Interferon-like protein zcyto21
FIELD: gene engineering, in particular method for treatment of viral infections.
SUBSTANCE: protein ZCYTO21 has amino acid sequence which is nearly similar to amino acid sequence of interferon-α. Protein and antibodies thereto have antiviral activity and are useful in treatment of hepatitis B and C as well as other diseases.
EFFECT: new protein with antiviral activity.
71 cl, 1 dwg, 6 tbl, 7 ex
BACKGROUND of the INVENTION
Differentiation of cells of multicellular organisms is regulated by hormones and polypeptide growth factors. These diffundere molecules allow cells to communicate with each other and act together for the formation of tissues and organs for repair and regeneration of damaged tissue. Examples of hormones and growth factors include, among others, steroid hormones, parathyroid hormone, follicle stimulating hormone, interferons, interleukins, platelet-derived growth factor, epidermal growth factor, granulocyte-macrophage colony-stimulating factor.
Hormones and growth factors affect cell metabolism by binding to the receptor proteins. Some receptors are integral membrane proteins that are associated with the hormone or growth factor outside the cell and are associated with the transmission of signals within the cell, such as a system of second messengers. Other classes of receptors are soluble intracellular molecules.
Cytokines usually stimulate the proliferation and/or differentiation of cells of hematopoietic direction of differentiation or participate in the mechanisms of immune and inflammatory reactions. Examples of cytokines that affect hematopoiesis, include retrop etin (EPO), which stimulates the development of red blood cells; thrombopoietin (TRO), which stimulates cell growth megakaryocytes line of differentiation; and granulocyte colony-stimulating factor (G-CSF), which stimulates the growth of neutrophils. These cytokines are applicable in the restoration of normal levels of blood cells in patients suffering from anemia, thrombocytopenia and neutropenia, or receiving chemotherapy for cancer. Cytokines play an important role in the regulation of haematopoiesis and immune reactions and can affect the development of lymphocytes.
The class II family of cytokines person includes subtypes of interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), IL-10, IL-19 (U.S. patent 5 985 614), MDA-7 (Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-20 (Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-22 (Xie et al., J.Biol. Chem. 275, 31335-31339, (2000)) and AK-155 (Knappe et al., J.Virol. 74, 3881-3887, (2000)). Most cytokines bind and transmit signals through the receptors of cytokines of Class I or Class II. Members of the family of receptors of cytokines of class II human include interferon-αR1 (IFN-α R1), interferon-γ-R2 (IFN-γ-R2), interferon-γR1 (IFN-γR1), interferon-γR2 (IFN-γR2), IL-10R (Liu et al., J.Immunol. 152, 1821-1829, (1994)), CRF2-4 (Lutfalla et al., Genomics 16, 366-373, (1993)), IL-20Rβ (Blumberg et al., Cell 104, 9-19, (2001)) (also known as zcytor7 (U.S. patent 5 945 511) and CRF2-8 (Kotenko et al., Oncogene 19, 2557-2565, (2000)), IL-20Rβ (Blumberg et al., ibid, (2001)) (t is also known as DIRS1 (PCT WO 99/46379)), IL-21 (IL-22 receptor-α1, presented in a HUGO for approval) (also known as IL-22R (Xie et al., J.Biol. Chem. 275, 31335-31339, (2000)), zcytor11 (U.S. patent 5 965 704) and CRF2-9 (Kotenko et al, Oncogene 19, 2557-2565, (2000)) and tissue factor.
Receptors of cytokines of class II are usually heterodimer, consisting of two different receptor chains, αand β-subunits of the receptor (Stahl et al., Cell 74, 587-590, (1993)). Usually α-subunit are the primary cytokinesis proteins, and β-subunit is required for the formation of binding sites of high affinity, as well as for signal transduction. The exception is the receptor for IL-20, in which both subunits are required for binding of IL-20 (Blumberg et al., ibid, (2001)).
Receptors of cytokines of class II identity is conservative cytokinesis domain of approximately 200 amino acids (D200) in the extracellular part of the receptor. This cytokinesis domain consists of two domains, fibronectin type III (Fnlll), of approximately 100 amino acids each (J.F. Bazan Proc. Natl. Acad. Sci. USA 87, 6934-6938, (1990); Thoreau et al., FEBS Lett. 282, 16-31, (1991)). Each FnIII domain contains conservative residues Cys, Pro, and Trp, which defines the characteristic configuration of stacking seven β-cords, similar to the constant domain of immunoglobulins (Uze et al., J. Interferon Cytokine Res. 15, 3-26 (1995)). These conserved structural members of a family of receptors of cytokines of class II shall allow to identify new members of this family based on homology of the primary amino acid sequence. Previously, the authors of this invention has successfully identified two new members of the family, receptors of cytokines of class II, zytor7 (U.S. patent 5 945 511) (also known as IL-20R α (Blumberg et al., ibid, (2001)) and zcytor11 (U.S. patent 5 965 704) (also known as IL-22R (Blumberg et al., ibid, (2001)) using this approach. Identification of additional new members of the family, receptors of cytokines of class II is of interest, since cytokines play a major role in the regulation of biological reactions.
IL-22, also known as IL-TIF (IL-10-related originating from T cells induced factor) (Dumoutier et al., J.Immunology 164, 1814-1819, (2000)), is a recently described a homologue of IL-10. Mouse IL-22 was originally identified as a gene induced by IL-9 in T cells and mast cells in vitro (Dumoutier et al., J.Immunology 164, 1814-1819, (2000)). Activity induction of the acute phase reactant was observed in the liver of mice by injection of IL-22, and the expression of IL-22 was rapidly induced after injection of lipopolysaccharide (LPS), suggesting that IL-22 contributes to the inflammatory response in vivo (Dumoutier et al., Proc. Natl. Acad. Sci. USA 97, 10144-10149, (2000)).
Interleukins are a family of cytokines, which mediate the immune response, including inflammation. Interleukins mediate a variety of inflammatory pathology. Central to the immune response is a T-cell, which produces many of qi is okine and creates an artificial (acquired) immunity to antigens. Cytokines produced by T-cells were classified as cytokines of Type 1 and Type 2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). The Type 1 cytokines include IL-2, IFN-γ, LT-α and participate in inflammatory reactions, antiviral immunity, immunity against intracellular parasites and allograft rejection. The Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13 and are involved in humoral responses, immunity against helminths and allergic reactions. There is some evidence to suggest that producing cytokines of Type 1 and Type 2 populations of T cells predominantly migrate to different types of inflamed tissue.
Of particular interest, from a therapeutic point of view, are the interferons (reviews by interferons provided De Maeyer and De Maeyer-'guignard, "Interferons," in The Cytokine Handbook, 3rdEdition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998) and Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages 158-188 (John Wiley & Sons 1998)). Interferons exhibit various biological activities and applicable for the treatment of certain autoimmune diseases, in particular cancers, and strengthen the immune response against infectious agents, including viruses, bacteria, fungi and protozoa. To date, identified six forms of interferons, which have been classified into two main groups. The so-called interferons "type I" include the impact interferon-α interferon-βinterferon-ωinterferon-δ and interferon-τ. Currently, interferon-γ and one subclass of interferon-α are the only interferon type II.
The type I interferons, which are thought to originate from the same ancestral gene, were kept fairly similar structure to act through the same receptor cell surface, α-chain of the receptor for interferon-α/β person contains the extracellular N-terminal domain, which has the characteristics of the cytokine receptor class-II. Interferon-γ does not have a significant overall homology with interferon type 1, or a subtype of interferon-α type II, but has a number of common biological activities with interferon type I.
People at least 16 nearely genes encode various subtypes of interferon-αwhereas interferon-β and ω encoded by single genes. Genes of type I interferons are collected in a cluster on the short arm of chromosome 9. In contrast to typical structural genes human genes interferon-α, interferon-β and interferon-ω do not have introns. The only gene for interferon-γ localized on chromosome 12 and contains three introns. To date, interferon-τ was described only in horned cattle and sheep, whereas interferon-δ was described only in pigs.
Clinicians use the advantage of the multiple activities of interferons through the use of these proteins for the treatment of a wide range of conditions. For example, one form of interferon-α was approved for use in more than 50 countries for the treatment of such medical conditions as reticuloendotheliosis, renal cell cancer (renal cell cancer), basal cell carcinoma (basal cell carcinoma), malignant melanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Jackinsky lymphoma, laryngeal papillomatosis, mushroom avium, genital warts, chronic hepatitis b, hepatitis C, chronic hepatitis D and chronic non-a, non-b/C hepatitis. The Department of quality control of food, drugs and cosmetics USA has approved the use of interferon-β for the treatment of multiple sclerosis, a chronic disease of the nervous system, interferon-γ used to treat chronic granulomatous disease, in which the interferon enhances the immune response of the patient to destroy infectious bacteria, fungi and belong to the simplest of pathogens. Clinical studies show that interferon-γ it may be applied in the treatment of AIDS, leishmaniasis and lepromatosis about the basics.
Demonstrated in vivo activity of this family of cytokines illustrate the enormous clinical potential of these cytokines and the need for other cytokines, agonists of cytokines and antagonists of cytokines. This invention satisfies these needs by providing a new cytokine that stimulates the hematopoietic cells of the line of differentiation, as well as related cytokine compositions and methods.
BRIEF description of the GRAPHICAL MATERIAL
Figure 1-5 shows the interferon-like protein Zcyto21.
Figure 6 presents the hydrophilicity profile Noor/Woods Zcyto21 protein sequence shown in SEQ ID NO:2. This profile is based on a sliding window of six residues. Hidden remains of G, S and T and remains open N, Y and W are not taken into account. These balances are shown on Fig.6 lowercase.
DETAILED description of the INVENTION
Before a detailed description of the present invention may be useful for understanding the definition of the following terms:
The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to the second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to the substrate. In principle, any peptide or protein, for which the available ant the body or other specific binding agent, can be used as affinity tags. Affinity tags include polyhistidine area (tract), protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S-transferase (Smith and Johnson, Gene 67:31, 1988), affinity tag, Glu-Glu (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag peptide™ (Hopp et al., Biotechnology 6:1204-10, 1988), strategicinitiatives peptide, or other antigenic epitope or binding domain. See, in General, Ford et al., Protein Expression and Purification 2:95-107, 1991. DNA encoding affinity tags that are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).
The term "allelic variant" is used here to denote any of the two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs through natural mutation and can lead to phenotypic polymorphism in populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is used here to refer to the proteins encoded by allelic variant of a gene.
The terms "amino-terminal" and "carboxyl-terminal" are used here to denote positions within the polypeptide. Where allows context, these terms are used with reference to concr is tnou sequence or part of a polypeptide to indicate proximity or relative position. For example, a certain sequence, located carboxyl-terminal relative to the reference sequence in the polypeptide that is located proximally relative to carboxyl-end reference sequence, but not necessarily located at carboxyl-end of the full polypeptide.
The term "pair complement/anticomplement" denotes non-identical parts of molecules, which form ecovalence associated, stable couple in suitable conditions. For example, Biotin and avidin (or streptavidin) are members-prototypes pairs complement/anticomplement. Other examples of pairs complement/anticomplement include a pair of receptor/ligand pairs antibody/antigen (or hapten or epitope), a pair of sense/antisense polynucleotide, etc. In the case where it is desired subsequent dissociation of pairs complement/anticomplement, a pair of complement/anticomplement preferably has a binding affinity of <109M-1.
The term "compliments polynucleotide molecule" refers to a polynucleotide molecule having a complementary sequence of bases and inverted orientation compared with the reference sequence. For example, the sequence 5′-ATGCACGGG-3′ complementary 5′-CCCGTGCAT-3′.
The term "degenerate nucleotide sequence" means, in sledovatelnot nucleotides, which includes one or more degenerate codons (as compared with a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e. triplets GAU and GAC, each encode Asp).
The term "expressing vector" is used to refer to a DNA molecule, linear or circular, which contains a segment that encodes an polypeptide that is functionally linked to additional segments that provide for its transcription. Such additional segments include promoter and termination sequences, and may also include one or more start points of replication, one or more breeding markers, enhancer, polyadenylation signal, etc. Expressing vectors usually made from plasmid or viral DNA, or may contain elements of both.
The term "isolated", when applied to polynucleotide, indicates that this polynucleotide has been removed from its natural genetic environment and, therefore, does not contain other extraneous or unwanted coding sequences, and is in a form suitable for use in genetically-engineered systems for the production of proteins. Such a dedicated mo is aculy are molecules who are isolated from their natural environment, and include cDNA clones and genomic clones. The selected DNA molecules of the present invention do not contain other genes with which they are usually associated, but may include naturally occurring 5′and 3′-untranslated regions such as promoters and terminators. Identification of related areas will be obvious to a person of ordinary skill in the art (see, for example, Dynan and Tijan, Nature 316:774-78, 1985).
"Isolated" polypeptide or protein is a polypeptide or protein, which is found in conditions other than its natural environment, such as apart from blood and animal tissue. In a preferred form, the selected polypeptide is essentially not containing other polypeptides, particularly other proteins of animal origin. Preferably provision of polypeptides in high purity form, i.e. having a purity of more than 95%, more preferably a purity of more than 99%. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternative glycosylated or derivationally form.
The term "neoplastic", when referred to in relation to cells refers to cells exposed to new and abnormal (anomaly is Oh) proliferation, in particular, in the tissue, where the proliferation is uncontrolled and progressive, leading to neoplasma. The neoplastic cells can be either malignant, i.e. invasive and metastatic or benign.
The term "functionally (resectable) related", when referring to DNA segments, indicates that these segments are arranged so that they operate together for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the coding segment to the terminator.
The term "ortholog" refers to a polypeptide or protein obtained from one species that is the functional copy of the polypeptide or protein from a different species. Differences in sequence among the orthologues are the result of speciation.
"Paralogy" are different, but structurally related proteins produced by the body. It is believed that paralogy arise from gene duplication. For example, α-globin, β-globin and myoglobin are paralogue each other.
The term "polynucleotide" means a single - or double-stranded polymer of deoxyribonucleotide or ribonucleotidic bases read from the 5′-end to 3′-the end. Polynucleotide include RNA and DNA can be isolated from natural sources, synthesized in vitro or p is obtained from a combination of natural and synthetic molecules. The size of polynucleotides are expressed as pairs of nucleotides (abbreviated as "BP"), nucleotide ("NT") or thousands of nucleotides ("TPN"). Where allows context, the last two terms can describe polynucleotide, which are single-stranded or double-stranded. When applying this term to the double-stranded molecules it is used to denote the total length, and it should be clear that it is equivalent to the term "nucleotide pairs". Specialists in this field will be clear that the two strands of double-stranded polynucleotide may differ slightly in length and that their ends can be located step in the enzymatic degradation; thus, not all of the nucleotides in the double-stranded polynucleotide molecule can be paired.
"Polypeptide" is a polymer of amino acid residues connected by peptide bonds, if he by natural or synthetic means. Polypeptides having less than about 10 amino acid residues, usually referred to as "peptides".
The term "promoter" is used here in its recognized in this field the value to denote a part of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are detected usually, but not always, 5߰ -non-coding regions of genes.
"Protein" refers to a macromolecule containing one or more polypeptide chains. Protein can include ones components, such as carbohydrate groups. Carbohydrates and other ones of the components may be attached to a protein by the cell producing the protein, and will vary depending on the cell type. Proteins are defined here in terms of their amino acid structures; alternates, such as carbohydrate groups, usually not specified, but nevertheless, they may be present.
The term "receptor" is used here to denote associated with a cell protein that binds to a bioactive molecule (i.e. a "ligand") and mediates the effect of this ligand on the cell. Membrane-bound receptors are characterized by multipeptide structure containing extracellular legendbase.ui domain and an intracellular effector domain that is typically involved in signal transduction. The binding of ligand to the receptor causes a conformational change in the receptor that causes the interaction between the effector domain and another molecule (molecules) in the cell. This interaction, in turn, leads to changes in cell metabolism. Metabolic events associated with the interactions of receptor-ligand include SEB is the transcription of genes phosphorylation, dephosphorylation, increasing the production of cyclic AMP, mobilization of cellular calcium, mobilization of membrane lipids, cellular adhesion, hydrolysis of insatalled and hydrolysis of phospholipids. Typically, the receptors can be membrane-bound, cytosolic or nuclear; Monomeric (e.g., the receptor for thyroid-stimulating hormone, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, the receptor growth hormone receptor, IL-3 receptor, GM-CSF, the receptor for G-CSF, erythropoietin receptor and the receptor for IL-6).
The term "secretory signal sequence" means a DNA sequence which encodes a polypeptide ("secretory peptide"), which, as a component of a larger polypeptide, directs this larger polypeptide through a secretory pathway cells in which it is synthesized. This larger polypeptide usually split with the destruction of secretory peptide during passage through the secretory pathway.
The term "splicing variant" is used here to refer to alternative forms of RNA transcribed from the gene. Splicing variation occurs through natural use of alternative splicing sites in the transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may lead to several them of mRNA transcribed from the same gene. Splicing variants may encode polypeptides having altered amino acid sequence. The term splicing variant used here to refer to the protein encoded splicing variant mRNA transcribed from the gene.
It should be clear that the molecular weight and length of the polymers, determined deprecationem analytical methods (e.g., gel electrophoresis), are approximate values. When this value is expressed as "about" or "approximately" X, the value X should be understood as defined accurate to ±10%.
All references cited here included by reference in their entirety.
Zcyto21 gene encodes a polypeptide of 200 amino acids shown in SEQ ID NO:2. The signal sequence for Zcyto21 can be predicted as containing amino acid residue 1 (Met) to amino acid residue 19 (Ala) of SEQ ID NO:2. The Mature protein for Zcyto21 begins at amino acid residue 20 (Gly).
Zcyto21 gene contained in the YOU-sequences AU 011445 and AC 018477, which are mapped in the chromosome 19q13.13. This region of chromosome 19 may also contain a cluster of interferon-like genes. The consensus cDNA showing polynucleotide sequence Zcyto21 shown in SEQ ID NO:6, and kodiruemye polypeptide shown in SEQ ID NO:7.
As described below, this invention provides the selected polypeptides having the amino acid sequence that is at least 70%, at least 80% or at least 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acid residues 20-200 SEQ ID NO:2 or amino acid residues 1-200 SEQ ID NO:2. This invention also provides a dedicated polypeptides having the amino acid sequence that is at least 70%, at least 80% or at least 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acid residues 20-219 SEQ ID NO:9, or amino acid residues 1-219 SEQ ID NO:9. This invention also provides a dedicated polypeptides having the amino acid sequence that is at least 70%, at least 80% or at least 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acid residues 20-203 SEQ ID NO:12, or amino acid residues 1-203 SEQ ID NO:12. The invention also includes a polypeptide that further comprises a signal secretory sequence that resides in an amino-terminal position relative to the first amino acid sequence, and this signal secretory sequence contains amino acid residues 1-19 amino acid sequence of SEQ ID NO:2.
In General foreseeable, ivalsa, that cytokines have a four-α-helical structure, and helix a, C, and D are the most important in ligand-receptor interactions and are more highly conserved among other members of this family. However, interferon (INF) and, in particular, interferon-alpha and interferon-Tau, are characterized as shestisparennymi beams. Spiral And interferon equivalent to A spiral Zcyto21; spiral interferon equivalent spiral With Zcyto21; spiral With interferon equivalent helix D Zcyto21 and helix D of interferon equivalent helix F Zcyto 21. Thus, the loop between the AB-loop, CD loop of interferon expanded in Zcyto21 thus to contain a short helix b and E Zcyto21.
Predicted that spiral Zcyto21 are as follows: the spiral And is defined by amino acid residues 49 (Ser) - 63 (Leu); helix B - amino acid residues 76 (Asn) - 84 (Val); helix C - amino acid residues 89 (Val) - 104 (Ala); helix D amino acid residues 111 (Glu) - 133 (Gln); helix E - amino acid residues 137 (Thr) - 158 (Lys) and helix F is amino acid residues 163 (Gly) - 189 (Leu); as shown in SEQ ID NO:2. Residues of cysteine are conservative between Zcyto21 and IFN-α and can form intermolecular disulfide bonds, in particular for education of homodimers with additional molecules Zcyto21. Additional analysis Zcyto21 on the basis set is the result of mapping predicts that cysteine in the provisions of the amino acid residues 34 and 131 and 68 and 164 (as shown in SEQ ID NO:2) will form an intramolecular disulfide bond. The cysteine at residue 190 is free and can form intermolecular disulfide bonds. Appropriate polynucleotide coding regions, domains, motifs, residues and sequences Zcyto21, described here, is shown in SEQ ID NO:1. Degenerate polynucleotide sequence of SEQ ID NO:2 is shown in SEQ ID NO:3. Degenerate polynucleotide sequence of SEQ ID NO:9 is shown in SEQ ID NO:10. Degenerate polynucleotide sequence of SEQ ID NO:12 is shown in SEQ ID NO:13.
Detailed mutational analysis of murine IL-2 (Zurawski et al., EMBO J. 12:5113-5119, 1993) shows that the residues in helices a and C are important for binding to IL-2Rβ; critical residues are Asp34, Asn99and Asn103, Multiple residues in the loop And/or spirals In the murine IL-2 are important for binding to IL-2Rαwhile only a single residue, Gln141in helix D, is essential for binding to IL-2Rα. Similarly, helix a and C are sites of interaction between IL-4 and IL-4Rα (structurally similar to IL-2Rα), and residues within helix D are critical for the interaction of IL-2Rα (Wang et al., Proc. Natl. Acad. Sci. USA 94:1657-1662, 1997; Kruse et al., EMBO J. 11:3237-3244, 1992). In particular, the ICC is of Tyr 124in Asp IL-4 person creates antagonist that binds to IL-4Rαbut not with IL-2Rα and, therefore, cannot transmit a signal (Kruse et al. ibid., 1992).
Cytokines with chetyrehspalnyh beams are grouped also by the length of the component-helices. Cytokines "linopirdine forms consist of spirals with 24-30 residues and include IL-6, ciliary atrofichesky factor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone (hGH). Cytokines "korotkoperiodnoi forms usually consist of helices from residues 18-21 and include IL-2, IL-4 and GM-CSF. Studies using CNTF and IL-6 demonstrate that the spiral CNTF can replace the equivalent helix in IL-6, giving the properties of the binding of CNTF to the Chimera. Thus, apparently, the functional domains chetyrehspalnyh cytokines are determined on the basis of structural homology, regardless of the identity of the sequence, and can maintain the functional integrity in the Chimera (Kallen et al., J.Biol. Chem. 274:11859-11867, 1999). Thus, the helical domains Zcyto 21 will be applied to obtain the chimeric fused molecules, in particular, with other interferons, to determine and modulate the binding specificity of the receptor. Of particular interest are fused proteins, which combine spiral and loop domains of interferons and cytokines, such as IFN-α, IL-10, th the mon human growth.
Zcyto21 mRNA was identified in brain tissue, islets, prostate, testis, pituitary, placenta, of an ovarian tumor, lung tumor, rectal tumor and tumor of the ovary, as well as an activated payline immune cells (CD3+) and lines of epithelial prostate cells that have been transformed by human papillomavirus IV (HPVS).
This invention provides polynucleotide molecules, including DNA and RNA that encode the polypeptides described herein Zcyto21. Specialists in this field will be clear that, due to degeneracy of the genetic code, there may be significant variation among these polynucleotide molecules. SEQ ID NO:3, SEQ ID NO:10 and SEQ ID NO:13 are degenerate DNA sequences that include all of the DNA that encode the Zcyto21 polypeptide SEQ ID NO:2, 9, and 12, respectively. Specialists in this field will be clear that the degenerate sequence of SEQ ID NO:3, for example, provides all RNA sequences encoding SEQ ID NO:2, by substituting U (uracil) T (thymine). Thus, encoding the polypeptide Zcyto21 polynucleotide containing the region from nucleotide 1 or 58 to nucleotide 603 SEQ ID NO:3, and their RNA equivalents are considered by the present invention. Table 1 gives the one-letter codes used within SEQ ID NO:3 to refer to the provisions of degenerate n is cleotides. Permissions represent the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide (complementary nucleotides). For example, the code Y denotes either C or T, and its complement R denotes a or G, and a is complementary to T and G is complementary to C.
Degenerate codons used in SEQ ID NO:3, 10, and 13, which includes all possible codons for a particular amino acid are presented in table 2.
|Amino-Acid||Single-letter code||Codons||Degenerate codon|
|Ser||S||AGC AGT TCA TCC TCG TSC||WSN|
|Thr||T||ACA ACC ACG ACT||CAN|
|Pro||P||CCA CCC CCG CCT||CCN|
|Ala||And||GCA GCC GCG GCT||GCN|
|Gly||G||GGA GGG GGG GGT||GGN|
|Arg||R||AGA AGG GGA GGG GGG GGT||MGN|
|lie||L||ATA ATC ATT||ATH|
|Leu||L||One HUNDRED CTC CTG CTT TTA TTG||YTN|
|Val||V||GTA GTC GTG GTT||GTN|
|Ter||.||TAA TAG TGA||TRR|
The person of ordinary skill in this field will be clear that some ambiguity is introduced in the definition of degenerate codon representing all possible codons encoding each amino acid. For example, the degeneracy of the codon for serine (WSN) may, in some circumstances the x, to encode arginine (AGR), and the degeneracy of the codon for arginine (MGN) may, in some circumstances, encode serine (AGY). A similar relationship exists between the codons encoding phenylalanine and leucine. Thus, some polynucleotides covered by the degenerate sequence may encode variant amino acid sequences, but the person of ordinary skill in this field will be able to easily identify such variant sequences by comparison with the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
The person of ordinary skill in this field will also be clear that the different types can be preferred (preferencyjne) using codons". In General, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al, Curr. Biol. 6:315-24,1996; Wain-Hobson, et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J.Mol. Biol. 158:573-97, 1982. In the application here, the term "preferred (preferencyjne) using codons" or "preferred (preferencyjne) codons" is the term for this area related to the codons of protein translation that is most frequently used in the cells of certain species, giving, therefore, a preference for one or just the representatives of the m possible codons, encoding each amino acid (See. table 3). For example, the amino acid threonine (Thr) can be encoded ACA, ACC, ACG, or ACT, but in mammals the most commonly used codon is ACC; in other species, for example, insect cells, yeast, viruses or bacteria, can be other preferred codons Tnr. Preferred codons for a particular species can be introduced into polynucleotide of the present invention in a variety of ways known in this area. Introduction the preferred sequence of codons in recombinant DNA may, for example, to increase the production of this protein, making more efficient protein translation in a particular cell type or form. Thus, the degenerate sequence of codons described in SEQ ID NO:3, serves as a matrix for optimization of expression of polynucleotides in various cell types and species commonly used in this field and are described here. Sequences that contain preferred codons can be tested and optimized for expression in different species and tested for functionality as described herein.
As previously noted, the selected polynucleotide of the present invention include DNA and RNA. Methods of obtaining DNA and RNA are well known in this field. Typically, RNA is extracted from tissue or cells to which I produces large quantities of RNA Zcyto21. Such tissues and cells identify Northern-blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980) or by screening, air-conditioned environment from different types of cells on the activity of the target cells or target tissue. After identifying the activity or producing RNA of cells or tissue, total RNA can be obtained using extraction isothiocyanato guanidine, followed by separation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+RNA is obtained from the total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) derived from poly (A)+RNA using known methods. Alternatively, it may be isolated genomic DNA. Then polynucleotide encoding polypeptides Zcyto21, identify and distinguish, for example, using hybridization or polymerase chain reaction (PCR).
The longer clone encoding Zcyto21, can be obtained by conventional cloning procedures. Clones complementary DNA (cDNA) are preferred, although for some applications (for example, expressive in transgenic animals) may be preferable to use a genomic clone or modification of the cDNA clone to include at least one genomic intron. Methods for producing cDNA clones and genomic clones of well known and is displayed within the ordinary skill in this field include the use described here, the sequence or its parts for probing or priming of the library. Expression libraries can be probed with antibodies to fragments of the receptor Zcyto21 or other specific binding partners.
Further, this invention provides copies of polypeptides and polynucleotides from other species (orthologues). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fishes, insects, and other species of vertebrates and invertebrates. Of particular interest are the Zcyto21 polypeptides from other mammalian species, including polypeptides mice, pigs, sheep, cows, dog, cat, horse species and polypeptides other primates. Orthologues Zcyto21 person can be cloned using information and compositions provided by the present invention in combination with conventional methods of cloning. For example, cDNA can be cloned using mRNA obtained from a tissue type or cells that Express Zcyto21, as described here. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences described here. Then prepare a library from mRNA positive tissue or cell line. Then the coding Zcyto21 polypeptide cDNA can be isolated in various ways, such as sensing full or partial human cDNA or one or the multiple sets of degenerate probes, based on the described sequences. This cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. patent No. 4 683 202), with a label primers designed as described here are representative sequence Zcyto21 person. In an additional method, the cDNA library can be used for transformation or transfection of host cells, and expression of the cDNA of interest can be detected with an antibody to the polypeptide Zcyto21, research associate or analysis activity. Such methods can be applied also to highlight genomic clones.
Specialists in this field will be clear that the sequence described in SEQ ID NO:1 represents a single allele DNA Zcyto21 person and it is expected that the existence of allelic variation and alternative splicing. Allelic variants of this sequence can be cloned by probing cDNA libraries or genomic libraries from different individuals in accordance with standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, including variants containing silent mutations and variants in which mutations lead to changes in amino acid sequence, are in the scope of the present invention, as well as proteins that is Vlada allelic variants of SEQ ID NO:2. cDNA generated from educated alternative splicing of mRNAs, which retain the properties of Zcyto21 polypeptide, are included in the scope of the present invention, as well as polypeptides encoded by these cDNA and mRNA. Allelic variants and splicing variants of these sequences can be cloned by probing cDNA libraries or genomic libraries from different individuals or tissues according to standard procedures known in the field. Examples of alternative playerowner options shown in SEQ ID NO:8 (SEQ ID NO:9 for the corresponding polypeptide) and in SEQ ID NO:11 (SEQ ID NO:12 for the corresponding polypeptide). Example allelic variant shown in SEQ ID NO:4, which corresponds to the polypeptide sequence shown in SEQ ID NO:5. There is a polymorphism between the polypeptide sequence shown in SEQ ID NO:1, and the sequence shown in SEQ ID NO:4, when the number of nucleotide 572. This polymorphism can create antagonist Zcyto21 or molecule reduced or modified functions, which can lead to a higher probability of susceptibility to the disease.
The invention also provides reagents which will find use in diagnostic applications. For example, Zcyto21 gene, a probe containing DNA or RNA Zcyto21 or suppositionally, can be used on the I definition, is there Zcyto21 gene on the chromosome, such as chromosome 19, or whether there was a gene mutation. Zcyto21 localized in the area q13.13 chromosome 19. Detected chromosomal aberrations in Zcyto21 gene locus include, but are not limited to, aneuploidy, changes in the copy number of the gene, loss of heterogeneity (LOH), translocations, insertions, deletions, and changes to the sites of the restriction and rearrangement. Such aberrations can be detected using polynucleotides of the present invention by application of molecular genetic methods, such as analysis of length polymorphism restriction fragments (RELP), analysis of short tandem repeats (STR) using PCR and other methods of genetic analysis of coupling known in the art (Sambrook et al., ibid; Ausubel et al., ibid; Marian, Chest 108:255-65, 1995).
Exact knowledge of the position of a gene may be useful for a number of purposes, including: 1) determine whether the sequence is part of an existing reference, and more surrounding genetic sequences in various forms, such as YAC, YOU or cDNA clones; 2) provide a possible gene candidates for hereditary diseases, which detects the clutch with the same chromosomal region; and 3) cross-reference model organisms such as the mouse, which can help the AMB definition, what function can have a specific gene.
For example, Delague et al. (Am. J.Hum. Genet. 67:236-243, 2000) identified that the disease Charcot-Marie-Toot (hereditary neural amyotrophy) localized in 19q13.1-13.3 (Delague et al., Am. J.Hum. Genet. 67:236-243, 2000).
Diagnostics can assist physicians in determining the type of disease and appropriate related therapy or aid in genetic counseling. Themselves antibodies against Zcyto21, polynucleotide and Zcyto21 polypeptides can be used to detect the polypeptide, Zcyto21 mRNA or antibody against Zcyto21, serving as markers and can be used directly for detection of genetic diseases or cancers, as described herein, using methods known in this field and are described here. Further, the polynucleotide probes Zcyto21 can be used to detect abnormalities or genotypes associated with deletions and translocations of chromosome 19 is associated with human diseases, or other translocations involved in malignant progression of tumors or other mutations 19q13.13 that, as expected, involved in chromosomal rurangirwa in malignancy; or in other cancers. Similarly, polynucleotide probes Zcyto21 can be used to detect abnormalities or genotypes associated with trisomy 19q13.13 and sweat the Rey chromosomes, associated with human diseases or spontaneous abortion. Thus, the polynucleotide probes Zcyto21 can be used to detect abnormalities or genotypes associated with these defects.
In General, the diagnostic methods used in the genetic analyses clutch for detection of genetic disorders or abnormalities in the patient's well-known in this field. Analytical probes will usually have a length of at least 20 BP, although can be used several shorter probes (e.g., 14-17 P.N.). PCR primers have a length of at least 5 NT, preferably 15 or more, even more preferably 20-30 NT. For macroscopic analysis of genes or chromosomal DNA polynucleotide probe Zcyto21 can contain the full exon or a large part of the gene. The exons can be easily determined by a specialist with expertise in this area by comparison of sequences of Zcyto21 (SEQ ID NO:1) with the genomic DNA for Zcyto21. Usually diagnostic methods used in the analysis of the energetic coupling to determine deviations from the norm or aberrate the patient, known to specialists in this field. In General, these diagnostic methods include the stage of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potentially not having a disease carrier p is casinogo allele of the disease; (b) receiving a first reaction product by incubating this genetic sample with a polynucleotide probe Zcyto21, and this polynucleotide will gibridizatsiya with complementary polynucleotide sequence, for example, RFLP analysis, or by incubation of this genetic samples from semantic or antimuslim primers in appropriate PCR reaction conditions; (iii) visualizing the first reaction product using gel electrophoresis and/or other known method, for example, visualizing the first reaction product with a polynucleotide probe Zcyto21, where this polynucleotide will gibridizatsiya with complementary sequence of the first reaction; and (iv) comparing the visualized first reaction product with the second product control reaction genetic sample from the patient the wild type. The difference between the first reaction product and the product of the control reaction indicates a genetic deviation from the norm in ill or potentially ill patient, or the presence of heterozygous recessive phenotype carrier for metabolises patient or the presence of a genetic defect in a tumor from a diseased patient, or the presence of genetic abnormalities in the fetus or preimplantation embryo. For example, the difference in the picture of restriction fragments, the length of the PCR is Reducto, the length of repetitive sequences in the genetic locus Zcyto21, etc. are indicators of genetic abnormalities, genetic aberration, or allelic difference in comparison with normal controls, wild-type. Controls can be unaffected family members or unrelated individuals, depending on the test and the availability of samples. Genetic samples for use in this invention include genomic DNA, mRNA and cDNA isolated from any tissue or other biological sample from the patient, such as, but not limited to, blood, saliva, semen, embryonic stem cells, amniotic fluid, etc. Polynucleotide probe or primer can be RNA or DNA, and it will contain a portion of SEQ ID NO:1, the complement SEQ ID NO:1 or its RNA equivalent. Such methods of analysis of the results of genetic clutch relative to the phenotypes of human diseases is well known in this field. In reference to the ways in diagnostics based on PCR, see, in General, Mathew (ed.) Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.) Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.) Clinical Applications of PCR (Humana Press, Inc. 1998) and Meltzer (ed.) PCR in Bioanalysis (Humana Press, Inc. 1998)).
Mutations associated with a locus Zcyto21, can be detected using nucleic acid molecules to the slot of the present invention through the application of standard methods for direct mutation analysis, such as the analysis of the polymorphism of the lengths of restriction fragments, the analysis of short tandem repeats using PCR, the amplication analysis system identification of mutations, detection of polymorphism single-stranded conformation, methods of cleavage by RNase, denaturing gradient gel electrophoresis, fluorescence analysis of incorrect mating and other genetic methods of analysis known in the art (see, for example, Mathew (ed.) Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995). Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.) Laboratory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al., (eds.), Genome Analysis, Vol.2: Detecting Genes (Cold Spring Harbor Laboratory Press, 1998), Dracopoli et al., (eds.), Current Protocols in Human Genetics (John Wiley & Sons, 1998) and Richards and Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998)). Direct analysis of Zcyto21 gene for a mutation can be performed using the genomic DNA of the subject. Methods amplification of genomic DNA, obtained, for example, from peripheral blood lymphocytes, well known to specialists with expertise in this area (see, for example, Dracopoli et al., (eds.), Current Protocols in Human Genetics, pages 7.1.6-7.1.7 (John Wiley & Sons 1998)).
In embodiments of the present invention, the selected encoding Zcyto21 molecules of nucleic acids can gibridizatsiya in stringent conditions with the nucleic acid molecules Ki the lot, having the nucleotide sequence of SEQ ID NO:1, nucleic acid molecules having the nucleotide sequence of nucleotides 58-603 SEQ ID NO:1, or nucleic acid molecules having a nucleotide sequence complementary to SEQ ID NO:1. Typically, stringent conditions are chosen so that the temperature was about 5°With lower than point thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tmis the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridize with exactly compatible probe.
A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can gibridizatsiya if these nucleotide sequences have some degree of complementarity. Hybrids can tolerate incorrectly paired base pairs in the double helix, but the degree of erroneous mating affects the stability of this hybrid. Tmmistakenly paired hybrid is reduced by 1°for every 1-1,5% erroneous mating grounds. Varying the stringency conditions of hybridization allows to control the degree of erroneous pairing, which will be present in the hybrid. The severity increases with the temperature of hybridization and decreasing ion is th power buffer for hybridization.
Specialist with expertise in this area may well be able to adapt these conditions to apply to a specific polynucleotide hybrid. Tmfor a particular target sequence is the temperature (under certain conditions), at which 50% of the target sequence will be gibridizatsiya exactly compatible probe. These conditions, which affect Tminclude the size and content of base pairs of polynucleotide probe, ionic strength of the hybridization solution and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating the Tmknown in this field and are specific for DNA, RNA, and DNA-RNA hybrids and polynucleotide sequences of the probes of varying length (see, for example, Sambrook et al. (Molecular Cloning. A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Software for sequence analysis, such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet are the available tools for the analysis of specific sequences and calculation of Tmbased on user-defined criteria. Such programs can also analyze the encoded specific sequence under certain conditions and to identify the appropriate sequence of probes. Typically, hybridization of a longer polynucleotide sequences, >50 base pairs, performed at temperatures of approximately 20-25°calculated below Tm. For smaller probes, <50 base pairs, hybridization is usually carried out at Tmor 5-10°calculated below Tm. It allows to reach a maximum speed of hybridization for hybrids, DNA-DNA and DNA-RNA.
After hybridization of the nucleic acid molecule can be washed to remove dehybridization molecules of nucleic acids in severe conditions or in conditions of high stringency. Typical stringent washing conditions include washing in a solution of 0.5x-2x SSC with 0.1% sodium dodecyl sulfate (LTOs) at 55-65°C. That is, nucleic acid molecule encoding a variant peptide Zcyto21, hybridize with nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement) under strict conditions of leaching, where the severity of flushing is equivalent to 0.5x-2x SSC with 0.1% LTOs at 55-65°including 0.5x SSC with 0.1% LTOs at 55°With or 2x SSC with 0.1% LTOs at 65°C. Specialist in this field can easily create equivalent conditions, for example, replacement of the SSC using the SSPE in the washing solution.
Typical conditions of high stringency include washing in a solution of 0,1-0,2x SSC with 0.1% dodecanal the blockhead sodium (LTOs) at 50-65° C. in Other words, the molecules of nucleic acids encoding the variant peptide Zcyto21, hybridize with nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement) under conditions of leaching high severity, the rigor washing equivalent to 0,1x-0,2x SSC with 0.1% LTOs at 50-65°including 0,1x SSC with 0.1% LTOs at 50°S, or 0,2x SSC with 0.1% LTOs at 65°C.
This invention also provides a dedicated Zcyto21 polypeptides, which have essentially the same sequence identity with the polypeptide SEQ ID NO:2 and their orthologues. The term "essentially similar sequence identity" is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95%or greater than 95%, 96%, 97%, 98%or 99%, sequence identity to the sequences shown in SEQ ID NO:2, or their orthologues. The invention also includes polypeptides that contain amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%or greater than 95%, 96%, 97%, 98%or 99%, sequence identity with the sequence of amino acid residues 1-200 or 20-200 SEQ ID NO:2. The invention includes includes additional molecules of nucleic acids, which encode such polypeptides. Methods for determining percent identity are described below.
This invention also considers a variant nucleic acid molecule Zcyto21, which can be identified using two criteria: a determination of the similarity between the encoded polypeptide and the amino acid sequence SEQ ID NO:2 and/or hybridization analysis described above. Such options Zcyto21 include molecules of nucleic acids: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement)under stringent washing conditions, where stringent washing is equivalent to 0.5x-2x SSC with 0.1% LTOs at 55-65°With; or (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%-ing to or greater than 95%, 96%, 97%, 98%or 99%, sequence identity with the amino acid sequence SEQ ID NO:2. Alternative options Zcyto21 can be characterized as molecules of nucleic acids: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement)under the washing conditions of high stringency, the rigor washing equivalent to 0,1x-0,2x SSC with 0.1% LTOs at 50-65°With; or (2) that encode polypeptide, having at least 70%, at least 80%, at least 90%, at least 95%or greater than 95%, sequence identity with the amino acid sequence SEQ ID NO:2.
The percentage sequence identity define common ways. See, for example, Altschul et al., Bull. Math, Bio. 48:603 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:of 10,915 (1992). Briefly, two amino acid sequences map building for optimization assessments mapping using a fine (penalty) opening gap 10 and the fine (penalty) extension gap 1 and the evaluation matrix "BLOSUM62" Henikoff and Henikoff (ibid.), a shown in table 3 (amino acids are represented using standard one-letter codes).
|V||0||-3td align="center"> -3||-3||-1||-2||-2||-3||-3||3||1||-2||1||-1||-2||-2||0||-3||-1||4|
Professionals in this field should be clear that there are many established algorithms for comparing two amino acid sequences. The search algorithm of similarity "FASTA" Pearson and Lipman is a suitable method for mapping proteins to study the level of identity, which is described here have in common amino acid sequences and amino acid sequences of possible options Zcyto21. The algorithm FASTA described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), and Pearson, Meth. Enzymol. 183:63 (1990).
Briefly, FASTA first characterizes sequence similarity by identifying regions shared the ask (query) sequence (for example, SEQ ID NO:2) and a test sequence that have the highest density of any of identities (if the ktup variable is 1)or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions or deletions. Then the ten areas with the highest density of identities re-evaluate by comparing the similarities of all pairs of amino acids using the W matrix of amino acid substitutions and the ends of these areas "straighten" to include only those residues, which contribute the highest evaluation. If there are several areas with grades lower than "limit" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the order given in the original trim areas are examined to determine whether or not these areas are to be connected with the formation of approximate matching with gaps. Finally, the areas with the highest rating of two amino acid sequences is correlated with the use of a modification of the algorithm of Needleman-Wunsch-Sellers (Needleman and Wunsch, J.Mol. Biol. 48:444 (1970); Sellers, SIAM J.Appl. Math. 26:787 (1974)), which makes possible insertions and deletions of amino acids. The preferred parameters for FASTA analysis are: ktup=1, the penalty (penalty) opening gap=10, penalty (penalty) extension gap=1 and the matrix of substitution=BLOSUM62. These parameters can be entered into the program FASTA-file modification evaluation matrix ("SMATRIX"), as explained in Appendix 2 Pearson, Meth. Enzymol. 183:63 (1990).
The program FASTA can be used to determine the sequence identity of the nucleic acid molecules using the above relations. For comparisons of nucleotide sequences of the ktup value can be between 1 and 6, preferably from 3 to 6, most preferably 3, with other parameters set p is the default.
Variant Zcyto21 polypeptides or polypeptides with essentially the same identity sequences are characterized as having one or more amino acid substitutions, deletions or accessions. These changes are preferably are of a minor nature, that is conservative replacement amino acids (see table 4) and other substitutions that do not affect significantly to the installation or activity of the polypeptide; small deletions, typically of one to ˜30 amino acids; and small amino - or carboxyl-terminal extension, such as amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or affinity label. Thus, the invention includes polypeptides of from about 149 to about 230 amino acid residues that comprise a sequence that is at least 70%, preferably at least 90% and more preferably at 95%, 96%, 97%, 98%, 99% or more identical to the corresponding region SEQ ID NO:2. Polypeptides containing affinity tags can further comprise the site of proteolytic cleavage between Zcyto21 polypeptide and this affinity tag. Preferred such sites include the sites of cleavage by thrombin and the sites of cleavage by factor XA.
|Conservative substitutions of amino acids|
Can be made identify amino acid residues that make up the regions or domains that are critical for the preservation of structural integrity. In these areas it is possible to identify specific residues, which will be more or less resistant to change and preserve the overall tertiary structure of the molecule. Methods of analysis of the structure of the sequence include, but are not limited to, mapping multiple p is sledovatelnot with high identity with amino acids or nucleotides, the tendency to form secondary structures, binary distribution, complementary packing and hidden polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). Usually, when designing modifications to molecules or to identify specific fragments of the structure definition will be accompanied by an estimation of activity of modified molecules.
Changes in amino acid sequence performed in the Zcyto21 polypeptides so as to minimize the destruction of the structure of a higher order, which are essential for biological activity. For example, when the Zcyto21 polypeptide contains one or more helices, changes in amino acid residues will be carried out in such a way as not to interfere with the geometry of the coils and other components of the molecule where changes in conformation reduce some critical function, for example, the binding of this molecule to its binding partners. The effects of changes in amino acid sequence can be predicted, for example, computer modeling, as described above, or determined by analysis of crystal structure (see, for example, Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other methods that are well known in the field, compare the packing variant protein with a standard molecule (e.g., native Bel who ohms). For example, can be made by comparing the distribution of cysteine in variance and standard molecules. Mass spectrometry and chemical modification using recovery and alkylation provide methods for determination of cysteine residues, which are linked by disulfide bonds or free from such relationships (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993, and Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is usually assumed that, if modified molecule does not have the same distribution cysteines of the standard molecule, stacking may be compromised. Another well-known and accepted method of measuring styling is a circular dichroism (CD). The measurement and comparison of the CD spectra generated by a modified molecule and the standard molecule is routine (Johnson, Proteins 7:205-214, 1990). Crystallography is another well-known method for the analysis of styling and patterns. Nuclear magnetic resonance (NMR), enzymatic peptide mapping and mapping of epitopes are also known methods for the analysis of similarity styling and patterns between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
Can be obtained profile hydrophilicity Hopp/Woods protein sequence Zcyto21 shown in SEQ ID NO:2 (Hopp et al., Proc. Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J.Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering 11:13-169, 1998). This profile is based on a sliding window of six residues. Hidden remains of G, S and T and exposed (open) residues of N, Y and W are not taken into account. For example, Zcyto21 hydrophilic regions include residues 155 (Glu) - 160 (Glu); residues 51 (Lys) - 56 (Ala); residues 50 (Phe) - 55 (Asp); the remains of 140 (Pro) - (145 (Arg); and residues 154 (Gln) - 159 (Lys), as shown in SEQ ID NO:2.
Specialists in this field will be clear that the hydrophilicity or hydrophobicity should be taken into account when designing modifications in the amino acid sequence of Zcyto21 polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest to replace represent hydrophobic residues selected from the group consisting of Val, Leu and Ile, or from the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.
The identities of essential amino acids can also be inferred from analysis of sequence similarity between IFN-α and other interferons. Using techniques such as analysis of "FASTA", described above, regions of high similarity identify the family of proteins and is used for the analysis of amino acid sequences for conservative districts. An alternative approach to the identification of variant polynucleotide Zcyto21 on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Poliny eated Zcyto21, to gibridizatsiya with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, as discussed above.
Other methods of identification of essential amino acids in the polypeptides of this invention are known in this field, such as site-directed mutagenesis or Leninskoye mutagenesis (Cunningham and Wells, Science 244:1081 (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering" in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter method, single alanine mutations introduced at every residue in the molecule, and the resulting mutant molecules are tested for biological or biochemical activity as described below, to identify amino acid residues that are critical for activity of this molecule. Cm. also Hilton et al., J.Biol. Chem. 271:4699 (1996).
This invention relates also to functional fragments of Zcyto21 polypeptides and nucleic acid molecules, encoding such functional fragments. "Functional" Zcyto21 or its fragment, defined here differs in its proliferative and differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to specifically bind with an antibody against Zcyto21 or receptor Zcyto21 (the solution is known or immobilized). As described previously, Zcyto21 characterized by the structure Shestopalova beam, comprising: a spiral And which is defined by amino acid residues 49 (Ser) - 63 (Leu); helix B - amino acid residues 76 (Asn) - 84 (Val); helix C - amino acid residues 89 (Val) - 104 (Ala); helix D amino acid residues 111 (Glu) - 133 (Gln); helix E - amino acid residues 137 (Thr) - 158 (Lys); and helix F is amino acid residues 163 (Gly) - 189 (Leu); as shown in SEQ ID NO:2. Thus, the invention additionally provides fused (hybrid) protein, comprising: (a) polypeptide molecules containing the above-described one or more coils; and (b) functional fragments containing one or more of these spirals. The other polypeptide portion of the fused protein may consist of another cytokine with a bunch of spirals or interferon, such as IFN-αor non-natural and/or an unrelated secretory signal peptide that facilitates secretion of this fused protein.
Zcyto21 polypeptides of the present invention, including full-sized polypeptides, biologically active fragments and fused polypeptides may be produced in accordance with conventional methods using cells, which was introduced in expressing vector encoding the polypeptide. In the application here, "cell, which was introduced in E. spressure vector include both cells, which directly manipulated through the introduction of exogenous DNA molecules and their offspring, which contains the introduced DNA. Appropriate cell hosts are those the types of cells that can be transformed or transliterowany exogenous DNA and grown in culture, and they include bacteria, fungal cells and cultivated in higher eukaryotic cells. Ways of manipulating cloned DNA molecules and introducing exogenous DNA into a variety of cell hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, (2nd ed.) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
In General, the DNA sequence encoding the polypeptide Zcyto21, functionally linked to other genetic elements required for its expression, generally including the promoter and terminator of transcription in expressing vector. This vector will typically contain one or more breeding markers and one or more points of initiation of replication, although experts in the field will be clear that in some systems of breeding markers may be provided on separate vectors, and the replication of exogenous DNA can be achieved by integration into the genome of the host cell. The choice of promoters, terminators, breeding markers in the Ktorov and other elements is a matter of routine design within the ordinary skill in this field. Many of these elements are described in the literature and are available through commercial suppliers.
For Zcyto21 polypeptide in the secretory path of the host cell, expressing the vector provide a secretory signal sequence (also known as a leader sequence, a pre-proposedvalue or pre sequence). The secretory signal sequence may be a signal sequence Zcyto21, or may be derived from another secreted protein (e.g., t-PA (tissue plasminogen activator, see U.S. patent No. 5 641 655) or synthesized de novo. The secretory signal sequence is functionally linked to a DNA sequence Zcyto21, i.e. these two sequences are joined in the correct reading frame and is correctly placed for the direction of the newly synthesized polypeptide into the secretory path of the host cell. Secretory signal sequences are usually located 5′ (left) from the DNA sequence that encodes an polypeptide, although certain signal sequences may be positioned elsewhere in the interest of the DNA sequence (see, e.g., Welch et al., U.S. Patent No. 5 037 743; Holland et al., U.S. Patent No. 5 143 830).
Cultured mammalian cells can be used in quality is as masters in this invention. Methods of introducing exogenous DNA into cells-mammalian hosts include mediated by calcium phosphate transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), mediated DEAE-dextran transfection (Ausubel et al., ibid.) and mediated by liposomes transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The preparation of recombinant polypeptides in cultured mammalian cells are described, for example, Levinson et al., U.S. Patent No. 4 713 339; Hagen et al., U.S. Patent No. 4 784 950; Palmiter et al., U.S. Patent No. 4 579 821; and Ringold, U.S. Patent No. 4 656 134. Suitable kulturama mammalian cells include cell lines COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J.Gen. Virol. 36:59-72, 1977) and cell line of Chinese hamster ovary (e.g., Cho-K1; ATCC No. CCL 61 or Cho DG44, Chasm et al., Som. Cell. Molec. Genet. 12:555, 1986). Additional suitable cell lines are known in this field and available from public depositories such as the American type culture collection (American Type Culture Collection, Manassas, VA). In General, preferred strong promoters of transcription, such as promoters from SV-40 or cytomegalovirus. See, for example, U.S. patent No. 4 956 288. Other suitable promoters include the promoters from the genes of metallothionein (U.S. Patent No. 4 579 821 and U.S. Patent No. 4 601 978) and the main on dni promoter of adenovirus. Expressing vectors for use in mammalian cells include pZP-1 and pZP-9, which were deposited with the American type culture collection (American Type Culture Collection, Manassas, VA) under access numbers 98669 and 98668, respectively, and their derivatives.
The selection of the drug is usually used for screening in cultured mammalian cells, in which was embedded with alien DNA. These cells commonly called "the transfectants". Cells that were cultured in the presence of the selective agent and is able to convey the interest of the gene to their offspring, called "stable transfectants". Preferred breeding marker is a gene encoding resistance to the antibiotic neomycin. The selection is carried out in the presence of drug type neomycin, such as G-418 or the like, the selection System may also be used to increase the level of expression of the gene of interest, method, called "amplification". Amplification spend the cultivation of transfectants in the presence of low levels of the selective agent and then increase the amount of selective agent for selection of cells that produce high levels of products introduced genes. Preferred amplificare breeding marker is dihydrotetrazolo that together the t cells resistant to methotrexate. Other genes of drug resistance (e.g. resistance to hygromycin, multiple drug resistance, parameterizedthreadstart) can also be used.
Adenovirus system can also be used to obtain the protein in vitro. By culturing infected with adenovirus cells-293 under conditions in which cells do not carry out rapid division, these cells can produce proteins for extended periods of time. For example, cells KSS grown to confluently in cell factories (cassettes bioreactors for large-scale production of cells) and then subjected to the action of adenoviral vector encoding interest secretory protein. Then these cells are grown in serum-free conditions that allow infected cells to survive for several weeks without significant cell division. In an alternative method, infected with adenoviral vector in 293 cells can be grown in the form of attached cells or in suspension culture with a relatively high density of cells to obtain significant quantities of the protein (See. Garnier et al., Cytotechnol. 15:145-55, 1994). Any Protocol expressed, heterologous secretory protein can be periodically extracted from superna the Anta, lysate or membrane fractions of cell culture depending on the placement of the expressed protein in the cell. In the Protocol producing infected 293 cells can also be obtained secretiruemy proteins.
Based on insect cells can be infected with recombinant baculovirus, usually produced from nuclear polyhedrosis virus Autographs californica (AcNPV) in accordance with methods known in this field. In a preferred method, the recombinant baculovirus obtained using system-based transposon described by Luckow et al. (J.Virol. 67:4566-4579, 1993). This system, which uses vectors-vectors, is commercially available in the form of a set (Bac-to-Bac™ Kit; Life Technologies, Rockville, MD). This vector is a vector (for example, pFastBac 1™; Life Technologies) contains the transposon TP to move DNA that encodes a protein of interest, in a baculovirus genome maintained in E. coli, as a large plasmid called a "bacmids". Cm. Hill-Perkins and Possee, J.Gen. Virol. 71:971-976. 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994 and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, vectors-vectors can contain in reading frame with the DNA encoding the polypeptide, elongation or affinity tag as described above. Using methods known in this field, vector-vector containing the coding Zcyto21 sequence, transform in to EDI host E. coli and the cells are screened for bacmid, which contain an interrupted lacZ gene indicative of recombinant baculovirus. Backmenu DNA containing the recombinant baculovirus genome, isolated by known methods and used for transfection of cells Spodoptera frugiperda, such as Sf9 cells. Then get the recombinant virus expressing the protein Zcyto21. Source materials recombinant virus prepared by the methods usually used in this field.
To obtain this protein recombinant virus is used to infect host cells insects, usually cell lines derived from autumn "field (military) worms, Spodoptera frugiperda (e.g., cells Sf9 or Sf21) or Trichoplusia ni (for example, cells High Five™, Invitrogen, Carlsbad, CA). See, for example, U.S. patent No. 5 300 435. For growing and maintaining these cells using serum-free medium. Suitable compositions environments known in the field and can be obtained from commercial suppliers. Cells grown from the inoculation density of approximately 2-5×105cells to a density of 1-2×106cells, at this point add the source material recombinant virus at a multiplicity of infection (MOI) of 0.1-10, more often about 3. Procedures generally known in this field.
Other higher securitiesa cells can also be used as hosts, t is the number of cells of plants and bird cages. Application Agrobactehum rhizogenes as a vector for gene expression in plant cells considered in the review Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
Fungal cells, including yeast cells, can also be used in this invention. The species of yeast, of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Methods of transformation of S.cerevisiae cells exogenous DNA and obtaining from them the recombinant polypeptides are described, for example, Kawasaki, U.S. Patent No. 4 599 311; Kawasaki et al., U.S. Patent No. 4 931 373; Brake, U.S. Patent No. 4 870 008; Welch et.al., U.S. Patent No. 5 037 743 and Murray et al., U.S. Patent No. 4 845 075. Transformed cells are selected based on phenotype, defined according to selected marker, usually in terms of resistance to drug or ability to grow in the absence of specific nutrients (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is a vector system NOT described by Kawasaki et al. (U.S. Patent No. 4 931 373), which allows to select the transformed cells on growth in containing glucose environment.
Suitable promoters and terminators for use in yeast include promoters and terminators of the genes of the glycolytic enzymes (see, for example, Kawasaki, U.S. Patent No. 4 599 311; Kingsman et at., U.S. Patent No. 4 615 974 and Bitter, U.S. Patent No. 4 977 092) and alcohol dehydrogenase genes. Cm. also Pat the options U.S. No. 4 990 446, 5 063 154, 5 139 936 and 4 661 454. System transformation for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa, known in this area. See, for example, Gleeson et al., J.Gen. Environ. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4 882 279 and Raymond et al., Yeast 14, 11-23, 1998. Cells of Aspergillus can be used in accordance with the methods McKnight et al., U.S. Patent No. 4 935 349. Methods of transformation of Acremonium chrysogenum described Sumino et al., U.S. Patent No. 5 162 228. Methods of transformation of Neurospora described Lambowitz, U.S. Patent No. 4 486 533. The preparation of recombinant proteins in Pichia methanolica is described in U.S. patent№№5 716 808, 5 736 383, 5 854 039 and 5 888 768.
Prokaryotic cells-owners, including strains of the bacteria Escherichia coli, Bacillus and other genera are also applicable cells masters in this invention. Ways of transforming these hosts and expression of cloned them alien DNA are well known in the art (see, for example, Sambrook et al., ibid.). When the expression of Zcyto21 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to periplasmatic space of a bacterial secretion sequence. In the first case, these cells are lysed and the granules are recovered and denatured using, for example, guanidinosuccinic or urea. Then dena is rirovanie polypeptide can be re-laid and dimerizer by diluting the product denaturation, for example, by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against buffered saline. In the latter case, the polypeptide can be extracted from periplasmatic space in a soluble and functional form by the destruction of these cells (for example, by sonication or osmotic shock) to release content periplasmatic space and extract this protein, whereby eliminating the need for denaturation and re-installation.
Transformed and transfetsirovannyh cell hosts are cultivated in accordance with conventional procedures in a culture medium containing nutrients and other components required for growth of the selected host cells. Various suitable environment, including the environment with a certain composition of the medium and complex environment, known in the field and typically include a carbon source, a nitrogen source, essential amino acids, vitamins and mineral compounds. The medium may also contain components such as growth factors or serum if required. The growing medium is usually to make a selection for cells containing exogenously added DNA, for example through selection using medical environments is TBA or deficiency of essential nutrients, complemented breeding marker carried on expressing vector or cotransfected cells of the host. Liquid cultures provide sufficient aeration using conventional means, such as shaking a small flasks or bubbling fermentors.
It is preferable to purify the polypeptides of the present invention to ≥80% purity, more preferably to ≥90% purity, even more preferably up to ≥95% purity, and particularly preferably up to pharmacologically pure state, i.e. to purity more than 99.9%in respect of contaminating macromolecules, particularly other proteins and nucleic acids, and infectious and pyrogenic agents. Preferably, the purified polypeptide is essentially not containing other polypeptides, particularly other proteins of animal origin.
Expressed recombinant Zcyto21 polypeptides (including chimeric polypeptides and multimeric proteins) are purified by using common methods protein purification, usually by a combination of chromatographic methods. See, in General, Affinity Chromatography: Principles &Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994. Proteins containing polyhistidine affinity tag (usually around 6 histidine residues), purified by affinity chromatography on a resin with Nickel chelate. See, e.g. the measures Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins containing the tag, Glu-Glu, can be cleaned immunoaffinity chromatography in accordance with generally accepted procedures. See, for example, Grussenmeyer et al., ibid. Malosorbatsiya proteins purified on amylose column in accordance with methods known in this field.
Zcyto21 polypeptides can also be obtained by chemical synthesis according to methods known in this field, including the use of exclusive (i.e. only) solid-phase synthesis, partial solid phase methods, condensation of fragments or classical synthesis in solution. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2ndedition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986 and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. Synthesis in vitro is particularly preferred to obtain a small polypeptides.
Using methods known in this field, Zcyto21 proteins can be obtained in the form of monomers or multimers; glycosylated or deglycosylated; paglierani or naegeliana and may contain or may not contain the original residue of the amino acid methionine.
Target cells for use in assays of activity Zcyto21 include, without limitation, vascular cells (in particular, endothelial cell and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, having numerous holes endothelial cells, Kupffer cells and ito cells), fibroblasts (including dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocyte, pericyte, chondrocytes, osteoblasts and epithelial cells of the prostate. Endothelial cells and hematopoietic cells are derived from a common ancestor cell, hemangioblast (Choi et al., Development 125:725-732, 1998).
Zcyto21 proteins of this invention are characterized by their activity, i.e. the modulation of cell proliferation, differentiation, migration, adhesion or metabolism of the respective cell types. The biological activity of Zcyto21 proteins analyzed using in vitro or in vivo, can be used to detect the proliferation, differentiation, migration or adhesion of cells; or changes in cellular metabolism (e.g., the production of other growth factors or other macromolecules). Numerous suitable assays known in the field and representative analyses are described here. Assays using cultured cells are most suitable for screening, for example, to determine the effects of amino acid substitutions, deletions or insertions. However, due to the complex nature of the process and the owls development (for example, angiogenesis, wound healing) usually must be used in vivo analyses for confirmation and further characterization of the biological activity. Some models in vitro, such as the model of a three-dimensional matrix of collagen gel Pepper et al. (Biochem. Biophys. Res. Comm. 189:824-831, 1992), are quite complicated to analyze histological effects. Analyses can be performed with the use of exogenous proteins obtained or may be conducted in vivo or in vitro using cells expressing interest polypeptide or polypeptides. Analyses can be conducted using Zcyto21 proteins alone or in combination with other growth factors such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative analyses are described below.
Activity Zcyto21 proteins can be measured in vitro using cultured cells or in vivo introduction of the molecules of the present invention to the appropriate animal models. Assays measuring proliferation or differentiation of cells, well known in this field. For example, assays measuring proliferation include such analyses, such as analysis of homocysteine to the dye neutral red (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radioactively labeled nucleotides (as described, for example, Raines andRoss, Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988, Cook et al., Analytical Biochem. 179:1-7, 1989), the inclusion of 5-bromo-2′-dose irradiation on neurogenesis (BrdU) into the DNA of proliferating cells (Porstmann et al., J.Immunol. Methods 82:169-179) and the use of salts of tetrazole (Mosmann, J.Immunol. Methods 65:55 to 63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995 and Scudiero et al., Cancer Res. 48:4827-4833, 1988). Differentiation can be tested using suitable precursor cells that can be induced to differentiation into more Mature phenotype. Tests that measure differentiation include, for example, measurement of cell surface markers associated with statespecific the expression of tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989, all incorporated herein by reference).
Activity Zcyto21 can also be detected using assays developed to measure induced Zcyto21 producing one or more additional growth factors or other macromolecules. Such preferred assays include assays to determine the presence of growth factor hepatocyte (HGH), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), interleukin-6 (IL-6), VEGF, acidic growth factor fibroblast is in (aFGF), angiogenin and other macromolecules produced by the liver. Suitable tests include tests of mitogenesis using target cells that respond to interest the macromolecule, analyses of receptor binding, analysis of competitive binding, immunological tests (e.g., ELISA) and other formats known in the field. Measure the secretion of metalloprotease of processed primary skin fibroblasts, synoviocytes and chondrocytes. The relative levels of collagenase, gelatinase and stromelysin produced in response to culturing in the presence of Zcyto21 protein, measured using zymography gels (gels for electrophoresis, in which the position of the enzyme is detected by the reaction, which depends on its enzymatic activity) (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990)). Synthesis of procollagen/collagen dermal fibroblasts and chondrocytes in response to the test protein is measured using include3H-Proline in nascent (emerging) secretory collagen,3H-labeled collagen visualize using electrophoresis in LTO-SDS page followed by autoradiography (Unemori and Amento, J.Biol. Chem. 265:10681-10685, 1999). The secretion of glycosaminoglycans and collagen (GAG) from dermal fibroblasts and chondrocytes was measured by analysis of binding dye 1,9-dimethylmethylene is inim (Farndale et al., Biochim. Biophys. Acta 883:173-177, 1986). Analyses of collagen and GAG carried out in the presence of IL-1α or TGF-α to test the ability of the protein Zcyto 21 to modify the set of responses to these cytokines.
Analyses of the activation of monocytes conduct (1) to determine the ability of Zcyto21 proteins additionally stimulate the activation of monocytes and (2) ability tests Zcyto21 proteins to modulate induced by attaching or induced by endotoxin activation of monocytes (Fuhlbrigge et al., J.Immunol. 138:3799-3802, 1987). The levels of IL-1α and TNFαproduced in response to activation, measured using ELISA (Biosource, Inc. Camarillo, CA). Monocytes/macrophages, thanks CD14 (LPS receptor), are extremely sensitive to endotoxin and proteins with moderate levels of endotoxin-like activity will activate these cells.
Hematopoietic activity Zcyto21 proteins may be tested on a variety of hematopoietic cells in culture. Preferred assays include assays of primary colonies of bone marrow and analyses linespecific restrictively colonies later stage of differentiation, which are known in this field (for example, Holly et al., WIPO Publication WO 95/21920). Bone marrow cells, seeded at a suitable semi-solid medium (for example, 50% methylcellulose containing 15% fetal calf serum, 10% bovine sivaraman the th albumin and 0.6% of a mixture of antibiotics PSN), incubated in the presence of a test polypeptide, and then examined microscopically for the formation of colonies. As controls use a known hematopoietic factors. Mitogenic activity of Zcyto21 polypeptides on the lines of hematopoietic cells can be measured as described above.
The migration of cells analyzed essentially as described Kä'hler et al., (Arteriosclerosis, Thrombosis and Vascular Biology 17:932-939, 1997). It is believed that the protein is chemotactic, if it induces the migration of cells from areas of low concentration of protein in the zone of high concentration of protein. A typical analysis performed using the modified camera Boyden with polystyrene membrane separating two chambers (Transwell; Corning Costar Corp.). The test sample is diluted in a medium containing 1% BSA, added to the lower chamber of a 24-hole tablet containing Transwell. The cells are then placed in the Transwell insert, which was previously treated with 0.2% gelatin. The migration of cells was measured after 4 hours incubation at 37°C. Non-migrating cells are washed with the upper surface of the Transwell membrane, a cell attached to the lower surface of the membrane, fixed and stained with 0.1% dye crystal violet. Then the stained cells extracted with 10% acetic acid, and the absorption was measured at 600 nm. Then the migration is calculated from the standard calibration curve. Mihr is tion of cells can also be measured using the method with Matrigel Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997).
Activity cell adhesion analyzed essentially as described LaFleur et al., (J.Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter tablets cover the test protein, non-specific sites blocked BSA and cells (e.g. smooth muscle cells, leukocytes or endothelial cells) were seeded at a density of approximately 104-105cells per well. The wells are incubated at 37°With (usually within approximately 60 minutes), then unattached cells are removed by gentle washing. Attached cells determine quantitatively the conventional methods (for example, crystal violet staining, the lysis of the cells and determining the optical density of lysate). Control wells cover the known adhesion proteins such as fibronectin or vitronectin.
Activity Zcyto21 proteins can be measured in the biosensor microphysiometer based on silicon, which measures the rate of extracellular acidification or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An example of such device is Microphysiometer Cytosensor™formulated Molecular Devices, Sunnyvale, CA. Using this method, can be measured different is shaped cell responses such as cell proliferation, ion transport, production of energy, inflammatory response, regulatory or receptor activation, etc. See for example, McConnel et al., Science 257:from 1906-1912, 1992; Pitchford et al., Meth, Enzymol. 228:84-108, 1997; Armilli et al., J.Immunol. Meth. 212:49-59, 1998 and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. Microphysiometer can be used for analysis attached or unattached eukaryotic or prokaryotic cells. By measuring changes in extracellular acidification in the cellular environment over time microphysiometer directly measures cellular responses to various stimuli, including Zcyto21 proteins, their agonists and antagonists. Preferably, microphysiometer used to measure reactions responsible for Zcyto21 eukaryotic cells compared to a control eukaryotic cell that does not respond to the Zcyto21 polypeptide. Responsible for Zcyto21 eukaryotic cells comprise cells into which was transfirieran receptor for Zcyto21, through which the received cell, which is responsible for Zcyto 21, as well as cells, natural answer Zcyto21. Differences, measured by this change, for example, increase or decrease in extracellular acidification, in the response of cells exposed to the action of Zcyto21 polypeptide, relative to a control not exposed to the action of Zcyto21, are the direct measured the eat modulated by Zcyto21 polypeptide cellular responses. In addition, such modulated Zcyto21 reactions can be analyzed under different incentives. Thus, this invention provides methods of identifying agonists and antagonists of Zcyto21 proteins, providing the cells responsible for Zcyto21 polypeptide, culturing a first portion of these cells in the absence of the test compound, culturing a second portion of these cells in the presence of test compounds and detecting changes, for example, increase or decrease in the cellular response of the second portion of the cells in comparison with the first portion of the cells. The change in cellular responses are in the form of the measured speed changes of extracellular acidification. Culturing a third portion of these cells in the presence of Zcyto21 protein and in the absence of the test compound provides a positive control for answering Zcyto21 cells and control to compare the agonist activity of test compounds with agonistic activity of Zcyto21 polypeptide. Antagonists Zcyto21 can be identified by exposure of cells to the action of Zcyto21 protein in the presence and in the absence of the test compound, whereby a decrease in stimulated Zcyto21 activity indicates antagonistic activity of the test compounds.
Expression of polynucleotides Zcyto21 in animals provides a model for further studies of biological engineering is x effects overproduction or inhibiting the activity of the protein in vivo. Encoding Zcyto21 polynucleotide and the antisense polynucleotide can be introduced into test animals such as mice using viral vectors or naked DNA, or can be obtained from transgenic animals.
One of the approaches in vivo analysis of the proteins of the present invention uses viral delivery. Examples of viruses for this purpose include adenovirus, herpes virus, retrovirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the most investigated vector gene transfer for delivery of heterologous nucleic acids. In respect of the review should watch Becker et al., Meth. Cell Biol. 43:161-89, 1994 and Douglas and Curiel, Science & Medicine 4:44-53, 1997. Adenovirus system provides several advantages. The adenovirus may (i) to accommodate relatively large insertion segments (inserts) DNA; (ii) be grown to high titer; (iii) to infect a wide range of types of mammalian cells, and (iv) be used with a large number of different promoters, including the ubiquitous, tissue-specific and regulated promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
By deletions of parts of the genome of an adenovirus can be provided over a large inserts (up to 7 TPN) heterol the same DNA. These inserts can be incorporated into viral DNA direct legirovaniem or homologous recombination with cotranslational the plasmid. In the example system, essential for adenovirus E1 gene deleteroute of the viral vector, and the virus will not be replicated until the E1 gene is not provided the host-cell (e.g. cell line 293 people). Intravenous intact animals adenovirus primarily affects the liver. If this adenoviral delivery system has a deletion of the E1 gene, the virus cannot replicate in the cells of the host. However, host tissue (e.g. liver) will Express and processional (and, if present secretory signal sequence, to secrete this heterologous protein. Secreted proteins will enter the bloodstream in wysokowykwalifikowana liver, and can be defined effects on the infected animal.
An alternative method of gene delivery involves taking cells from the body and the inclusion of the vector in these cells as naked DNA plasmid. Then these transformed cells are re-implanted in the body. Naked DNA vectors are introduced into cells of the host by methods known in this field, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextranomer SPO is obom, calcium-phosphate precipitation, use of a gene gun (shotgun method) or using a vector-vector DNA. See, Wu et al., J.Biol. Chem. 263:14621-14624, 1988; Wu et al., J.Biol. Chem. 267:963-9677, 1992, and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
Can be also obtained mouse, "engineered" for Zcyto21 gene expression, and mouse, which detect the complete absence of gene function Zcyto21, called "mice with knockout (gene)" (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-742, 1993). These mice can be used to study Zcyto21 gene and the proteins encoded by them, in the system in vivo. Transgenic mice applicable, in particular, to study the role of Zcyto21 proteins in early development due to the fact that they make it possible to identify deviations from the norm or block originating from overexpression or lack of expression of a particular factor. Cm. also Maisonpierre et al., Science 277:55-60, 1997, and Hanahan, Science 277:48-50, 1997. Preferred promoters for transgenic expression include the promoters from the genes of metallothionein and albumin.
The loss of normal inhibitory control of muscle contraction was associated with damage or perturbation selected secreting gamma-aminobutyric acid neurons. For example, the syndrome Stiff Man (numb person) includes substantial rigidity of the muscles, which is reputed to be mediated by the disruption of the functioning of producing gamma-aminobutyric acid (GABA) neurons in these patients. Other related neuromuscular disorders include myotonia, metabolic myopathy syndrome Isaac, dystonia and tehnicheskie (tetanus) spasms (Valldeoriola, J.Neurol 246:423-431, 1999).
Similarly, direct measurement of Zcyto21 polypeptide or loss of its expression in tissues can be performed in tissue or cells when they are exposed to the progression of the tumor. Increase the invasiveness and motility of cells or the increase or loss of expression of Zcyto21 in precancerous or cancerous condition, in comparison with normal tissue, can serve as diagnostic indicators for the transformation of tumor invasion or metastasis in tumor progression. The mere knowledge of the stage of progression or metastasis of the tumor will assist the Clinician in choosing the most appropriate therapy or aggressiveness of treatment for individual cancer patient. Methods of measuring gain or loss of expression (mRNA or protein) are well known in this field and described herein and can be applied to the expression of Zcyto21. For example, the appearance or disappearance of polypeptides that regulate the motility of the cells, can be used to aid in diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999). As effector mobility tile is to or as a specific for liver marker the increase or loss of expression of Zcyto21 can serve as a diagnostic indicator for brain cancer and other cancers. In addition, analogues of specific antigen of the prostate (PSA), increased levels of Zcyto21 polypeptides or antibodies against Zcyto21 in the patient relative to the normal control, can be indicators of brain cancer and other cancers (See, for example, Mulders, TMT, et al., Eur. J.Surgical Oncol. 16:37-41, 1990). Strong expression of Zcyto21 in tissue that usually does not detect the expression of Zcyto21, could serve as a diagnostic indicator of deviation from the norm in this type of cells or tissue invasion or metastasis of cancer of the liver tissue in vnepechenochnoe fabric and could help the physician in the direction of further testing or research or to assist in the selection of therapy.
In addition, polynucleotide probes Zcyto21, antibodies against Zcyto21 and the detection of the presence of Zcyto21 polypeptides in tissue can be used to assess whether brain tissue or other fabric, which is detected normally expresses Zcyto21, for example, after surgery on the occasion of excision of abnormal or cancerous liver or neural tissue. Thus polynucleotide, polypeptides and antibodies according to this invention can be used to determine whether all the fabric cut after x is rugii, for example, brain surgery and other cancer. In such cases it is especially important to remove all potentially infected tissue to maximize cure cancer and to minimize relapse. Preferred variants include fluorescent or radioactively labeled or calorimetrically labeled antibodies against Zcyto21 and partners specific binding Zcyto21, which can be used histologically in situ.
In addition, activity and action Zcyto21 on tumor progression and metastasis can be measured in vivo. Have developed several syngeneic mouse models to study the effect of polypeptides, compounds or other treatments on the progression of the tumor. In these models, tumor cells, passaged in culture, implanted in mice of the same strain as the donor tumors. These cells will develop into tumors that have similar characteristics in the recipient mice, and in some of these models will also be observed metastasis. Suitable models of tumors for research authors include Lewis lung cancer (ATSS No. CRL-1642) and B16 melanoma (ATSS No. CRL-6323), among others. Both are commonly used tumor lines, syngeneic relative to C57BL6 mouse, which are easily cultured and manipulated in vitro. Tumors arising from impl is ncacii any of these cell lines, can metastezirutaya easy in C57BL6 mice. The model of Lewis lung cancer has recently been used in mice to identify an inhibitor of angiogenesis (O′'reilly MS, et al., Cell 79:315-328, 1994). Mice C57BL6/J is treated with an experimental agent or daily injections of recombinant protein, agonist or antagonist, or a single injection of recombinant adenovirus. Three days after this treatment, 105-106cells are implanted under the skin of the back. Alternatively, these cells can be infected with recombinant adenovirus, such as expressing Zcyto21, before implantation so that the protein is synthesized at the tumor site or intracellularly, and not systematically. These mice usually develop visible tumors within 5 days. To allow tumors to grow in the period up to 3 weeks, and during this period of time they can reach the size of 1500-1800 mm3in the treated control group. Tumor size and body weight are subjected to accurate observation throughout the experiment. While killing the tumor removed and weighed together with the liver and lungs. It was shown that the weight of the lung was well correlated with the metastatic tumor burden. As an additional criterion considered metastases to the lung surface. Then the tumor, lungs and liver were prepared for histop is tological studies immunohistochemistry and in situ hybridization using methods known in this field and are described here. Thus, it was possible to evaluate the influence of this expressed polypeptide, for example, Zcyto21, on the ability of the tumor to recruit vasculature and undergo metastasis. In addition, along with the use of adenovirus, the implanted cells can be temporarily transliterowany Zcyto21. The use of stable transfectants Zcyto21, and the use of inducible promoters to activate expression Zcyto 21 in vivo are known in this field, and they can be used in this system to assess the induction of metastasis Zcyto21. In addition, purified Zcyto 21 or air-conditioned Zcyto 21 environment can directly inetservices in the same mouse model, and hence be used in this system. In respect of General references, see O′'reilly MS, et al., Cell 79:315-328, 1994 and Rusciano D, et al., Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
Antisense methodology can be used to inhibit transcription of the gene Zcyto21 to study the effects of such inhibition in vivo. Construct polynucleotides that are complementary to a segment encoding Zcyto21 of polynucleotide (for example, polynucleotide presented in SEQ ID NO:1) for binding to the coding Zcyto21 mRNA and inhibiting translation so the th mRNA. Such antisense oligonucleotides can also be used for inhibiting the expression of the coding Zcyto21 polypeptide genes in cell culture.
The majority of cytokines and other proteins produced by activated lymphocytes, play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body. It is expected that Zcyto21 and inhibitors of activity Zcyto21 will have a variety of therapeutic applications. These therapeutic applications include the treatment of diseases that require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, severe psevdomatematicheskoe myasthenia gravis, systemic lupus erythematosus, and diabetes. Zcyto21 may be important in the regulation of inflammation and, therefore, may be useful in the treatment of rheumatoid arthritis, asthma and sepsis. The possible role of Zcyto21 in mediating oncogenesis, resulting antagonist Zcyto21 would be useful in the treatment of cancer. Zcyto21 may be useful in modulating the immune system, resulting in Zcyto21 and antagonists Zcyto21 can be used to reduce transplant rejection, prevention of reaction of graft-versus-host, boosting immunity to infectious diseases, treatment of patients with a weakened immune system (for example, HIV-Polo is sustained fashion patients) or in improving vaccines.
As interferon-like polypeptide in brain tissue, islets, prostate, testis, pituitary, placenta, of an ovarian tumor, lung tumor, rectal tumor and tumor of the ovary, as well as CD3+ cells and infected with the virus line of epithelial cells of the prostate gland, Zcyto21 applicable to modulate viral infection, tumorigenesis and metastasis in these and other tissues. In such cases, the interferon-like molecule can be released by cells at the site of infection or abnormal cell growth, or in the form of secreted molecules, it can migrate to this area from the backside of the fabric.
Antiviral properties Zcyto21 particularly applicable in the treatment of infection with papilloma viruses in vitro and in vivo. For example, tumors caused by the human papilloma virus, induce benign tumors (i.e. genital genital warts), and malignant tumors, such as squamous cell carcinoma. Treatment for these conditions is usually surgery or destruction of tissue. However, currently it is shown that some anti-virus/immune modulating drugs, including interferon alpha, are effective in reducing tumor size. Cm. Baker, G.E. et al., Dermatol. Clin. Apr. 15:331-340, 1997. In addition, as discussed Rockley, P.F. et al. (Pharmacol. Ther. 65 ():265-287, 1995), immunological therapy with interferon may be directed against all sites of infection, including clinical, podchinyonnogo and latent diseases. In this example, IFN-alpha, IFN-beta and IFN-gamma have been used as monotherapy and in combination with other therapies for the treatment of anogenital genital warts. Zcyto21 will be a useful tool, similar to IFN-alpha, IFN-beta and IFN-gamma in the treatment. In addition, there was a close Association between certain types of human papillomavirus and cervical cancer. Zcyto21 can be used for detection, monitoring and treatment of cancers of the cervix.
As a small, secreted protein in islet cells Zcyto21 can modulate the growth and differentiation of these cells. In addition, Zcyto21 can be used in the treatment of diabetes and immunological conditions associated with growth and differentiation of these cells.
The presence of Zcyto21 in the cells of the brain and pituitary suggests that it may also find application in the growth and differentiation of these cells. In addition, molecules of this invention can be responsible for the homeostasis of power, including behavioral disorders associated with nutrition and decreased appetite. In addition, molecules Zcyto21 can find application in the treatment of reproductive disorders the population in General.
Zcyto21 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF. When using Zcyto21 in combination with an additional agent, these two compounds can be administered simultaneously or sequentially depending on the specific subject to treatment status.
Zcyto21 will be applicable in the treatment of carcinogenesis and, therefore, could be useful in the treatment of cancer. Inhibition using Zcyto21 anti-lgM-stimulated normal b cells and a similar effect is observed in b-cell tumor lines, I presume that may be of therapeutic benefit in the treatment of patients Zcyto21 for the induction of cell-cell tumors in less proliferative state. This ligand could be implemented in combination with the already used by other agents, including conventional chemotherapeutic agents, and immunomodulators such as interferon alpha. It was shown that the interferon alpha/beta are effective in the treatment of some leukemias and models of animal diseases, and inhibiting the growth effects of interferon-alpha and Zcyto21 can be additive derived from b-cell tumor cell lines.
This invention provides a method of reducing proliferation of neoplastic B - or T-cells, providing for the introduction mlekovita the mu - or T-cell neoplasms amount of the composition Zcyto21, sufficient to reduce proliferation of neoplastic B - or T-cells. Stimulation by Zcyto21 lytic natural killer cells from precursor cells of the bone marrow and proliferation of T cells after activation of receptors antigens could enhance the treatment of patients receiving allogeneic bone marrow transplants, and, therefore, Zcyto21 will enhance the generation of protivoopuhulevyh reactions infusion or no infusion of donor lymphocytes.
In another aspect, the invention provides a method of reducing proliferation of neoplastic B - or T-cells, involving the administration to a mammal with a B - or T-cell neoplasms amount of the composition antagonist Zcyto21, sufficient to reduce proliferation of neoplastic B - or T-cells. In addition, antagonist Zcyto21 can be fused protein ligand/toxin.
Merged toxin Zcyto21-saporin can be used against a similar number of leukemias and lymphomas, which expands the range of leukemias that can be treated Zcyto21. Indirect merged toxin activation of the receptor Zcyto21 provides two independent way of inhibiting the growth of target cells, the first of which is identical to the effects observed with the use of one ligand, and the second is due to the delivery of the toxin through the internalization of the receptor.
On the basis of the written here interferon-like molecules Zcyto21 of the present invention will be applicable for the detection, monitoring and treatment of such diverse conditions as reticuloendotheliosis, renal cell cancer (renal cell cancer), basal cell carcinoma (basal cell carcinoma), malignant melanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Jackinsky lymphoma, laryngeal papillomatosis, mushroom avium, genital warts induced by papillomavirus the warty epidermodysplasia, chronic hepatitis b, hepatitis C, chronic hepatitis D and chronic non-a, non-b/C hepatitis.
The Department of quality control of food, drugs and cosmetics USA has approved the use of interferon-β for the treatment of multiple sclerosis, a chronic disease of the nervous system. Interferon-γ used to treat chronic granulomatous disease, in which the interferon enhances the immune response of the patient to destroy infectious bacteria, fungi and belong to the simplest of pathogens. Clinical studies show that interferon-γ it may be applied in the treatment of AIDS, leishmaniasis and lepromatous leprosy.
For pharmaceutical applications Zcyto21 proteins are prepared for local or parenteral, in particular, within the military or subcutaneous, delivery in accordance with conventional ways. Typically, the pharmaceutical compositions will include Zcyto21 polypeptide in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like, the Composition can optionally contain one or more fillers, preservatives, solubilization, bafarawa agents, albumin to prevent loss of protein on the surfaces of the bottles, etc. Methods for making compositions are well known in this field and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19thed., 1995. Zcyto21 will preferably be used at a concentration of from about 10 to 100 μg/ml of total volume, although can be used in concentrations in the range of 1 ng/ml - 1000 μg/ml For local use, for example, to accelerate wound healing, protein will be applied in the range from 0.1 to 10 µg/cm2the area of the wound, and the exact dosage should be determined by the physician according to accepted standards, taking into account the nature and gravity of the subject to treatment status, patient, etc. dose Determination is within the ordinary skill of a person skilled in the art. The dose is a daily or periodically during the treatment period. Intravenous Bud is t to run the bolus-injection or infusion during the normal period of one or several hours. Can also be used in the composition of prolonged action. Typically, a therapeutically effective amount Zcyto21 is a quantity sufficient to produce clinically significant changes in the treated condition, for example, clinically significant changes in hematopoietic or immune function, significant decrease in pain or significantly increased histological grade.
Proteins, agonists and antagonists Zcyto21 applicable for modulation stretching, proliferation, activation, differentiation, migration, metabolism answering these types of cells, which include both primary cells and cultured cell lines. Of particular interest in this respect are hematopoietic cells, mesenchymal cells (including stem cells and Mature myeloid and lymphoid cells), endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, hepatocytes, neural cells and embryonic stem cells. Zcyto21 polypeptides added to the medium for tissue culture of these cell types in a concentration of about 10 PG/ml, about 100 ng/ml of Professionals with expertise in this area will be clear that Zcyto21 proteins can be advantageously combined with other growth factors in the culture medium.
In the field of laboratory studies b the CTL Zcyto21 can also be used as molecular mass standards or as reagents in assays to determine the levels of this protein in the bloodstream, for example, in the diagnosis of disorders characterized by overproduction or insufficient production of Zcyto21 protein or in the analysis of cell phenotype.
Zcyto21 proteins can also be used to identify inhibitors of their activity. Test compounds are added to the above-described tests for the identification of compounds inhibiting the activity of protein Zcyto21. In addition to these above tests, samples can be tested for inhibition of activity Zcyto21 in numerous assays designed to measure receptor binding or the stimulation/inhibition of Zcyto21-dependent cellular responses. For example, xpressimage Zcyto21 cell lines can be transliterowany design with a reporter gene that is driven Zcyto21 pathway. Design with reporter gene of this type are known in this area and will typically contain Zcyto21-activated answering serum element (SRE), is functionally associated with the gene coding for the analyzed protein, such as luciferase. Candidate connections, solutions, mixtures or extracts are tested for the ability to inhibit the activity of Zcyto21 on the target cells, defined by a decrease stimulation Zcyto21 the expression of the reporter gene. Tests of this type will detect compounds that directly block the binding is of Zcyto21 with receptors on the cell surface, as well as compounds that block processes in the cellular pathway after binding of the receptor-ligand. The alternative, compounds or other samples can be tested for direct blocking of binding of Zcyto21 with the receptor using Zcyto21, labeled detectable label (e.g.,125I, Biotin, horseradish peroxidase, FITC, and the like). In analyses of this type, the ability of a test sample to inhibit the binding of labeled Zcyto21 with receptor indicates inhibitory activity, which can be confirmed by secondary analyses. The receptors used in the analysis of binding can be cellular receptors or dedicated immobilized receptors.
In the application here, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, their antigennegative fragments, such as F(ab′)2and Fab fragments, single-chain antibodies and the like, including genetically-engineered antibodies. Antibodies of the person can be "humanized" (humanitarian) by grafting the CDR is not the person on the framework and constant region of human immunoglobulin or the inclusion of a complete variable domains of a person (not necessarily a "wrapping" them with a human-like surface by replacement of exposed residues, resulting in a "tiled" antibody). In some SL is teas "humanized" antibodies can save the remnants of immunoglobulin could frame the domain of the variable region of a person to enhance the characteristics of the correct binding. Through humanitarian" antibodies can be increased biological half-time of existence in the body, and the potential for adverse immune response when administered to humans is reduced. The person skilled in the art can generate humanized antibodies with specific and different constant domains (i.e. different subclasses of Ig) to facilitate or inhibit various immune functions associated with a particular constant domains of antibodies. Antibodies are, by definition, specifically binding if they bind to a polypeptide or protein Zcyto21 with an affinity at least 10-fold higher than the binding affinity of control (non-Zcyto21) the polypeptide or protein. The affinity of monoclonal antibodies can be readily determined by the person of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci., 51:660-672, 1949).
Ways to obtain polyclonal and monoclonal antibodies are well known in the art (see for example, article Hurrel, J.G.R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, incorporated herein by reference). Of particular interest is the generation of antibodies to the hydrophilic antigenic sites, which include, for example, residues 155 (Glu)- 160 (Glu); residues 51 (Lys) - 56 (Ala); residues 50 (Phe) - 55 (Asp); the remains of 140 (Pro) - 145 (Arg) and residues 154 (Gln) - 159(Lys), as shown in SEQ ID NO:2. As should be obvious to a person of ordinary skill in this field, polyclonal antibodies can be obtained from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats. Immunogenicity of Zcyto21 polypeptide may be increased by applying an adjuvant, such as alum (aluminum hydroxide) or a full or partial beta-blockers. Polypeptides that are applicable for immunization also include fused (hybrid) polypeptides, such as hybrids Zcyto21 polypeptide or part thereof with the immunoglobulin polypeptide or with malesurvivor protein. Polypeptide immunogen can be a full-length molecule or a part of it. If this part of the polypeptide is "hapten-like", such portion may preferably be connected or linked to a macromolecular carrier (such as hemocyanin fissurella (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
Alternative ways of generating or selecting antibodies include exposure in vitro lymphocyte action of Zcyto21 polypeptides and selection of libraries presentation antibodies in phase or similar vectors (for example, through use of immobilized or labeled Zcyto21 polypeptide). Human antibodies can be obtained from the transgene is s, non-human animals, which were "designed" to obtain genes of human immunoglobulin, as described in WIPO Publication WO 98/24893. Preferably, the endogenous immunoglobulin genes in these animals were inactivated or eliminated, for example, homologous recombination.
A variety of assays known to those with skill in this field can be used to detect antibodies that specifically bind polypeptides Zcyto21. Examples of the analyses described in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Typical examples of such analyses include: competitive immunoelectrophoresis, radioimmunoassays, radioimmunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
Antibodies to Zcyto21 can be used for affinity purification of the protein, in diagnostic assays to determine the levels of this protein in blood; for the detection or quantitative determination of soluble Zcyto21 polypeptide as a marker hidden pathology or disease; for immunolocalization in whole animals or tissue sections, including immunodiagnostics applications; for immunochemistry and as antagonists for inhibiting the activity of a protein in vitro and in vio. Antibodies to Zcyto21 can also be used for tagging cells expressing Zcyto21; for affinity purification of polypeptides and proteins Zcyto21; in analytical methods using FACS; for screening expression libraries and to generate antiidiotypic antibodies. Antibodies may be associated with other compounds, including therapeutic and diagnostic agents, using known methods to provide targeting of these compounds in cells expressing receptors for Zcyto21. For some applications, including diagnostic applications in vitro and in vivo, it is preferable to use labeled antibodies. Suitable direct tags (markers) or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags (markers) or labels can withdraw in the first place the use of pairs of Biotin-avidin or other pairs complement/anticomplement as intermediate compounds. Antibodies of the present invention may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates can be used for diagnostic or therapeutic applications in vivo (e.g., inhibition of cell proliferation). Cm. in General amakrishnan et al., Cancer Res. 56:1324-1330, 1996.
The polypeptides and proteins of this invention can be used to identify and isolate receptors. Receptors Zcyto21 may participate in the regulation of growth in the liver, the formation of blood vessels and other development-related processes. For example, proteins and Zcyto21 polypeptides can be immobilized on a column and membrane preparations is passed through this column (as described in General in Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can be radioactively labeled (Methods Enzymol., vol.182, "Guide to Protein Purifications", M.Deutscher, ed., Academic Press, San Diego, 1990, 721-737) or photouplink labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biocem. Pharmacol. 33:1167-1180, 1984) and used to label specific proteins on the cell surface. Similarly, radioactively labeled proteins and Zcyto21 polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with the expression cDNA library.
Zcyto21 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure at the atomic level, spectroscopy, nuclear magnetoresonance to reveal the structure of proteins in solution. For example, the set containing Zcyto21, may be provided to the student for analysis. Since the amino acid sequence is known to the instructor, the protein can be given to the student as a test to determine his skill or to develop skills of the student, the instructor will know whether the student has analyzed the polypeptide. Since every polypeptide is unique, the educational applicability Zcyto21 could be unique in itself.
Antibodies that bind specifically with Zcyto21, can be used as a teaching tool to instruct students how to prepare columns for affinity chromatography for the purification of Zcyto21, cloning and sequencing of polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies. Zcyto21 gene, polypeptide, or antibody would then be packaged selling reagent companies and sold to educational institutions so that students qualified in the field of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in the laboratory. Such sets for training, containing the gene, polypeptide Zcyto1 or antibody, are within the scope of this invention. The invention described thus in General, will be more understandable with reference to the following examples which are given for illustration and not to limit the present invention.
Expressing plasmid containing the entire polynucleotide encoding Zcyto21, or part thereof, designed using homologous recombination. The cDNA fragment Zcyto21 isolated by using PCR with the use of polynucleotide sequence SEQ ID NO:1 with flanking regions at the 5′and 3′-ends corresponding to the vector sequences flanking the injection point Zcyto21. Primers for PCR, each include from 5′-end to 3′-end: 40 BP of flanking sequence from the vector and 17 BP corresponding to the amino end and carboxyl-end of the open reading frame Zcyto21.
Ten μl of the 100 μl PCR reaction mixture is subjected to electrophoresis on 0.8% melting at a low temperature agarose (SeaPlaque GTG®; FMC BioProducts, Rockland, ME) gel 1 × TBE-buffer for analysis. The remaining 90 μl of the reaction mixture is precipitated by adding 5 μl of 1 M NaCl and 250 μl of absolute ethanol. Plasmid pZMP6, which was cut Smal, used for recombination with this PCR fragment. Plasmid pZMP6 is expressing vector mammals, with the expression containing a series of the cartridge, having immediate early cytomegalovirus promoter, multiple restriction sites for installation of coding sequences, a stop codon and a terminator of human growth hormone; the replication origin E. coli; expression unit of breeding marker mammals, containing the promoter, enhancer and the starting point of the SV40 replication, a DHFR gene, and the SV40 terminator; and sequence of the URA3 and CEN-ARS, required for selection and replication in S. cerevisiae. It was constructed from pZP9 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 under access number No. 98668) with yeast genetic elements taken from pRS316 (deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 under access number No. 77145), element internal customers occurrences of ribosomes (IRES) of poliovirus and the extracellular domain of CD8, shortened at the C-terminal side of the transmembrane domain,
One hundred microlitres competent yeast cells (S. cerevisiae) independently combined with 10 μl of various mixtures of DNA from above and transferred to a cuvette for electroporation see 0,2 Mix the yeast/DNA is subjected to electrical pulses using the source power (BioRad Laboratories, Hercules, CA) of 0.75 kV (5 kV/cm) ∞ Ohms, 25 μf. In each cuvette add 600 μl of 1.2 M sorbitol and the yeast sown in the form of two aliquot of 300 μl in two URA-D-tablet and incubated at 30°Szpuszta approximately 48 hours yeast transformants Ura +one tablet resuspended in 1 ml of H2O and out quickly for the deposition of yeast cells. Cellular precipitate resuspended in 1 ml lisanova buffer (2% Triton X-100, 1% LTOs, 100 mm NaCl, 10 mm Tris, pH 8.0, 1 mm EDTA). Five hundred microlitres lizinoj mixture is added to the Eppendorf tube containing 300 μl acid washed glass granules and 200 ál of a mixture of phenol-chloroform, shaken on a vortex for 1 minute intervals two or three times and turn off for 5 minutes in an Eppendorf centrifuge at maximum speed. Three hundred microlitres the aqueous phase transferred to a fresh tube and the DNA precipitated with 600 μl ethanol (EtOH), followed by centrifugation for 10 minutes at 4°C. the precipitated DNA resuspended in 10 μl of N2O.
Transformation electrocompetent host cells E. coli (cells Electromax DH10B™; obtained from Life Technologies, Inc., Gaithersburg, MD) performed with 0.5-2 ml yeast DNA and 40 μl of cells. Cells subjected to electrical pulses at 1.7 kV, 25 μf and 400 Ohms. After electroporation, 1 ml SOC (2% Bacto™ Tripton (Difco, Detroit, Ml), 0.5% of yeast extract (Difco), 10 mm NaCl, 2.5 mm KCI, 10 mm MgCl2, 10 mm MgSO4, 20 mm glucose) are sown in the form of aliquot 250 ál of four tablets with LB AMR (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/l Ampicillin).
Individual clones carrying the correct expression construct for Zcyto21, ID is tefillot splitting restrictable to confirm the presence of inserts Zcyto21, and to confirm, that the various DNA sequences have been joined correctly to one another. The inserts of positive clones subjected to sequence analysis.
Large-scale isolation of plasmid DNA is performed using a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen, Valencia, CA) according to the manufacturer's instructions. The correct design is named pZMP6/Zcyto21.
Cells Cho DG44 (Chasin et al., Som. Cell, Molec. Genet. 12:555-666, 1986) were seeded into plates for tissue culture 10 cm and allow them to grow to approximately 50-70% of confluently over night at 37°C, 5% CO2in the environment of Ham F12/FBS (Ham′s F12 medium (Life Technologies), 5% fetal calf serum (Hycione, Logan, UT), 1% L-glutamine (JRH Biosciences, Lenexa, KS), 1% sodium pyruvate (Life Technologies)). These cells then transferout the plasmid Zcyto21/pZMP6 mediated by liposomes by transfection using a 3:1 (mass/mass) liposomal compositions of poly-lipid 2,3-tileorasi-N-[2-(sprintersexual)ethyl]-N,N-dimethyl-1-propanamine-trifenatate and neutral lipid dioleoylphosphatidylcholine in the filtered water through the membrane (reagent Lipofectamine™Life Technologies) in the composition of serum-free (SF)medium (Ham F12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml of fetuin, 1% L-glutamine and 1% sodium pyruvate). Zcyto21/pZMP6 diluted in tubes of 15 ml to a total final volume of 640 μl of SF medium. 35 μl Lipof stamina™ mixed with 605 μl of SF medium. The resulting mixture was added to the DNA mixture and allowed to incubated for approximately 30 minutes at room temperature. Five ml of SF-environment add to the mixture of DNA:Lipofectamine™. Cells are washed once with 5 ml of SF environment, sucked off and add the mixture of DNA:Lipofectamine™. Cells are incubated at 37°C for five hours, then in each Cup add to 6.4 ml of medium Ham F12/10% FCS, 1% PSN. Cup incubated at 37°during the night and the mixture of DNA:Lipofectamine™ replace with fresh medium with 5% FCS/Ham the next day, on day 3 after transfection cells distribute in bulb T-175 in the growing medium. At day 7 after transfection cells stained with monoclonal antibodies FITC-anti-CD8 (PharMingen, San Diego, CA) followed by the addition of anti-FITC-conjugated magnetic granules (Miltenyi Biotec). CD8-positive cells separated using commercially available columns (mini-MACS columns; (Miltenyi Biotec) according to the manufacturer's recommendations and placed in DMEM/Ham F12/5% FCS without nukes, but with 50 nm methotrexate (selective medium).
Cells were seeded for sublimirovanny at a density of 0.5, 1 and 5 cells per well in 96-well tablets in selective medium and allow to grow for approximately two weeks. Wells screened for evaporation of environment and bring again to 200 µl per well as necessary during this processed a large percentage of the colonies in the tablet become close to confluently, 100 µl of the environment taken from each well for analysis using dot-blot and cell serving fresh selective medium. The supernatant is applied on the nitrocellulose filter device for dot-blot analysis and this filter is treated with 100°With vacuum thermostat for denaturation of the protein. The filter is incubated in 625 mm Tris-glycine, pH of 9.1, 5 mm β-mercaptoethanol at 65°C, 10 minutes, then in the Western buffer And with 2.5% fat-free dried milk (of 0.25% gelatin, 50 mm Tris-HCl pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.05% of Igepal CA-630) overnight at 4°C on a rotary shaker. The filter is incubated with the conjugate antibody-HRP Western-buffer And with 2.5% non-fat dry milk for 1 hour at room temperature on a rotating shaker. Then the filter is washed three times at room temperature in SFR plus 0.01% tween-20, 15 minutes per wash. Filter show chemiluminescent reagents (set for direct labeling ECL™; Amersham Corp., Arlington Heigts, IL) according to the manufacturer's recommendations and exhibit on film (Hyperfilm ECL, Amersham Corp.) for about 5 minutoli clones trypsinized of the 96-hole tablet and transferred into 6-hole Cup in selective medium to zoom in culture and analysis by Western blot.
Full Zcyto21 protein get in KSS-cells transfected pZMP6/Zcyto21 (the example 1). Cells VNC ADS CRL-10314) were seeded into plates for tissue culture 10 cm and allow them to grow to approximately 50-70% of confluently over night at 37°C, 5% CO2in the DMEM/FCS (DMEM, Gibco/BRL High Glucose; Life Technologies), 5% fetal calf serum (Hyclone, Logan, UT), 1 mm L-glutamine (JRH Biosciences, Lenexa, KS), 1 mm sodium pyruvate (Life Technologies). These cells then transferout the plasmid pZMP6/Zcyto21 mediated by liposomes by transfection (using Lipofectamine™; Life Technologies), in serum free (SF) medium (DMEM, supplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml of fetuin, 1% L-glutamine and 1% sodium pyruvate). Plasmid diluted in tubes of 15 ml to a total final volume of 640 μl of SF medium. 35 μl of the lipid is mixed with 605 μl of SF medium and the resulting mixture is allowed to incubated for approximately 30 minutes at room temperature. Five ml of SF-environment add to the mixture of DNA:lipid. Cells are washed once with 5 ml of SF environment, sucked off and add the mixture of DNA:lipid. Cells are incubated at 37°C for five hours, then in each Cup add to 6.4 ml of medium DMEM/10% FCS, 1% PSN. Cup incubated at 37°during the night and the mixture of DNA:lipid replaced with fresh medium with 5% FCS/DMEM for the next day. At day 5 after transfection cells distribute in bulb T-162 in selective medium (DMEM+5% FCS, 1% L-Gln, 1% sodium pyruvate, 1 μm methotrexate. After approximately 10 days after transfer the AI two cultural cups 150 mm resistant to methotrexate colonies from each transfection trypsinized and these cells are combined and seeded into a flask T-162 and transferred to large-scale culture.
For construction of adenoviral vectors encoding protein region Zcyto21 man amplified using PCR using primers that add restriction sites Pmel and Ascl 5′- and 3'-ends, respectively. Amplification is performed with cDNA-matrix full-Zcyto21 in a PCR reaction as follows: one cycle at 95°C for 5 minutes; then 15 cycles at 95°C for 1 min, 61°C for 1 min and 72°C for 1.5 min; then 72°C for 7 min, then soaking at 4°C. the PCR reaction Product is applied by 1.2% melting at a low temperature agarose gel in the Tae buffer (0.04 M Tris-acetate, 0.001 M EDTA). The PCR product Zcyto21 cut out from the gel and purified using a commercially available kit containing a column of silica gel with a membrane (the cleaning kit QIAquick PCR® PCR Purificaton Kit and kit for treatment gel; Qiagen, Inc.), in accordance with the instructions set. Then the PCR product digested Pmel and AscI, extracted with a mixture of phenol/chloroform, precipitated by EtOH and rehydration in 20 ml TE (Tris/EDTA, pH 8). Then the fragment Zcyto21 are ligated in sites Pmel-AscI transgenic vector pTG12-8 and transformed into competent cells of E. coli DH10B™ by electroporation. Vector pTG12-8 was obtained from RV (Palmiter et al., Mol. Cell Biol. 13:5266-5275, 1993) insertional intron insulin II rats (approximately 200 BP) and polylinker (Fsel/Pmel/Acl) in the Nrul site. This vector contains the promoter of the mouse metallothionein (MT-1) (approximately 750 BP) and untranslated region and polyadenylation signal of the human growth hormone (hGH) (approximately 650 BP), flanked 10 TPN 5′-flanking sequences of MT-1 and 7 TPN 3′-flanking sequences of MT-1. This cDNA built between sequences insulin II and hGH. Clones containing Zcyto21, identify using minipreparation plasmid DNA and subsequent cleavage of Pmel and AscI. A positive clone is sequenced to ensure that there have been no deletions or other abnormalities in this structure.
DNA is obtained using a commercially available kit (Maxi Kit, Qiagen, Inc.) and cDNA Zcyto21 isolated from the vector pTG12-8 using enzymes Pmel and Ascl. cDNA allocate 1% melting at a low temperature agarose gel and cut out from the gel. Cut the gel, melted at 70°and DNA extracted twice with equal volumes of buffered with Tricom phenol, precipitated by EtOH and resuspended in 10 μl of N2O.
cDNA Zcyto21 clone in sites EcoRV-AscI modified plasmid pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998). This structure contains the marker gene green fluorescent protein (GFP). CMV, triggering the expression of GFP, replace the SV40 promoter, and the SV40 polyadenylation signal replace signal poly is generirovaniya of human growth hormone. In addition, the natural linker replaces the sites Fsel, EcoRV and Ascl. This modified form of pAdTrack-CMV named pZyTrack. Ligation is carried out using a commercially available kit for ligation and DNA screening (Fast-Link® kit; Epicentre Technologies, Madison, Wl). Clones containing Zcyto21, identify the splitting minipreparation DNA Fsel and Ascl. To linearize the plasmid, approximately 5 μg of the obtained plasmids pZyTrack Zcyto21 split Pmel. Approximately 1 μg of the linearized plasmid cotransformation with 200 ng super helical conformation was pAdEasy (Not et al., ibid.) in cells of E. coli BJ5183 (Not et al., ibid.). Cotransformation carried out using gene pulser, Bio-Rad at 2.5 kV, 200 Ω and 25 μf. The entire mixture to cotransformation plated on 4 LB-cups containing 25 μg/ml kanamycin. The smallest colony vyskrabat and propagated in LB/kanamycin and recombinant adenoviral DNA identify standard procedures for obtaining minipreparation DNA. Minireport DNA recombinant adenovirus transformed into competent cells of E. coli DH10B™ and DNA obtained using set, Maxi Kit (Qiagen, Inc.) in accordance with the instructions set.
Approximately 5 µg of recombinant adenoviral DNA were cleaved by the enzyme Pad (New England Biolabs) for 3 hours at 37°in a reaction volume of 100 µl, containing 20-30 E Pacl. Cleaved DNA extracted twice equally the m by volume mixture of phenol/chloroform and precipitated with ethanol. Precipitate DNA resuspended in 10 μl of distilled water. The flask KZT25 cells QBI-293A (Quantum Biotechnologies, Inc. Montreal, Qc. Canada), inoculated the day before and grown to 60-70% of confluently, transferout split Pacl DNA. Split Pacl DNA was diluted to a total volume of 50 µl with sterile HBS (150 mm NaCl, 20 mm HEPES). In a separate tube, 20 μl 1 mg/ml salts of N-[1-(2,3-tileorasi)propyl]-N,N,N-trimethylammonium (DOTAP) (Boehringer Mannheim, Indianapolis, IN) was diluted to a total volume of 100 μl HBS. DNA is added to DOTAP, mix gently by pipetting up and down and leave at room temperature for 15 minutes. The medium is removed from the cells A and washed with 5 ml serum-free minimum essential medium (MEM) alpha, containing 1 mm sodium pyruvate, 0.1 mm non-essential amino acids MEM and 25 mm HEPES buffer (all reagents were obtained from Life Technologies, Gaithersburg, MD). 5 ml of serum-free MEM is added to the cells A and incubated them at 37°C. a Mixture of DNA/lipid is added dropwise into the flask KZT25, with cells A, gently mixed and incubated at 37°C for 4 hours. After 4 hours the medium containing a mixture of DNA/lipid, sucked off and replaced with 5 ml complete MEM containing 5% fetal calf serum. Transfetsirovannyh cells are monitored for GFP expression and the formation of foci (viral plaques).
Seven days after transfection cells A recombinant viral DNA these to EDI Express a protein GFP (green fluorescent protein) and begin to form foci (viral plaques"). The crude viral lysate harvested using a cell scraper (scraper) to collect all cells A. This lysate is transferred into a conical flask and 50 ml For the release of the majority of viral particles from these cells perform three freeze/thaw in the bath with a mixture of ethanol and dry ice in a water bath at 37°C.
The crude lysate amplified (primary (1° (C) amplification) to obtain a working "stock" lysate Zcyto21-rAdV. Ten of cups 10 cm almost confluent (80-90%) of cells A cook for 20 hours before this, 200 ml of the crude rAdV lysate added to each Cup 10 cm and these cups see within 48-72 hours for cytopathic effect (CPE) under a light microscope (white light) and on the expression of GFP under fluorescent microscope. When all cells A find CPE, this original lysate is collected and execute cycles of freezing/thawing, as described above.
Then perform secondary (2° (C) amplification Zcyto21-rAdV. Twenty-cups for tissue culture 15 cm cells A prepared so that the cells were 80-90% confluently. The entire amount, except 20 ml of 5% medium MEM, and remove each Cup inoculant 300-500 ml of the amplified if 1°With rAdv lysate. After 48 hours cells A are lysed produced from virus lysate collected in polypropylene centrifuge bottles of 250 ml and purified rAdV.
Doba is given in the detergent NP-40 to a final concentration of 0.5% in the vials of the crude lysate for lysis of all cells. The vials are placed on a rotating platform for 10 minutes with possible rapid agitation without tipping bottles. Debris precipitated by centrifugation at 20000×g for 15 minutes. The supernatant is transferred to a polycarbonate centrifuge bottles of 250 ml and add 0.5 volume of solution 20% PEG/2.5 M NaCI. The vials shaken overnight on ice. The bottles are centrifuged at 20000×g for 15 minutes and the supernatant discarded in decolorizing the solution. Using a sterile cell scraper (scraper) white precipitate virus/PEG two bottles resuspending 2.5 ml SPR. The resulting solution of the virus is placed in microcentrifuge tubes in 2 ml and centrifuged at 14000×g in microcentrifuge for 10 minutes to remove additional quantities of cellular debris. The supernatant from the centrifuge tubes, 2 ml is transferred into a polypropylene test tube 15 ml with a snapping lid and bring to a density of 1.34 g/ml using CsCl. This solution is transferred to a polycarbonate thick-walled centrifuge tubes 3.2 ml, and turn off when 348000×g for 3-4 hours at 25°C. the Virus forms a white stripe. Stripe virus remove the tips of the pipettes with a wide aperture.
For desalting of the preparation of the virus using commercially available ion-exchange column (e.g. column PD-10,pre-Packed Sephadex® G-25M (Pharmacia Biotech, Piscataway, NJ). Column balance 20 ml SPR. Put the virus and allow him to pass into the column. In column add 5 ml SPR and collect fractions from 8-10 drops. Optical density of 1:50 dilutions of each fraction is determined at 260 nm on a spectrophotometer. Fraction peaks unite and determine the optical density (OD) dilution 1:25. The value of OD is converted into the concentration of the virus using the formula: (OD at 260 nm)(25)(1,1×1012)=virions/ml
For storage of the virus to a purified virus add glycerol to a final concentration of 15%, gently, but effectively mixed and stored in aliquot at -80°C.
The Protocol developed Quantum Biotechnologies, Inc. (Montreal, Canada), used to measure the infectivity of the recombinant virus. Briefly, two 96-well plate to tissue culture seeded with 1×104cells A per well in MEM containing 2% fetal calf serum, for each of the tested recombinant virus. After 24 hours, to prepare 10-fold dilution of each virus 1×10-2up to 1×10-14in MEM containing 2% fetal calf serum. 100 μl of each dilution is placed in each of 20 wells. After 5 days at 37°With holes read as either positive or negative in relation to the ENVIRONMENT and calculate the value of the "plaque-forming units/ml (PFU).
Transgenic W the animals expressing the genes for Zcyto21, obtained using adult fertile males (suitable for mating) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown, HY)), vasectomany males (unsuitable for mating) (CD1, 2-8 months of age (Taconic Farms), prepubertal fertile females (donors) (B6C3f1, 4-5 weeks of age (Taconic Farms)) and adult fertile females (recipients) (CD1, 2-4 months of age (Taconic Farms)).
Donors will have acclimatised for 1 week and then injected about 8 IU/mouse serum gonadotropin foals mares (Sigma, St. Louis, MO) I.P. and through 46-47 hours 8 IU/mouse hereticism the gonadotropin human (hCG (Sigma)) for the induction of superovulation. Donors pair with suitable for mating animals after injection of hormones. Ovulation usually occurs within 13 hours after the hCG injection. The copulation confirm the presence of mucous plugs in the morning after mating.
Fertilized eggs are collected under a surgical microscope (Leika MZ12 Stereo Microscope, Leica, Wetzlar, DE). The fallopian tubes are collected and egg release in glass slides for analysis of urine containing hyaluronidase (Sigma). Egg washed once in hyaluronidase and twice in the environment Witten W640 (table 4), which were incubated with 5% CO25% of O2and 90% N2at 37°C. Then the egg is incubated with 37°C/5% CO2before Mick is injectii.
10-20 micrograms of plasmid DNA containing a cDNA of the gene Zcyto21, linearized, purified from the gel and resuspended in 10 mm Tris pH 7.4, 0.25 mm EDTA pH 8.0 at a final concentration of 5-10 nanograms per microliter for microinjection.
Plasmid DNA microinjection in the collected oocytes contained in a drop of medium W640 covered with warm CO2balanced mineral oil. DNA is pulled into the needle for injection (using a capillary made of borosilicate glass 0,75 EXT. diameter, 1 mm OD) and injected in a separate eggs. Each egg comes with a needle for injection, in one or in both haploid pronucleuses.
DNA in the number of picolitres injected in these pronuclei and a needle for injection retard without bringing it into contact with nucleoli. This procedure is repeated until you have injected all the eggs. Successfully microinjection egg is transferred into the Cup for tissue culture bodies with pre-aerated environment W640 to cure over night in an incubator with 37°C/5% CO2.
The next day 12-17 healthy 2-cell embryos from injection of the previous day is transferred into a recipient. Determine the position of the swollen capsules and keep the oviduct between the ampoule and the Bursa, produce rupture of the oviduct needle 28 G near Bursa, trying not to break the ampoule or Bursa. The embryos are implanted cereseto gap and by holding on peritoneal wall of the reproductive organs sent back into the abdominal cavity.
Recipients return in cell pairs, and allow it to pass 19-21 days of pregnancy. After childbirth allow 19-20-day postpartum period before weaning calves. The weaners determine the floor and put them in separate cells on the floor and take 0.5 cm-biopsy (used for genotyping of the tail using clean scissors.
Genomic DNA obtained from tail pieces with a set of Qiagen Dneasy in accordance with the manufacturer's instructions. Genomic DNA is analyzed by means of PCR using primers designed regarding 3′-UTR of the human growth hormone (hGH) this transgenic vector. The area is unique to the sequence of a person, identified from mapping 3′-UTR DNA sequences of human growth hormone and mouse, providing the condition that the PCR reaction is not amplificare murine sequence. The primers, which amplified a fragment of hGH 368 P.N., and primers that hybridize with the vector sequences and amplified this cDNA insert, often used together with primers hGH. In these experiments, DNA from animals positive in respect of this transgene, will generate two lanes, lane 368 BP corresponding to the 3′-UTR-hGH fragment, and a strip of variable size corresponding to the cDNA-insert.
After confirmation, well the animals are transgenic (TG) they can back-to interbreed in inbred line location TG females with male wild-type or TG-male with one or two females of the wild type. After the birth of their young ones are divided by sex and their tails cut off pieces for genotyping.
Analysis of the expression level of mRNA of each transgenic animal is carried out using analysis of hybridization solution RNA or real-time PCR instrument (ABI Prism 7700 (PE Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions.
|WEDNESDAY 640 WITTEN|
|mg/200 ml||mg/500 ml|
|EDTA||100 µl||250 ál|
|5% phenol red||200 ál||500 ál|
All reagents are available from Sigma.
1. Stimulation of the expression of interferon-responsive promoter
In one series of experiments, conditioned medium (CM)containing Zcyto21 protein, generated by infection of cells A recombinant adenovirus containing the cDNA for Zcyto21 (AdZy-Zcyto21), at a multiplicity of infection of 400 particles per cell. CM collected at several time points within 40 hours after infection and stored at -20°C. SEE also receive from infection with recombinant adenovirus without cDNA (AdZy-source). Before using the CM portion was concentrated in 14 times in the centrifuge filter Millipore Ultrafree-15 (nominal limit ("clipping") of molecular weight 5000)) and then filtered through a centrifugal filter Millipore Ultrafree-15 (nominal limit ("clipping") of molecular weight equal to 100000)) to reduce the number of viral particles present in the rede, and finally filtered through a syringe filter, Millipore 0.2 μm for sterilization CM. Samples of concentrated CM diluted 1:2 in buffer for binding and incubated with cells from a mouse cell line for 5 hours at 37°C.
2. Antiviral activity Zcyto21
Another series of experiments feels antiviral activity Zcyto21. In these studies, antivirus analysis performed by seeding cells L929 (ATSS No. CCL-1) in culture medium RPMI 1640 containing 10% fetal calf serum, penicillin, streptomycin and L-glutamine in 96-well format at 50,000 cells per well. Adenovirus CM from cells A, infected or AdZy-Zcyto21, or AdZy-source, as described above, incubated with the cells during the night. The positive control in this assay provide using murine interferon-α, serially diluted 1:10, starting with 100 ng/ml Cells L929 only with culture medium provided a negative control. Treated cells incubated for 24 hours. Medium throw, add fresh medium and injected the virus encephalomyocarditis ATS No. vr129b) at multiplicity of infection of 0.1 (i.e. one viral particle for every ten cells L929). Cells incubated in the presence of the virus within 24 hours, and then the wells evaluated for cytopathic effect in percent (CPE).
3. Analysis of antiproliferative the th activity using cell line BaF3
BaF3 used to determine whether Zcyto21 antiproliferative properties. Kidney cells cub Chinese hamster (KSS) was stable transfusional expressing vector containing the CMV promoter plus intron a to the left (against the course of transcription from cDNA Zcyto21 or unrelated cDNA, named Zα30, using lipofectamine BRL. Stable transfetsirovannyh cells were seeded into cell factory in serum-free environment and allow them to grow for three days before collecting the conditioned medium and its concentration in the filter 5K to 10-fold concentration. Samples of the concentrated conditioned medium was stored at 4°C.
The following analysis is used to test antiproliferation BaF3. In 96-hole tablet perform 1:2 serial dilution only culture medium (RPMI 1640, 10% fetal calf serum, 1 mm sodium pyruvate, 2 mm L-glutamine) or murine IL-3 (starting with 50 PG/ml in culture medium) for a final volume of 100 μl. Fifty microlitres add the following composition as a divorced only culture medium and diluted mlL-3 tracks: interferon-α human (100 ng/ml, 10 ng/ml or 1 ng/ml diluted in the culture medium), interferon-β human (100 ng/ml, 10 ng/ml or 1 ng/ml diluted in the culture medium), murine interferon-β (100 ng/m is, 10 ng/ml or 1 ng/ml diluted in the culture medium), murine interferon-β (100 ng/ml, 10 ng/ml or 1 ng/ml diluted in the culture medium), Zcyto21 (2,5x, 0,5x or 0,1x) and murine Zα30 (2,5x, 0,5x or 0,1x).
Cell line BaF3 washed three times in culture medium, precipitation resuspended in the culture medium, the cells are considered and diluted in culture medium to 5000 cells/50 μl. Then fifty microliters diluted cells are added to each dilution of the samples. Test tablets ingibiruet in thermostat at 37°within three to four days. Then twenty microlitres dye Alomar blue added to each well and the plate incubated overnight at 37°C. the Tablets read on a fluorescent tablet reader with the wave excitation 544 nm and the wave emission 590 nm.
Tissue distribution Zcyto21 person in the panel of tissues using
A panel of cDNA samples from human tissues were subjected to screening for the expression of Zcyto21 using PCR. The panel was prepared in the laboratory and it contained 77 samples marathon cDNA (marathon cDNA and cDNA from a variety of healthy and cancerous human tissues and cell lines, as shown in table 6. The cDNA samples were from libraries available in the laboratory, or drugs the marathon cDNA from RNA, which was received in the laboratory, or were obtained from commercial the ski supplier, for example, Clontech (Palo Alto, CA) or Invitrogen (Carlsbad, CA). The marathon cDNA was obtained with the use of the kit for the amplification of the marathon cDNA (Clontech). To guarantee the quality of the samples panel conducted three tests on the quality control (QC): (1) To assess the quality of RNA used for libraries, obtained in the laboratory cDNA was tested on the average size of the inserts using PCR with vector oligo that were specific against vector sequences for individual cDNA library; (2) Standardization of the concentration of cDNA in the samples panel were obtained using standard PCR methods to amplify the full-size cDNA alpha-tubulin or G3PDH; and (3) the sample was sent for sequencing to check for possible contamination ribosomal or mitochondrial DNA. This panel was set up in 96-well format, which included a positive control sample of human genomic DNA (Clontech). Each well contained approximately 0.2 to 100 PG/ál cDNA. The first PCR reaction was set up using oligo ZC39270 (SEQ ID NO:14) and ZC39272 (SEQ ID NO:15), a mixture of DNA polymerase Advantage 2 (Clontech), and Rediload dye (Research Genetics, Inc., Huntsville, AL). Amplification was carried out as follows: 1 cycle at 94°C for 1 min, then 35 cycles of 94°C, 10 seconds; 67°C, 45 seconds and finished 3-minute final elongation at 72°C. the Correct size DNA is ragment observed in the brain, islet cells, prostate gland, testis, pituitary gland, placenta, of an ovarian tumor, lung tumor, CD3+cells and HPVS. Another PCR reaction was set up using oligo ZC39270 (SEQ ID NO:14) and ZC39271 (SEQ ID NO:16), a mixture of DNA polymerase Advantage 2 (Clontech), and Rediload dye (Research Genetics). Amplification was carried out as follows: 1 cycle at 94°C for 1 min, then 35 cycles of 94°C, 10 seconds; 65°C 30 seconds, 72°C, 30 seconds and finished 3-minute final elongation at 72°C. the Right size of the DNA fragment was observed in the pituitary gland, rectal cancer and tumors of the ovary.
|Cloth||the number of test samples||Cloth||the number of test samples|
|the adrenal gland||1||bladder||1|
|bone marrow||3||the brain||2|
|the brain of the fetus||3||the heart of the fetus||2|
|the kidney of the fetus||1||the liver of the fetus||2|
|easy fruit||1||the skin of the fetus||1|
|heart||2||muscle PLO is and||1|
|the pituitary gland||2||placenta||3|
|the prostate gland||3||rectum||1|
|salivary gland||2||skeletal muscle||1|
|the small intestine||1||spinal cord||2|
|stomach||1||library of adipocytes||1|
|the thymus||1||prostate SMC||1|
|thyroid gland||2||RPMI 1788 (ATS # CCL-156)||1|
|trachea||1||WI38 (ATS # CCL-75)||1|
|tumor of the esophagus||1||the lung tumor||1|
|tumor pecen the||1||tumor of the ovary||1|
|rectal tumor||1||tumor of the stomach||1|
|tumor of the uterus||2||selected from CD3+ library RVMS (stimulated)||1|
|library Nasal||1||library HPV (ATS # CRL-2221)||1|
|library of HPVS (ATS #CRL-2221) - selected||1||library MG63||1|
|K562(ATCC # CCL-243)||1|
From the foregoing it should be clear that, although there have been described specific variants of the present invention for the purpose of illustration, may be made of various modifications without deviating from the idea and scope of this invention. Thus, this invention is not limited by anything except the attached claims.
1. The selected polypeptide having antiviral activity, which includes amino acid residues 20-200 SEQ ID NO:2.
2. The selected polypeptide according to claim 1, further comprising a secretory signal sequence.
3. The selected polypeptide according to claim 2, where the secretory signal sequence comprises amino acid residues 1-19 SEQ ID NO:2.
4. The selected polypeptide comprising the amino acid sequence that is at least 90% identical to either amino acid residues 20-200 SEQ ID NO:2 or amino acid residues 1-200 SEQ ID NO:2, where specified, the selected polypeptide has antiviral activity.
5. The selected polypeptide according to claim 4, where amino acid sequence is at least 95% identical to either amino acid residues 20-200 SEQ ID NO:2 or amino acid residues 1-200 SEQ ID NO:2.
6. The selected polypeptide according to claim 4, having antiviral activity against the hepatitis virus.
7. The selected polypeptide according to claim 6, having antiviral activity against the virus of hepatitis C.
8. The selected polypeptide according to claim 6, having antiviral activity against the virus of hepatitis C.
9. The selected polypeptide according to claim 4, which is a recombinant polypeptide.
10. The selected polypeptide according to claim 4, where the polypeptide isolated from prokaryotic cells.
11. Vydeleny the polypeptide of claim 10, where the polypeptide isolated from Escherichia coli.
12. The selected polypeptide according to claim 4, where the polypeptide isolated from eukaryotic cells.
13. The selected polypeptide according to item 12, where the polypeptide isolated from mammalian cells.
14. The selected polypeptide according to item 13, where the polypeptide isolated from ovary cells Chinese hamster.
15. The selected polypeptide according to claim 4, attached to the polyethylene glycol.
16. The selected nucleic acid molecule which encodes a polypeptide, where the encoded polypeptide includes an amino acid sequence that is at least 90% identical to amino acid residues 20-200 SEQ ID NO:2, and where the encoded polypeptide has antiviral activity.
17. The selected nucleic acid molecule according to clause 16, where amino acid sequence is at least 95% identical to amino acid residues 20-200 SEQ ID NO:2.
18. The selected nucleic acid molecule according to clause 16, where the encoded polypeptide has an antiviral activity against the hepatitis virus.
19. The selected nucleic acid molecule according p, where the encoded polypeptide has an antiviral activity against the virus of hepatitis C.
20. The selected nucleic acid molecule according p, where the encoded polypeptide has an antiviral activity against the virus of hepatitis C.
21. The selected nucleic acid molecule according to the .16, where the encoded polypeptide is a recombinant polypeptide.
22. The selected nucleic acid molecule according to clause 16, where the encoded polypeptide isolated from prokaryotic cells.
23. The selected nucleic acid molecule according to item 22, where the encoded polypeptide isolated from Escherichia coli.
24. The selected nucleic acid molecule according to clause 16, where the encoded polypeptide isolated from eukaryotic cells.
25. The selected nucleic acid molecule according to paragraph 24, where the encoded polypeptide isolated from mammalian cells.
26. The selected nucleic acid molecule according A.25, where the encoded polypeptide isolated from cells of the ovary of the Chinese hamster.
27. The selected nucleic acid molecule according to clause 16, where any difference between the encoded polypeptide and the corresponding amino acid residues 20-200 SEQ ID NO:2 is due to a conservative substitution of amino acids.
28. The selected nucleic acid molecule according to item 16, comprising nucleotides 58-600 SEQ ID NO:1.
29. The selected nucleic acid molecule according to item 16, comprising nucleotides 58-603 SEQ ID NO:1.
30. The selected nucleic acid molecule according to item 16, comprising nucleotides 1-603 SEQ ID NO:1.
31. The selected nucleic acid molecule according to item 16, comprising nucleotides 1-600 SEQ ID NO: 1.
32. The selected nucleic acid molecule according to item 16, which encoded it the polyp is the Chida attach the peg.
33. The expression vector comprising the following functionally linked elements: a promoter of transcription; a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence that is at least 90% identical to amino acid residues 20-200 SEQ ID NO:2, where the encoded polypeptide has antiviral activity; and a transcription terminator.
34. The expression vector for p, where the encoded polypeptide comprises amino acid sequence which is at least 95% identical to amino acid residues 20-200 SEQ ID NO:2.
35. The expression vector for p, where the encoded polypeptide comprises amino acid residues 20-200 SEQ ID NO:2.
36. The expression vector for p, where the encoded polypeptide comprises amino acid residues 1-200 SEQ ID NO:2.
37. The expression vector for p, where the nucleic acid molecule includes the nucleotide 58-600 SEQ ID NO: 1.
38. The expression vector for p, where the nucleic acid molecule includes the nucleotide 58-603 SEQ ID NO: 1.
39. The expression vector for p, where the molecule is a nucleic acid also encodes a secretory signal sequence, is functionally associated with the polypeptide.
40. The expression vector according to § 39, where the encoded secretory signal sequence comprises amino acid residues 1-19 SEQ ID NO:2.
41. The expression vector for p can transform or transferout cell host.
42. A method of obtaining a polypeptide comprising amino acid sequence being at least 90% identical to either amino acid residues 20-200 SEQ ID NO:2 or amino acid residues 1-200 SEQ ID NO:2, and the method comprises the stage of culturing recombinant host cells that contain the expression vector according p and that produce the polypeptide, where the polypeptide has antiviral activity.
43. The method according to § 42, where amino acid sequence is at least 95% identical to either amino acid residues 20-200 SEQ ID NO:2 or amino acid residues 1-200 SEQ ID NO:2.
44. The method according to § 42, additionally providing for the extraction of the polypeptide from the cultured recombinant host cells.
45. The antibody or antibody fragment that specifically bind to a polypeptide according to claim 4 and obtained by a process comprising use of a polypeptide according to claim 4.
46. The composition containing the selected polypeptide comprising amino acid sequence being at least 90% identical to either amino acid residues 20-200 SEQ ID NO:2 or amino acid residues 1-200 SEQ ID NO:2, provided the polypeptide has antiviral activity, and a pharmaceutically acceptable carrier.
47. The composition according to item 46, where amino acid sequence is at least n is 95% identical to either amino acid residues 20-200 SEQ ID NO:2, or amino acid residues 1-200 SEQ ID NO:2.
48. The composition according to item 46, where the polypeptide is a polypeptide that is attached to the peg.
49. The selected nucleic acid molecule comprising the nucleotide 58-600 SEQ ID NO:1, where the molecule encodes a polypeptide having antiviral activity.
50. The selected nucleic acid molecule according to § 49, where the selected nucleic acid molecule includes the nucleotide 58-603 SEQ ID NO:1.
51. The selected nucleic acid molecule according to § 49, where the selected nucleic acid molecule includes the nucleotide 1-600 SEQ ID NO:1.
52. The selected nucleic acid molecule according to § 49, where the selected nucleic acid molecule includes the nucleotide 1-603 SEQ ID NO:1.
53. The selected polypeptide having antiviral activity, which includes amino acid residues 20-200 SEQ ID NO:5.
54. The selected nucleic acid molecule comprising the nucleotide 58-600 SEQ ID NO:4, wherein the molecule encodes a polypeptide having antiviral activity.
55. The selected nucleic acid molecule according to item 54, where the selected nucleic acid molecule includes the nucleotide 58-603 SEQ ID NO:4.
56. The selected nucleic acid molecule according to item 54, where the selected nucleic acid molecule includes the nucleotide 1-600 SEQ ID NO:4.
57. The selected nucleic acid molecule according to item 54, where the selected molecule well Lanovoy acid includes a nucleotide 1-603 SEQ ID NO:4.
58. The selected nucleic acid molecule encoding a polypeptide comprising amino acid residues 20-200 SEQ ID NO:5, where the encoded polypeptide has antiviral activity.
59. The selected nucleic acid molecule according to § 58, where the encoded polypeptide has an antiviral activity against the hepatitis virus.
60. The selected nucleic acid molecule according to § 58, where the encoded polypeptide isolated from eukaryotic cells.
61. The selected nucleic acid molecule according p, where the encoded polypeptide isolated from mammalian cells.
62. The selected nucleic acid molecule encoding a polypeptide containing amino acid residues 20-200 SEQ ID NO:7, where the encoded polypeptide has antiviral activity.
63. The selected nucleic acid molecule according to item 62, where the nucleic acid molecule contains a nucleotide 135-700 SEQ ID NO:6.
64. The selected nucleic acid molecule according to item 62, where the encoded polypeptide contains amino acid residues 1-200 SEQ ID NO:7.
65. The selected nucleic acid molecule according p, where the nucleic acid molecule contains a nucleotide 98-700 SEQ ID NO:6.
66. The selected nucleic acid molecule according to item 62, where the encoded polypeptide has antiviral activity relative to the hepatitis virus.
67. The selected nucleic acid molecule according to item 62, where kopirovany the polypeptide is a recombinant polypeptide.
68. The selected nucleic acid molecule according p, where recombinant polypeptide selected from prokariotic cells.
69. The selected nucleic acid molecule according p, where recombinant polypeptide isolated from Escherichia coli.
70. The selected nucleic acid molecule containing the nucleotide 155-700 SEQ ID NO:6 encoding the polypeptide having antiviral activity.
71. The selected polypeptide containing amino acid residues 20-200 SEQ ID NO:7 having antiviral activity.
30.06.2000 - claims 1 and 2;
20.04.2001 - PP-5, 9-17, 21-52;
29.06.2001 - PP-8, 18-20, 53-71.
FIELD: biotechnology, biochemistry.
SUBSTANCE: sequence of polynucleotide is determined by change of nonlinear optical signal or linear optical signal associated with nonlinear signal recorded in interaction of enzyme immobilized in fixed position with separate base of the targeted polynucleotide that can interact with nucleotide chain and move continuously along it. Using the invention allows carrying out to determine the sequence of polynucleotides of large size with high degree by automatically controlled process. Invention can be used in sequencing of polynucleotide sequences.
EFFECT: improved method of sequencing.
24 cl, 2 dwg
FIELD: medicine diagnosis, molecular biology.
SUBSTANCE: invention relates to method for detection of DNA of human Lyme borreliosis excitant in peripheral blood, cell culture and mites. Claimed method includes DNA isolation from biological material; synthesis of primers; PCR with using synthesized primers and evaluation of obtained data. In method BBs14.F/BBs14.R - 5'-AAGAATACATTAAGTGCGATANN-3', 5'-CAATCCACTTAATTTTGTGTTAT-3' primers to gene site encoding 16S rRNA are used. Kit for this method also in disclosed.
EFFECT: high-specific, sensitive and simplified method.
2 cl, 2 dwg, 5 tbl
FIELD: medicinal microbiology, molecular biology, biochemistry.
SUBSTANCE: invention describes a method for assay of virulence loss by strains of plague microorganisms because of loss of the pigmentation region. Method involves carrying out the polymerase chain reaction (PCR) by using primers IH 1- 5'-TTT-ACC-GCA-ACA-ACA-TCA-TCC-3' and IY 4- 5'-GGG-CTG-AAA-CCA-CTG-AGA-TG-3' in the reaction mixture of the following composition: 1 X PCR-buffer, MgCl2, 2.5 mM; dNTP mix, 0.3 mM; primer -1H, 3.52 pmole; primer IY-4-, 0.645 pmole; Tag DNA polymerase, 0.2 U for 35 cycles of amplification (denaturation at 94°C for 1 min, annealing at 60C for 1 min, elongation at 72°C for 4 min). Virulence of strain is determined by the presence of specific amplicons of size 2468 nucleotide pairs for strains of the main subspecies and 2666 nucleotide pairs for non-main strains of subspecies of the main subspecies indicating the absence of the pigmentation region. Using proposed methods provides rapid and precise detection of avirulent strains of plague microorganism and to avoid danger, non-humane and expensive experiments in animals.
EFFECT: improved assay method.
3 cl, 4 ex
FIELD: animal science, genotyping in cattle.
SUBSTANCE: the present innovation deals with genotyping in cattle by the alleles of BoLA DRB 3 gene of the main histocompatibility complex (MHC) class II to detect animals predisposed to persistent lymphocytosis. Exon2 of BoLA DRB 3 gene should be amplified, (the size of PCR-product corresponds to 284 pn) followed by the analysis of restriction polymorphism of this area due to endonucleases Rsa I, Hae III, BstYI. If at restriction by Rsa I it is possible to detect the fragments 141, 54-51, 39 pn, amplificate should be similarly treated with restrictase Bst 2U I. In this case, allelic variant BoLA DRB 3*7 forms the fragments 24, 57, 89 and 111 pn; allelic variant BoLA DRB 3*8 - 24, 80 and 180 pn; allelic variant BoLA DRB 3*9 - 24, 57 and 203 pn. Thus, it is possible to successfully differentiate alleles BoLA DRB 3*7, BoLA DRB 3*8 and BoLA DRB 3*9. The innovation enables to detect leucosis-sensitive animals at allel BoLA DRB 3*8.
EFFECT: higher accuracy of detection.
SUBSTANCE: the present innovation deals with laboratory diagnostics of multiple curative tuberculosis. For solving the task it is necessary to detect specific mutations in gene rpoB with polymerase chain reaction with allel-specific primers for codons 516, 526,531. At the absence of mutations in the given codon it is possible to observe a codon-dependent allel-specific PCR-amplification of gene rpoB fragment; availability of mutation in position correspondent to primer's 3'-terminal leads to nonmating of primer and matrix and polymerase's incapacity for the synthesis of complementary fragment. Thus, availability of mutation (RIF-resistant phenotype) leads to the absence of indicator fragment. Simultaneously, it is necessary to conduct three PCR reactions oriented for three codons 516, 526, 531, and PCR result should be analyzed due to electrophoresis in horizontal agarose mini-gel. At the lack of one out of indicator fragments 167, 181, 214 it is possible to conclude upon mutation and mycobacterial resistance to rifampicin.
EFFECT: higher accuracy of quick test detection.
3 dwg, 2 ex
SUBSTANCE: the present innovation deals with diagnostics in vitro hypertension diathesis caused with genetic factors due to detecting in a sample (a) SNP which should be detected in exon 8(C→T) of hsgk1 gene and presented in SEQ ID NO:1, and also (B) SNP which should be detected in intron 6(T→C) of hsgk1 gene and localized in a fragment of 551 size being opposite the transcription against SNP in exon 8 according to item (a), or both SNP according to items (a) and (b). The innovation refers, also, to application of such SNP for obtaining a diagnostic kits and sets for carrying out diagnostics of hypertension diathesis caused with genetic factors. The method provides concrete sequences of hsgk1 gene that cause hypertension diathesis.
EFFECT: higher accuracy and efficiency of diagnostic analysis.
3 cl, 2 ex, 5 tbl
SUBSTANCE: the present innovation deals with isolating DNA out of lymphocytes of peripheral venous blood due to polymerase chain reaction technique, synthesis of DNA amplification of genes PON1 and CETP fragments within the limits of which polymorphous nucleotide substitutions are localized, carrying out the analysis of polymorphism in the lengths of restriction fragments of amplicons and at detecting genotypes PON*BB and CETP*II one should predict the risk in development of hypertonic disease.
EFFECT: higher accuracy of prediction.
4 ex, 4 tbl
SUBSTANCE: the present innovation deals with hybridization of chimeric amplified fluorescently labeled product consisted of gene sequences that participate in certain chromosomal leucosis-associated translocation with oligonucleotide microchip. The method deals with reverse transcription, two-stage "socket" multiplex PCR; labeling chimeric PCR-product in the second round of PCR; hybridization upon a biochip that contains original set of oligonucleotides. The innovation enables to accelerate and simplify the technique applied.
EFFECT: higher accuracy and efficiency of identification.
16 cl, 4 dwg, 8 ex, 3 tbl
SUBSTANCE: method involves separating DNA from peripheral venous blood leukocytes using DNA synthesis polymerase chain reaction method, class II DQB1 HLA gene specificity amplification method. When detecting DQB1*0302/07/08 allele and DQB1*02/DQB1*03 genotype in Bashkirian people and DQB1*0302/08 allele in Russian people, DQB1*02 specificity and DQB1*0302/08 allele in Tatar people, type 1 diabetes mellitus risk is predicted to take place.
EFFECT: high accuracy of prognosis.
FIELD: molecular biology, biochemistry.
SUBSTANCE: invention proposes a method for amplification of nucleic acid (NA) involving carrying out a primer reaction elongation on matrix in the presence of at least one nucleotide as a precursor of DNA recognized by a cleaving agent, digestion of elongated primer but retaining a bind with matrix NA by indicated agent to form fragments comprising free OH-group at 3'-end and elongation of prepared fragment on matrix. The distinctive feature of a method is the combining all reactions in a single vessel and using the reaction mixture containing all necessary components, and DNA polymerase, a cleaving agent and the reaction conditions are selected by taking into account their simultaneous functioning. Invention can be used in analytical methods of different designation associated with determination of quantitative and qualitative indices of NA.
EFFECT: improved method for amplification.
28 cl, 2 dwg, 8 ex
FIELD: biotechnology, medicine.
SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.
EFFECT: valuable properties of new cytokine.
18 cl, 5 tbl, 1 dwg, 55 ex
FIELD: preparative biochemistry, medicine, pharmacology.
SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.
EFFECT: improved purifying method.
8 cl, 1 tbl, 6 ex
FIELD: preparative biochemistry, medicine, pharmacology.
SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.
EFFECT: improved purifying method.
8 cl, 1 tbl, 6 ex