1, is one suitable way to check additional bacteria, fungi and viruses, using known in the art methods, such as any of the ways outlined here. Non-limiting examples include Epstein-Barr,Staphylococcus aureus,Aspergillus niger, Enterococcus, Pseudomonas,Haemophilus influenzaeand Salmonella.

Example 6: Clinical data

Test was performed phase I/II dose is increased to assess the maximum tolerated dose (MTD) of recombinant histone N person (rhH1.3) in patients with recurrent or refractory acute myeloid leukemia (AML) and patients who refuse chemotherapy, or patients who are not suitable for chemotherapy. Criteria for inclusion patients had signed informed consent, any race, both anatomical sex, the age of at least 18 years of age, cytologically proven AML with at least 20% of blast cells in the bone marrow, unsuccessful attempt of standard chemotherapy, discrepancy or her rejection of it, adequate General condition (index Tarnovskogo >60%) and life expectancy, component, at least 30 days. The criteria that led to the exclusion of patients were significant organ failure, known infectious disease caused by HIV, a known infectious disease caused by the hepatitis C virus or virus hepatitis is In, pregnancy or breastfeeding, other malignancy, circulating antibodies against H1, treatment with heparin within two weeks prior to visit 1 or during the study, active disease, which is known that they can interfere with the treatment rhH1.3, such as rheumatoid arthritis or systemic lupus erythematosus (SLE), as well as the abuse of alcohol and/or drugs.

Study design

Patients received 3 infusions per week for three consecutive weeks. The initial dose was 38 mg/m2. Scheme used increasing doses shown in table 3.

Table 3
The scheme of increased dose
Level doseDose in mg/m2The planned number of patientsTestedThe treatment
13837Full
26037Full
3/td> 9633Full
415333Full
The new increase in dose
5, 5, 6245, 245, 3921 (cycle 1)1Full
6, 6, 7392, 392, 628(cycle 2, the same patient as above)Full
6, 6, 7392, 392, 62811Full

Clinical trial phase I/II was conducted at Saarland University Hospital in Homburg in patients with AML (acute myeloid leukemia). Used in this test experimental batches of the drug product were B1, B2, B3 and B4. The characteristics of these four experimental series and one experimental series GLP, used the Oh in Toxicological research, presented below in table 4.

Table 4
Characteristics of the experimental series of the polypeptide used in this study
Experimental seriesDescriptionEndotoxin [in this case/mg]assessment of the level of endotoxinpeaks MS rhH1.3 [Yes]anticancer activity in cell research IC50 [μm]
L-H1A-03B07*5below ALN + bis-Met1,7-2,7
M-H1A-P02B112above ALN + bis-Met3,2
M-NA-PolB295above ALmainly bis-Met3,1
M-NA-PolB30,8below ALChapter the main way bis-Met 3,1
M-NA-PolB41,7below ALmainly bis-Met2,2
* used in Toxicological research, but not in a clinical trial.
AL: the maximum permissible level level
MS-peaks: 22221 Danes Met22481 Davis-MetN-terminal Met-Met

6.1. Preliminary assessment

Table 5 summarizes the preliminary clinical results of the first 22 patients with AML treated with recombinant histone N person (rhH1.3, “Oncohist”) at levels of increasing doses (so-called scheme of Fibonacci).

Each patient WW13 and WW27 received two cycles of treatment (one cycle consisted of 3 infusions per week for three weeks, a total of 9). For WW27 used to increase the dose in each cycle, i.e. 5-5-6: level 5 doses within two weeks, the dose level 6 on the third week and similarly in the second cycle dose levels: 6-6-7.

td align="justify"> poorly tolerated
Table 5
Obtained during the preliminary assessment of the clinical outcome of patients with AML treated with recombinant histone N person (“Oncohist”)
In ciali patients and No. Level doseCommentsThe experimental series of the medicinal productThe composition in accordance with MS (N, bis-Met)Contamination with endotoxin [in this case/mg]Side effects
Ass 011TTIB1N + bis-Met12medium portable
NM 021TTIB1N + bis-Metpoorly tolerated
HS 031TLIB1N + bis-Metwell-tolerated
RH 041TTIB1N + bis-Metwell-tolerated
MF 05TTI, TLIB1N + bis-Metpoorly tolerated
RS 071TTIB1N + bis-Metwell-tolerated
B1-21 bubbles
PS 101TTIB2-4 bubblesN + bis-Metwell-tolerated
MT 112B2mainly bis-Met95poorly tolerated
MG 122TLIB2mainly bis-Met
WW 132PR, TNB2mainly bis-Metpoorly tolerated
AH 152TTI, TLIB2mainly bis-Metpoorly tolerated
GB 162TN, TLIB2mainly bis-Metpoorly tolerated
HF 182TTI, TLIB2mainly bis-Metpoorly tolerated
The beginning of the introduction of drug - histone without endotoxin

PR, TTI
ES 192TLIB3mainly bis-Met0,8NSE
IG 203B3mainly bis-MetNSE
BG 213TTIB3mainly bis-MetNSE
GR 223B3mainly bis-Met
EL 234TTI, TLIB3mainly bis-MetNSE
EW 244 B3mainly bis-MetNSE
BH 264B3mainly bis-MetNSE
C1:
5, 5, 6;
C2:
WW 276, 6, 7PR, TTIB3/B4mainly bis-MetB4: 1,7NSE
PF 286, 6, 7B3 mainly bis-Met
preliminary analysis:
PR: partial remission
TTI: a temporary increase in platelet count
TN: platelets at a normal level
TLI: temporary increase of leukocytes
NSE: no side effects
H1.3: Mature recombinant histone N person; bis-Met: N-Met-Met-H1.3

As can be seen from table 5, the side effects of drugs are found only in the result of contamination by endotoxin (see also table 3). And naturally occurring histone N, and the derived bis-Met" show similar properties when considering clinical evidence of effectiveness.

Immunogenicity

All patients were subjected to screening for the presence of antibodies against histone N before, during and after treatment. During treatment, the antibodies did not develop in any of the treated patients, nor those who were subjected to one cycle of treatment, nor those who received two cycles. Histone H1 is a very conservative evolutionary protein, and was not expected and has not been proven that he immunogenes. Clinical data confirm that the immunogenic activity can be observed using either naturally occurring histone N, or derived bis-Met".

therapeutic effect is

Approximately 50% of patients demonstrated an increase in platelets, and in some of them - also the increase of leukocytes, both very important biomarkers of AML. These patients had partial remission (reduction of tumor cells to less than 50% of the initial value). The patient WW13 have demonstrated an increase in platelet count to normal levels (210 × 109/l), which lasted 18 months. The platelet count before treatment Oncohist was equal to 47 × 109/HP

6.2. Final evaluation of the clinical results

Table 6 summarizes the clinical results obtained after a detailed analysis of 22 patients with AML treated with recombinant histone N person (rhH1.3, “Oncohist”) at levels of increasing doses (Fibonacci).

As described above, each of the patients WW13 and WW27 received two cycles of treatment (one cycle consisted of 3 infusions per week for three weeks, a total of 9). For WW27 used to increase the dose in each cycle, i.e. 5-5-6: level 5 doses within two weeks, the dose level 6 on the third week and similarly in the second cycle dose levels: 6-6-7.

Table 6
Received the final assessment of the clinical outcome of patients with AML treated with recombinant histone N man (“Oncohist”)
The initials of the patients and No.Level doseCommentsThe experimental series of the medicinal productThe composition in accordance with MS (N, bis-Met)*Contamination with endotoxin [in this case/mg]Side effects
Ass 011B1N + bis-Met12medium portable
NM 021TI, LIB1N + bis-Metpoorly tolerated
HS 031TLIB1N + bis-Metmedium portable
RH 041B1N + bis-Metwell tolerated the
MF 051B1N + bis-Metpoorly tolerated
RS 071B1N + bis-Metwell-tolerated
B1-21 bubbles
PS 101B2-4 bubblesN + bis-Metwell-tolerated
MT 112B2mainly bis-Met95poorly tolerated
MG 122LIB2poorly tolerated
WW 132PR, TNB2mainly bis-Metpoorly tolerated
AH 152LIB2mainly bis-Metpoorly tolerated
GB 162B2mainly bis-Metpoorly tolerated
HF 182B2mainly bis-Metpoorly tolerated
The beginning of the introduction of drug - histone without endotoxin
ES 192PR, TI mainly bis-Met0,8well-tolerated
IG 203B3mainly bis-Metwell-tolerated
BG 213TIB3mainly bis-Metwell-tolerated
GR 223B3mainly bis-Met

EL 234TIbLIB3mainly bis-Metwell-tolerated
EW 244B3primary education is bis-Met well-tolerated
BH 264B3mainly bis-Metwell-tolerated
C1: 5, 5, 6;PR, TI
WW 27C2: 6, 6, 7LIB3/B4mainly bis-MetB4: 1,7well-tolerated
day 8**, day 19
PF 286, 6, 7 B3mainly bis-MetSAE***
* preliminary analysis
** after one of 9 infusions AE, fixed
*** during the last infusion SAE, atrial fibrillation 74-year-old patient, causing doubt on the link
PR: partial remission
TI: increase platelets
TN: platelets at a normal level
LI: the number of leucocytes
AE: side effects; SAE: serious adverse actions
H1.3: Mature recombinant histone N person; bis-Met: N-Met-Met-H1.3

Therapeutic effects

In accordance with final rating in seven of the 22 demonstrated an increase in platelets, and in some of them - also the increase of leukocytes, both very important biomarkers of AML. These patients had partial remission (reduction of tumor cells to less than 6-25%, with a concomitant improvement in other indicators of blood). The patient WW13 have demonstrated an increase in platelet count to normal levels (210 × 109/l), which lasted 18 months. The platelet count before treatment Oncohist was equal to 47 × 109/HP

Safety assessment

In the clinical study report demonstrates that rhH1.3 (Oncohist) safe in the doses used for treatment so far. Take the EIT side effects were observed except for one atrial fibrillation in infusion rhH1.3, which, as it is associated, perhaps, with the investigational drug. Seventeen (17) patients completed one course of therapy (8-9 injections), and two responding patients received a second course without side effects. DLT (dose limiting toxicity was not determined, and the maximum tolerated dose was not reached at 628 mg/m2.

Most importantly, clean, free of endotoxin investigational drug was well tolerated by the patients, i.e. without side effects, in contrast to cytotoxic drugs. This result is consistent with prior studies demonstrating that recombinant derived histone N person does not cause damage to healthy blood cells and does not cause resistance.

Example 7: Determination of the presence of bis-Met-hH1.3 sample

With MS "bis-Met-histone hH1.3 easily distinguished from endogenous histone H1. This analysis can be done directly using identification using ESI-QTOF in the source is not subjected to the processing solution of a medicinal product rhH1.3 or method RP-HPLC-ESI-MS with chromatographic separation using HPLC with reversed phase (RP-HPLC) and subsequent identification using ESI-MS. As can be seen in figure 2, the experimental series B1 contains the histone N and N-Met-Met-derived. Figure 3 shows one of the three e is sperimentali series (B2), which consist mainly of N-Met-Met-N. Regardless of the composition of the different experimental series show comparable cytotoxic activity against leukemia cells (table 3).

The following spectra were obtained using tandem mass spectrometry (QTOF, the combination of quadrupole and time-of-flight spectrometry in one device). As can be seen in figure 2, the experimental series B1 contains a histone N, and N-Met-Met-derived. Figure 3 shows one of the three experimental series (B2), which consist mainly of N-Met-Met-N. Regardless of the composition of the different experimental series show comparable cytotoxic activity against leukemia cells (table 3).

1. The nucleic acid molecule encoding a polypeptide having the function of histone, which
(a) encodes a polypeptide consisting of
(aa) two methionine residues in the first and second N-terminal amino acid residues connected via a peptide bond with
(ab) Mature the eukaryotic histone;
(b) encodes a polypeptide consisting of
(ba) of the two methionine residues in the first and second N-terminal amino acid residues connected via a peptide bond with
(bb) the Mature eukaryotic a polypeptide sequence which is at least 80% identical to the Mature sequence is th eukaryotic histone (a) and substantially retains its biological activity; or
(c) hybridized in stringent conditions with the complementary chain of the molecule is a nucleic acid that encodes a polypeptide (a) or (b)with the specified nucleic acid molecule encodes a polypeptide that has at least two N-terminal methionine residue and substantially retains the biological activity of the polypeptide (a) or (b).

2. The nucleic acid molecule according to claim 1, where the histone selected from the group consisting of histone H1.0, H1.1, H1.2, N, H1.4, H1.5 and H1t.

3. The sequence of the nucleic acid that is complementary to the nucleic acid molecule according to claim 1 or 2.

4. Antisense oligo - or polynucleotide the nucleic acid molecule according to claim 1 or 2, which includes the nucleotides complementary triplets of nucleotides coding for the first two N-terminal methionine residue (ab), (bb) or (C), and has a minimum length, which is 10 nucleotides.

5. The expression vector comprising the nucleic acid molecule according to claim 1 or 2.

6. A host cell transformed by the vector according to claim 5, for expression of the polypeptide encoded by the nucleic acid according to claim 1 or 2, and a host cell is not a person and not a human embryo.

7. A host cell according to claim 6, which is a bacterium, yeast cell, insect cell, a cell of a fungus, a cell of a mammal or plant cell.

8. Methods for the production of the polypeptide, encoded by a nucleic acid according to claim 1 or 2, comprising culturing the host cell according to claim 6 or 7 in suitable conditions, and the allocation of the produced polypeptide.

9. The polypeptide having the activity of Mature eukaryotic histone, recombinante expressed with high efficiency and is detectable in the presence of endogenous histones encoded by the nucleic acid molecule according to claim 1 or 2 or produced using the method according to item 8.

10. Pharmaceutical composition for treating cancer, bacterial, viral or fungal infections, containing the polypeptide according to claim 9, and optionally additionally contains a pharmaceutically acceptable carrier and/or diluent.

11. The composition of claim 10 additionally containing Mature eukaryotic histone.

12. Composition for the diagnosis of the patient in terms of response to the pharmaceutical composition of claim 10 or in respect of the treatability with it containing the polypeptide of claim 9.

13. The composition according to item 12, optionally containing Mature eukaryotic histone.

14. A method of treating cancer, bacterial, viral or fungal infections, comprising introducing the composition of claim 10 to the needy in this subject.

15. The use of the nucleic acid molecule according to claim 1 or 2 or the vector according to claim 5, or a host according to claim 6 or 7, or polypeptide according to claim 9 in preparation for the notizie for the treatment of cancer, bacterial, viral or fungal infections.

16. Method for checking the presence of the nucleic acid molecule according to claim 1 or 2 or of the polypeptide according to claim 9, including the analysis of the sample obtained from the subject, the presence of a specified nucleic acid molecule or polypeptide.

17. The method according to clause 16, in which the specified sample is blood, serum, plasma, saliva, urine, tissue mucosa, mucus.



 

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