SUBSTANCE: there is offered application of group of survival-improving polypeptide cone cells originated from rod cells and designated as RDCF, and also coding molecules of nucleic acid to prepare medicines, particularly pharmaceutical compositions used to treat retinal dystrophy. Methods for preparing RDCF by recombinant DNA technologies, and required aids, as well as preparation of antibodies distinguishing said polypeptides are described.
EFFECT: improved clinical effectivenesses.
12 cl, 19 dwg, 1 ex
The technical field of the invention
The present invention relates to methods and compositions for the detection and treatment of degenerative diseases of the retina. In particular, the invention relates to a protein having a protective function against degeneration of cones, the molecules of nucleic acid encoding such a protein, antibodies that recognize the protein, and methods for diagnosing degenerative diseases of the retina.
Background of invention
Photoreceptors are specialized subset of neurons of the retina responsible for vision. The photoreceptors consist of rods and cones, which are photosensitive cells in the retina. Each stick and cone produce specialized cilia, referred to as the outer segment, which has the ability to phototransduction. Sticks contain specific light-absorbing visual pigment rhodopsin. In humans there are three classes of cones, characterized by the ability to Express different visual pigments: pigments of the cones, specific against blue color, specific in terms of its green color and specific in relation to red. Each type of visual pigment protein characterized by the ability to absorb light in a certain range is the area of the wavelengths, other than the wavelength range of other types of protein. Rhodopsin sticks mediates scotophase vision in dim light), while the pigments of the cones are responsible for photocopies vision (in bright light). Red, blue and green pigments are also the basis of color vision in humans. Visual pigments in rods and cones respond to light and generate bioelectric potential in the cell, generating an output signal, i.e. in the bipolar neurons of the sticks, which is then transmitted ganglion neurons of the retina, causing visual stimulus to the visual areas of the cerebral cortex.
In humans, many diseases of the retina are accompanied by progressive degeneration and eventually the death of photoreceptors, which inevitably leads to blindness. All diseases associated with degeneration of photoreceptors, such as hereditary dystrophy of the retina (e.g., retinitis pigmentosa), age related macular degeneration and other maculopathy, or retinal detachment, characterized by progressive atrophy and loss of function of photoreceptor outer segments. In addition, photoreceptor death or loss of photoreceptor function in patients suffering from retinal degeneration leads to a partial differentiation of the secondary neurons of the retina (bipolar cells in the horizontal cells of sticks), what causes a decrease in overall efficiency of propagation of an electrical signal generated by the photoreceptors. Secondary changes in glue and pigment epithelium caused by the degeneration of photoreceptors, cause changes in blood vessels, leading to ischemia and gliosis. For treatment of these conditions, you can apply the methods based on the use of trophic factors, which have the ability to protect the photoreceptors from cell death and/or restore the function of dysfunctional (atrophic or dystrophic) photoreceptors.
The progressive development of these States shows the consecutive loss of two classes of photoreceptors: first perish sticks due to the direct genetic damage or disturbance caused by the environment, or have an unknown origin, which leads to night blindness and reduced field of view, then the inevitable death of cone cells, leading to complete blindness. Thus, the loss cone is the result of indirect actions, because they are not exposed to the primary damage.
Currently, not all identified genes associated with retinal degeneration. The identification of these genes may allow as make the diagnosis, and to develop effective methods of therapeutic the ski of treatment.
Summary of the invention
The invention generally relates to a new family of genes derived from Bacillus factor viability of cone cells (Rod-derived Cone Viability Factor (Rdcvf). The first object of the invention is selected polypeptide having the amino acid sequence represented in SEQ ID NO: 2 or SEQ ID NO: 4. It is established that such polypeptide or its fragments are present in the eye of the person suffering from retinal degeneration, in substantially smaller quantities than in the human eye, which does not suffer from retinal degeneration. Fragments selected polypeptide having the amino acid sequence represented in SEQ ID NO: 2 or SEQ ID NO: 4 include polypeptides containing from about 5 to 10 amino acids, preferably from about 10 to about 20 amino acids, more preferably from about 20 to about 100 amino acids, and most preferably from about 20 to about 50 amino acids. The object of the invention relates to a new polypeptide originating from the body of a mammal, in particular of the body of the mouse or human, as well as its fragments, variants and derivatives, variants and derivatives of the fragments and their analogs, which can be used in biological, diagnostic or therapeutic purposes. Under the volume from which retene are also polypeptides, almost identical to the polypeptide having the amino acid sequence represented in SEQ ID NO: 2 or SEQ ID NO: 4, for example, amino acid sequence represented in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.
The second object of the invention is selected nucleic acid molecule having the nucleotide sequence represented in SEQ ID NO: 1 or SEQ ID NO: 3. Under the scope of the invention also includes nucleic acids, almost the same as the nucleic acid having the nucleotide sequence represented in SEQ ID NO: 1 or SEQ ID NO: 3, for example, the nucleotide sequence represented in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. The preferred embodiment of the invention is selected molecule is a nucleic acid that encodes a polypeptide selected from the group comprising the polypeptide presented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, for example, nucleotides 45-374 SEQ ID NO: 1, nucleotides 26-676 SEQ ID NO: 3, nucleotides 24-353 SEQ ID NO: 5, nucleotides 48-686 SEQ ID NO: 7, nucleotides 265-570 SEQ ID NO: 9, nucleotides 300-770 SEQ ID NO: 11 or nucleotides 331-738 SEQ ID NO: 13. In a preferred embodiment of the invention selected DNA is in the form of a vector molecule containing the DNA represented in SEQ ID NO: 1 or SEQ ID NO: 3.
The third object of the present image is a shadow is a method of diagnosis of retinal dystrophy in humans, provide for the identification of reducing the transcription of messenger RNA transcribed on the basis of Rdcvf-1 - or Rdcvf'2-coding DNA in the eye of a mammal, preferably human, where such decrease transcription is a diagnostic indication that the patient is suffering from retinal degeneration and / or pathological aging (ARMD). Another variant embodiment of the invention relates to a method of diagnosis of retinal dystrophy in the body of a mammal, preferably human, providing a measurement of the amount of the polypeptide Rdcvf1 or Rdcvf2 or its fragments in the eye of the person in respect of whom there is the assumption that he was suffering from retinal degeneration, appraisal reduction amount of the polypeptide or its fragments compared to the amount of the polypeptide or its fragments in the human eye, does not suffer from degeneration of the retina, which serves as a diagnostic sign of the presence of the patient's retinal dystrophy.
The next object of the present invention are antisense polynucleotide, which regulate the transcription of the gene Rdcvf1 or Rdcvf2; another embodiment of the invention is double-stranded RNA, which has the ability to regulate gene transcription Rdcvf1 or Rdcvf2.
Another object of the invention is a method of obtaining the above polypeptides,fragments of polypeptides, variants and derivatives, fragments, variants and derivatives and their analogues. The preferred embodiment of this object of the invention are methods of obtaining the above polypeptides Rdcvf1 involving culturing host cells that have built-in expression vector containing obtained by exogenous polypeptide encoding Rdcvf1 or Rdcvf2, under conditions suitable for expression of the polypeptides Rdcvf1 or Rdcvf2 in the host, and the subsequent allocation of the expressed polypeptide.
Another object of the invention is the products, compositions, processes and methods, which apply the above polypeptides and polynucleotide including scientific, biological, clinical and therapeutic purposes.
Other specific preferred objects of the invention are antibody or its fragment with the ability to specifically bind to a polypeptide having the amino acid sequence represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, i.e Rdcvf1 or SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, i.e. Rdcvf2. In this regard, the most preferred objects are antibodies having a high selectivity in respect of the polypeptides Rdcvf1 or Rdcvf2 mammal, preferably a mouse, and most preferably human, or fragments of such is polypeptides Rdcvf1 or Rdcvf2. Related with this object of the invention is an antibody or its fragment, which is(s) associated with a fragment or part of the amino acid sequence represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.
The next object of the invention are methods of treating disease in a patient, where the disease is mediated by or associated with changes in gene expression Rdcvf1 or Rdcvf2, for example, by reducing the level of polypeptide RDCVF1 or RDCVF2 in the eye, involving the administration to a patient in need, a therapeutically effective amount of protein RDCVF1 or RDCVF2 presented in SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, or a related protein or its fragment or part. The invention also proposed methods of diagnosis using immunoassay disease or condition associated with decreased expression of the gene Rdcvf1 or Rdcvf2 or with a decrease in the number of polypeptide RDCVF1 or RDCVF2, the patient, involving the application of antibody, which binds to the polypeptide having the amino acid sequence represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, or a fragment or part.
Another object of the invention are cells that can proliferate in vitro, preferably vertebrate cells, which are capable of when airasian in culture to produce the polypeptide, having the amino acid sequence represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 or its fragments, where cells contain controls the transcription of DNA sequences other than mouse or human transcription-controlling sequences Rdcvf1 or Rdcvf2, and controlling the transcription of the sequence controls the transcription of DNA which encodes a polypeptide having the amino acid sequence represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 or its fragments.
A related object of the present invention is a method of producing polypeptides Rdcvf1 or Rdcvf2 involving culturing the host cell containing embedded in her expression vector, which contains obtained by exogenous polynucleotide encoding Rdcvf1 or Rdcvf2, under conditions suitable for expression of the polypeptides Rdcvf1 or Rdcvf2 in the cage-owner, with receipt of the expressed polypeptide, and the allocation of the expressed polypeptide.
Another object of the present invention are methods of analysis and kits comprising the components necessary to detect abnormal expression, for example, reduced compared to the normal expression level of polynucleotides Rdcvf1 or Rdcvf2 or polypeptides or f is of Ahmetov in tissue samples, obtained from the patient, where such kits contain, for example, antibodies that have the ability to bind with Rdcvf1 or Rdcvf2, or oligonucleotide probes that hybridize with polynucleotide according to the invention. In a preferred embodiment of the invention, such kits contain instructions describing in detail the processes of application component set.
The next object of the invention is a polypeptide Rdcvf1 or Rdcvf2 intended for treatment of a human or animal. A related object of the invention is the use of the polypeptide Rdcvf1 or Rdcvf2 or its fragment, the nucleotide coding Rdcvf1 or Rdcvf2, or its fragment, or antibody, which binds to Rdcvf1 or Rdcvf2 or its fragment, for manufacturing a medicinal product intended for the treatment of retinal dystrophy.
Another object of the invention is having a protective effect against retinal agent containing polypeptide selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, and optionally a pharmaceutically acceptable carrier. A related object of the invention relates to pharmaceutical compositions containing the polypeptide Rdcvf1 or Rdcvf2 or its fragment, nucleotide encoding Rdcvf1 or Rdcvf2 or its part, intended to ensure distri the retina. Another related object of the invention is a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, and a pharmaceutically acceptable carrier.
Another related object of the invention is a method of treating retinal degeneration, which consists in the introduction to a patient in need, a therapeutically effective amount of a polypeptide selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, in combination with a pharmaceutically acceptable carrier.
Another object of the invention are methods of identifying molecules that have the ability to contact Rdcvf1 or Rdcvf2 and/or modulate the activity of Rdcvf1 or Rdcvf2, or molecules that have the ability to bind to nucleotide sequences that modulate transcription or translation Rdcvf1 or Rdcvf2. Such methods are described, for example, in US patents 5541070, 5567317, 5593853, 5670326, 5679582, 5856083, 5858657, 5866341, 5876946, 5989814, 6010861, 6020141, 6030779 and 6043024, the contents of all of these patents are included in full in the present description by reference. Molecules identified by such methods also fall under the scope of the present invention.
The following object of the invention relative to the tsya to methods of introducing nucleic acids according to the invention in one or more types of tissue of the patient, in need of such treatment, so that the inside fabric was the expression and/or secretion of the cells of one or more proteins encoded by nucleic acids.
Another object of the invention is a method of obtaining intended for implantation of photoreceptor cells, providing for the cultivation of photoreceptor cells together with the RdCVF1 or RdCVF2.
Other purposes, features, advantages and objects of the present invention will be obvious to specialists on the basis of the following description. It should be understood that the following description and specific examples regarding preferred embodiments of the invention presented only for illustrative purposes. For specialists in this field after studying the following description and explore the other sections of the present invention will be apparent that it is possible to make various changes and modifications within the scope and essence of the invention.
Brief description of drawings
The drawings show:
figure 1 is the nucleotide sequence of murine Rdcvf1 obtained by expression cloning and amino acid sequence of murine RdCVF1;
figure 2 - amino acid sequence of murine Rdcvf1L;
figure 3 is the nucleotide sequence of the human Rdcvf1 and aminos the PCI-e slot sequence of the human Rdcvf1;
figure 4 is the nucleotide sequence of the human Rdcvf1L and amino acid sequence of human Rdcvf1L;
figure 5 is the nucleotide sequence of murine Rdcvf2 and amino acid sequence of murine Rdcvf2;
figure 6 is the nucleotide sequence of murine Rdcvf2L and amino acid sequence of murine Rdcvf2L;
7 is the nucleotide sequence of the human Rdcvf2 and amino acid sequence of human Rdcvf2;
on Fig - comparison of amino acid sequences of short forms Rdcvf: (SEQ ID NO 2, 6, 10 and 14) and long forms of Rdcvf: (SEQ ID NO 4, 8, 12 and 14);
figure 9 - primers for GST-Rdcvf1;
figure 10 - multiple comparisons of the sequences of RDCVF1/RDCVF2;
figure 11 - results of comparative sequence analysis of mouse and human RDCVF2;
on Fig - the results of multiple comparative sequence analysis of mouse Rdcvf2 and sequences of EST clones be552141, bi517442, bg707818 and bi603812;
on Fig - the results of multiple comparative sequence analysis Rdcvf1 and sequences of EST clones bg299078, ai716631, bg294111, be108041 and bg395178;
on Fig - EST-sequence bg299078 adjusted to match the sequence of Rdcvf1;
on Fig - EST-sequence bg294111 adjusted to match posledovatelnostyu Rdcvf1L;
on Fig - the results in real-time RT-PCR analysis of expression of arrestin sticks (a) and RdCSF1 (B) a 5-week retina of C57BL/6 and 5-week N (marked in grey) and SN/NO and N (red);
on Fig - results FROM RT-PCR analysis, suggesting that the expression of Rdcvf2 depends on the presence of rods and its expression occurs in other parts of the CNS;
on Fig - results of PCR analysis, suggesting that the expression of RdCVF1 depends on the presence of sticks.
Detailed description of the invention
All the cited patent applications, patents and literary publications included in this description in full by reference.
When implementing the present invention apply numerous suitable for this purpose, methods in molecular biology, Microbiology and recombinant DNA. These methods are well known and described, for example, in Current Protocols in Molecular Biology, volumes I, II and III (Ed. by F.M.Ausubel), 1997; Sambrook and others, 1989, Molecular Cloning: A Laboratory Manual, published by Cold Spring Harbor Laboratory Press, 2nd ed., Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, volumes I and II (edited by D.N.Glover.), 1985; Oligonucleotide Synthesis (Ed. by M.L.Gait.), 1984; Nucleic Acid Hybridization (edited by Hames and Higgins), 1985; and reduced Translation (edited by Hames and Higgins), 1984; Animal Cell Culture (Ed. by R.I.Freshney), 1986; Immobilized Cells and Enzymes, published by IRL Press, 1986; Perbal, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology, and the d-Academic Press, Inc., 1984; Gene Transfer Vectors for Mammalian Cells (Ed. J.H.Miller and M.P.Calos published by Cold Spring Harbor Laboratory, 1987; Methods in Enzymology, volume 154 and vol 155 (Ed. by Wu and Grossman, and Wu, respectively).
In the context of the present description, the term "differentially expressed gene" refers to (a) a gene containing at least one of the above in the present description of DNA sequences (e.g., presented in figure 1 and SEQ ID NO: 1, or presented in figure 2 and SEQ ID NO: 3); (b) any DNA sequence that encodes the amino acid sequence that is encoded by the DNA sequences presented in this description (e.g., presented in figure 1 and SEQ ID NO: 2 or presented in figure 2 and SEQ ID NO: 4); or (C) any DNA sequence that is almost similar to that shown in the present description coding sequences.
In the context of the present description, the term "virtually the same" as applied to the nucleotide sequence in its broadest sense, refers to a nucleotide sequence corresponding to the nucleotide sequence, with which comparison is made, where the corresponding sequence encodes a polypeptide that has almost the same structure and function as the polypeptide encoded by the nucleotide sequence, with which comparison is made, for example, in case the e, if replacement of amino acids do not affect the function of the polypeptide. Preferably almost the same as the nucleotide sequence encodes the polypeptide encoded by the nucleotide sequence, with which comparison is made. Preferably, the percentage identity between almost the same as the nucleotide sequence and the nucleotide sequence, with which comparison is made, is at least 90, more preferably at least 95, more preferably at least 99%. The comparison of sequences is carried out using the algorithm of comparison sequences Smith-Waterman (see, for example, M.S. Waterman, Introduction to Computational Biology: Maps, sequences and genomes, published by Chapman & Hall., London: 1995, ISBN 0-412-99391-0, or on the website http://www-hto.usc.edu/software/seqaln/index.html). In the program the localS, version 1.16, use the following parameters: match: 1, penalty for mismatch: 0,33, the penalty for opening a gap: 2, the penalty for extending gap: 2. Nucleotide sequence that is "almost the same" nucleotide sequence with which comparison is made, hybridized with nucleotide sequence with which comparison is made, in 7% sodium dodecyl sulfate (LTOs), 0.5 m NaPO4, 1 mm add at 50°C With washing 2X SSC, 0.1% of LTOs at 50°C, more predpochtitelno 7% sodium dodecyl sulfate (LTOs), 0.5 m NaPO4, 1 mm add at 50°C With washing 1X SSC, 0.1% of LTOs at 50°C, more preferably in 7% sodium dodecyl sulfate (LTOs), 0.5 m NaPO4, 1 mm add at 50°C With washing 0.5x SSC, 0.1% of LTOs at 50°C, preferably in 7% sodium dodecyl sulfate (LTOs), 0.5 m NaPO4, 1 mm add at 50°C With washing 0,1X SSC, 0.1% of LTOs at 50°C, more preferably in 7% sodium dodecyl sulfate (LTOs), 0.5 m NaPO4, 1 mm add, 0.5 M NaPO4, 1 mm add at 50°C With washing 0,1X SSC, 0.1% of LTOs at 65°C, while it still retains the ability to encode a functionally equivalent gene product.
Presented in this description of differentially expressed genes expressed in the tissue of the eye and, in particular, they are produced in the cells of the sticks, but in the tissues of a person suffering from retinal degeneration, such as retinitis pigmentosa, age related macular degeneration syndrome Barda, syndrome Bessin-Kornzweig, the disease best, choroidea, atrophy of the cerebral convolutions, congenital Amoros, syndrome Resana, syndrome Stargardt and usher syndrome, they are produced in small quantities, i.e. the level of expression is lower than in the corresponding tissues of people who are not suffering from retinal degeneration. Levels of messenger RNA, transcribed on the basis of differentially expressed genes, and the protein translated from this mRNA, which is constituted is contained in the tissues of sticks and/or associated with such tissues, at least twice, preferably at least five times, more preferably at least ten times, most preferably at least about one hundred times lower than the levels of mRNA and protein detected in the corresponding tissues of people who do not suffer from retinal degeneration. In the present description, such a decrease in the transcription of mRNA Rdcvf1 or Rdcvf2 labeled as "reduced transcription".
In the context of the present description, the term "a host cell" refers to a prokaryotic or eukaryotic cell, bearing heterologous DNA that has been integrated into the cell by, for example, electroporation, precipitation with calcium phosphate, microinjection, transformation, viral infection, etc.
In the context of the present description, the term "heterologous" means "originating from different natural sources" or refers to the condition not occurring under natural conditions. For example, if the cell host transformed with DNA or a gene from another organism, in particular, from an organism of another species, the gene is heterologous to the cell master, and against the offspring of the host cells carrying the gene. Similarly, the term "heterologous" refers to a nucleotide sequence obtained and integrated into the cell of the same Ref is underwater type, occurring in natural conditions, but which is not naturally occurring, for example, is present in a different number of copies, or is under the control of other regulatory elements.
Vector molecule is a molecule of nucleic acid, which can be integrated heterologous nucleic acid and which can then be entered in the appropriate cell of the host. Preferably the vectors have one or multiple sites of initiation of replication and one or more web sites in which you can embed recombinant DNA. Typically, the vectors have certain characteristics that cells carrying the vector, can be distinguished from cells that do not have vectors, for example, they encode genes that contribute to drug resistance. Usually used such vectors as plasmids, viral genomes, and (especially in the case of yeast and bacteria) "artificial chromosomes".
In the present description "plasmids", usually denoted by a capital letter "p"preceded or followed by capital letters and/or numbers in accordance with the standard rules of signs known to specialists in this field. Specified in the present description plasmids, either go on sale or are publicly available without ogran the values, or they can be constructed from available plasmids using standard well known published methods. Many plasmids and other cloning and expression vectors that can be used according to the present invention, are well known experts in this field can easily use them. In addition, experts in this field can easily construct any number of other plasmids suitable for use according to the invention. The person skilled in the art based on the present description, it is easy to understand the properties, design and application of such plasmids and other vectors of the present invention.
The term "isolated" means that the product is isolated from its original environment (for example, from the natural environment if it is naturally occurring). For example, naturally occurring polynucleotide or polypeptide present in the body of the living animal is not selected, but the same polynucleotide or polypeptide, separated from some or all of the collateral materials present in natural conditions (natural system)is selected, even if it then being introduced to the natural system. Such polynucleotide can be a part of the vector and/or these polynucleotide or polypeptides can be a part of the song and they still fall under this concept, if such vector or composition is not located in their natural environment.
In the context of the present description, the term "controlling the transcription of the sequence" refers to DNA sequences, such as initiating sequence, enhancer sequence and the promoter sequence, which induce, inhibit or otherwise control transcription of protein coding nucleotide sequences with which they are functionally linked.
In the context of the present description, the term "sequence that controls transcription of Rdcvf1 or sequence controlling the transcription of Rdcvf2" refers to any of such regulatory transcription of sequences that normally associated with the gene of Rdcvf1 or Rdcvf2 mammals, preferably with Rdcvf2 gene present in the genome of mouse or human.
In the context of the present description, the term "obtained from the body in addition to human sequence that controls transcription" refers to any sequence that controls transcription, which is not present in the human genome.
In the context of the present description, the term "polypeptide" is used interchangeably with the terms "polypeptide" and "protein(s).
In the context of the present description, "chemical derivative" p is dipeptide according to the invention means a polypeptide according to the invention, which contains additional chemical fragments that are not usually contained in the molecule. Such fragments can improve the solubility, absorption, biological half-life of molecules, etc. In an alternative embodiment, the fragments can reduce the toxicity of the molecule, eliminate or reduce any undesirable side effects of molecules, etc. Fragments, which are able to exert such actions are described, for example, in Remington''s Pharmaceutical Sciences, 16th ed., published by Mack Publishing Co., Easton, Pa. (1980).
In the context of the present description "neuroprotective agent" means a compound that prevents the degeneration of nerve cells or protect them from it. "Agent that protects the retina" refers to a compound that prevents the degeneration of retinal cells or protect them from it.
Under the scope of the invention covered by the nucleic acid molecules, preferably DNA molecules, such as (1) a separate molecule having the nucleotide sequence represented in SEQ ID NO: 1 or SEQ ID NO: 3, (2) a selected nucleic acid molecules having nucleotide sequences that hybridize under strict conditions with the selected DNA is presented in SEQ ID NO: 1 or SEQ ID NO: 3, and (3) nucleotide sequences that hybridize to the sequences specified above in (1) or (2). As indicated above, Takayasu hybridization can be strict or not strict (relaxed). In cases where the nucleic acid molecules are deoxyoligonucleotide ("oligonucleotides"), stringent conditions may indicate, for example, washing in 6X SSC/0,05% sodium pyrophosphate at 37°C (for oligonucleotides, consisting of 14 bases)at 48°C (for oligonucleotides, consisting of 17 bases), at 55°C (for oligonucleotides, consisting of 20 bases) and at 60°C (for oligonucleotides, consisting of 23 bases). Suitable ranges for these stringent conditions to nucleic acids of different composition are described in Krause and Aaronson, Methods in Enzymology, 200, 1991, cc.546-556, as well as the above-quoted reference Maniatis and other
Such nucleic acid molecules can act as antisense molecules of the target genes that can be applied, for example, for the regulation of the target genes and/or as antisense primers in amplification reactions of the nucleotide sequences of the target genes. Furthermore, such sequences can be used as part of ribozyme and/or sequences triple helical structures that can also be used for regulation of the target genes. In addition, such molecules can be used as components of diagnostic methods, making it possible to detect the presence of an allele that causes a disease associated with RdCVF1 or RdCVF2.
Under volume izobreteniya (a) vectors, containing any of the above coding sequences (i.e. semantic sequence) and/or their complements (i.e. antisense sequence); (b) expression vectors that contain any of the above coding sequence functionally linked to a regulatory element that controls expression of the coding sequences; and (C) constructed by methods of genetic engineering cell-hosts that contain any of the above coding sequence functionally linked to a regulatory element that controls expression of the coding sequences in the cell host. In the context of the present description regulatory elements include, but are not limited to inducible and penduline promoters, enhancers, operators and other well-known experts in this field elements that control and regulate expression.
Under the scope of the invention covered fragments of any of the above in the present description of the nucleotide sequences. Fragments of the full gene Rdcvf1 or Rdcvf2 can be used as a probe hybridization to cDNA library to isolate the full-size gene and selection of other genes with a high level of similarity with the gene of Rdcvf1 or Rdcvf2 and similar biological asset is awn. Probes of this type preferably contain at least about 30 bases and may contain, for example, from about 30 to about 50 bases, from about 50 to about 100 bases, from about 100 to about 200 bases or more than 200 bases (e.g., 300). The probe can also be used to identify a cDNA clone corresponding to the primary transcript and a genomic clone or clones that contain the complete gene Rdcvf1 or Rdcvf2, including regulatory and promotor regions, exons and introns. Example of screening is the allocation of the coding region of the gene Rdcvf1 or Rdcvf2 using the known DNA sequence to synthesize oligonucleotide probe or accidental premirovany selected sequence represented in figure 1-8. Labeled oligonucleotides having a sequence complementary to the sequence of the gene of the present invention, is used for screening libraries of human cDNA, genomic DNA to identify individual clones from the library, which hybridize with the probe.
In addition to the above gene sequences is possible without numerous experiments to identify and can easily be distinguished using molecular biology techniques orthologues of such sequences that may be present, such as the er, in other species. In addition, other genetic loci of the genome can be genes that encode proteins with a high degree of homology (homologues) with one or more domains of such gene products. Such genes can be identified also by using similar methods. Examples of orthologues or homologues presented on Fig, 10, 11, 12 or 13.
For example, in a dedicated downregulation of the gene sequence you can enter a label and use it for screening the cDNA library derived from mRNA of interest of the body. You can use relaxed hybridization conditions, if a cDNA library derived from an organism different from the type of organism from which the removed carrier label sequence. In an alternative embodiment, for screening of the genomic library derived from the interest of the body, you can apply a labeled fragment using and in this case, the corresponding relaxed conditions. Such relaxed conditions well known to specialists in this field and they can deliberately vary depending on the phylogeny of specific organisms, which are the library and the labeled sequence. Instructions regarding the choice of such conditions can be found, for example, in the above manual, Sambrook and others
Chrome is also you can select a previously unknown expressed sequence gene type, performing PCR using two pools of degenerate oligonucleotide primers designed based on the amino acid sequences encoded gene of interest. As a matrix for the reaction can serve as a cDNA obtained by reverse transcription of mRNA isolated from cell lines or tissue of a person or body other than the person for which it is known or there is the assumption that they Express homologues or obtained as a result of splicing variants.
The PCR product can be subclinical and sequenced to confirm that amplificatoare sequences are sequences similar to the nucleotide sequences of the expressed gene. Then the PCR fragment can be used to highlight the full-length cDNA clone using different methods. For example, in amplificatory fragment, you can enter the label and used for screening the cDNA library of bacteriophage. Alternatively, the labeled fragment can be used for screening of the genomic library.
PCR can also be used to highlight the full-length cDNA sequences. For example, using standard methods, you can select the NC from the respective sources, representing a cell or tissue. During the synthesis of the first chain reaction reverse transcription can be carried out on the basis of RNA using oligonucleotide primers specific for most 5'-end of the amplified fragment. Then the resulting hybrid DNA/RNA can "grow" guanine residues using standard processing end sections transferase, a hybrid can be split by RNase H, and then it is possible to carry out the synthesis of the second chain by premirovany using a poly-C primer. Thus, it is possible to easily select cDNA sequence located against the course of transcription relative to the amplified fragment. A review of cloning strategies which may be used for this purpose is shown, for example, Sambrook and others, 1989, above.
In those cases, when the identification of differentially expressed gene is a normal gene or wild-type gene, this gene can be used for selection of mutant alleles of the gene. The selection is preferably carried out in the case of processes and disorders, which are known or believed to have a genetic basis. Mutant alleles can be distinguished from the body of patients, which are known or believed to have the genotype that causes the symptoms is zabolevaniya. Mutant alleles and the products of mutant alleles can then be used in the following diagnostic systems analysis.
cDNA mutant gene can be selected, for example, using well known to specialists in this field method RT-PCR. In this case, the first chain cDNA can be synthesized by hybridizing an oligo-dt oligonucleotide (or random hexamers) with mRNA isolated from tissues of a patient, which presumably carries the mutant allele, which is known or assumed that it is the expression of the mutant allele, and by extending the new circuit using reverse transcriptase. Then synthesize the second circuit cDNA using the oligonucleotide, which specifically hybridizes with the 5'-end of the normal gene (or by any other means). After that, the product amplified by PCR using the two primers clone in a suitable vector and perform DNA sequencing using methods well known to specialists in this field. By comparing the DNA sequence of a mutant gene with the DNA sequence of the normal gene can be identified mutation(s), cause(s) loss or alteration of function of the mutant gene product.
Alternatively, you can create a library of genomic or cDNA and the wasp is estolate screening using DNA or RNA, isolated from the patient's tissue, which presumably carries the mutant allele, which is known or assumed that it is the expression of the gene of interest. Then in the normal gene or any suitable fragment, you can enter the label and use as a probe to identify the corresponding mutant allele in the library. After that, the clone containing this gene can be cleaned by standard methods used in this field, and to subject the above sequencing.
In addition, you can create a library of expressed sequences using DNA isolated from the patient's tissue, which presumably carries the mutant allele, which is known or assumed that it is the expression of the gene of interest, or cDNA synthesized from such tissue. Thus, the gene products produced cloth, presumably carrying the mutation, it is possible to Express and be subjected to screening using standard methods of screening using antibodies using, as described below, antibodies to the normal gene product (see the description of the methods of screening, for example, Antibodies: A Laboratory Manual". /Ed. by Harlow, E. and Lane, published by Cold Spring Harbor Press, Cold Spring Harbor, 1988). In cases where the mutation leads to the fact that expressio the p gene product has a modified function (for example, in the result of missense mutations), apparently, should be subjected to cross-react with the mutant gene product set polyclonal antibodies. Included in the library clones, identified on the basis of their reaction with such labeled antibodies can be purified and subjected to sequencing according to the above method.
Differentially expressed gene products include proteins encoded by a nucleotide sequence represented in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, in particular, a polypeptide constituting or comprising the amino acid sequence represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, or its fragments.
In addition, downregulation of gene products may include proteins that represent functionally equivalent gene products. Such an equivalent gene product may have deletions, additions or substitutions of amino acid residues in the amino acid sequence encoded by the above-described sequences of differentially expressed gene, but those which lead to "silent" change, resulting in a functionally equivalent product of differentially expressed gene (polymorphism). Replacement of amino acids can be made on the basis of similarity on which arnosti, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of the investigated residues and on the basis of comparison with amino acid sequences of other species.
For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, Proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include spartanova acid and glutamic acid. In the context of the present description, the term "functional equivalent" may refer to a protein or polypeptide, which have almost the same activity in vivo or in vitro, as endogenous products of differentially expressed gene encoded by the above sequences of differentially expressed genes. The term "functional equivalent" can also refer to proteins or polypeptides having the ability to interact with others inside the cells or extracellular molecules in the same way as the corresponding portion of the product of the endogenous differentially expressed gene. For example, the concept of "functionale equivalent" refers to a peptide, which according to the immunoassay has the ability to reduce the binding of an antibody with the corresponding peptide (i.e. the amino acid sequence of a peptide which has been modified for "functionally equivalent peptide) endogenous protein, or to the endogenous protein, where the antibody is an antibody to the corresponding peptide endogenous protein. Equimolar concentration of functionally equivalent peptide can reduce the above binding of the corresponding peptide of at least about 5%, preferably about 5-10%, more preferably about 10-25%, even more preferably about 25-50%, and most preferably about 40-50%.
The products of differentially expressed gene can be obtained by using methods of recombinant DNA, known in this area. Thus, in the present description provides methods of producing the polypeptides and peptides encoded by differentially expressed genes according to the invention, based on the expression of nucleic acid coding sequences of differentially expressed genes. To create expression vectors containing coding sequences of the products expressed gene and the corresponding signals Kon the control of transcription/translation. You can apply well-known methods, including, for example, methods of recombinant DNA in vitro, methods of synthesis and the method of recombination/genetic recombination in vivo (see, for example, the techniques described in Sambrook and others, 1989, supra, and Ausubel and others, 1989, above). Alternatively, RNA, or cDNA, with the ability to encode the sequence of the protein expressed by the gene can be synthesized by chemical means, for example, using synthesizers (see, for example, the techniques described in "Oligonucleotide Synthesis". Ed. Gait, M. J., published by IRL Press, Oxford, 1984, the publication is fully incorporated into the present description by reference).
For expression of coding sequences of differentially expressed gene according to the invention it is possible to use a different vector system, expressed in the host. Such expressed in the host system are the media through which you can get and then clear interest sequence, they also include cells that after their transformation or transfection of the corresponding coding nucleotide sequences that Express a protein differentially expressed gene according to the invention in situ. They include (but are not limited to microorganisms such as bacteria (for example E. coli, .subtilis), which transformera the s by using recombinant DNA bacteriophage, expression vectors based on the plasmid DNA or kosmidou containing DNA encoding the protein sequences of differentially expressed gene; yeast (for example Saccharomyces, Pichia), which are transformed with recombinant expression vectors of yeast containing the coded protein sequences of differentially expressed gene; systems based on insect cells infected with recombinant viral expression vectors (e.g. baculovirus)containing the coding protein sequences of differentially expressed gene; system-based plant cells infected with recombinant viral expression vectors (for example, on the basis of the cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)or transformed with recombinant plasmid transformation vectors (for example, Ti-plasmid)containing the coding protein sequences of differentially expressed gene; or system on the basis of mammalian cells (for example, COS, Cho, KSS, 293, T)carrying a recombinant expression system containing promoters derived from the genome of mammalian cells (for example, the promoter metallothionein) or from mammalian viruses (e.g., the late promoter of adenovirus; the promoter of the vaccinia virus C; early promo is the PR gene of cytomegalovirus).
You can also make the expression of RDCVF1 or RDCVF2 in the cell using a gene Rdcvf1 or Rdcvf2, which are native to the cell. Methods of implementation of this expression are described, for example, in US 5641670; 5733761; 5968502; and 5994127, the contents of all patents are fully incorporated in the present description by reference. Cells subjected to induction for the expression of RDCVF1 or RDCVF2, using any of the methods described in patents US 5641670, 5733761, 5968502 and 5994127, could be implanted in the corresponding tissue of a living animal in order to increase the local concentration of RDCVF1 or RDCVF2 in the tissue.
For bacterial systems depending on the desired application, you can choose numerous expression vectors designed for expression of the protein encoded by differentially expressed genes. For example, when you want to produce a large quantity of such a protein, it may be appropriate to apply to generate antibodies or to screen peptide libraries, for example, the vectors providing for the expression of high levels of products in the form of a fused protein, which is easy to clean. Such vectors include, but are not limited to) expressed in E. coli expression vector pUR278 (Ruther and others, EMBO J. 2, 1983, s), in which encoding the protein sequence of the differential expressive the CSOs gene can be embedded by ligation into the vector in frame reading containing the coding region of the lac Z, resulting in a fused protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13, 1985, cc.3101-3109; Van Heeke and Schuster, J. Biol. Chem. 264, 1989, cc.5503-5509); etc. you Can also use the PGEX vectors for expression of foreign polypeptides in the form of a fused protein with glutathione S-transferase (GST). Typically, these fused proteins are soluble and can be easily isolated from lysed cells using absorption to glutathione-sepharose granules and subsequent elution in the presence of free glutathione. The PGEX vectors are creating so that they contain the sites of cleavage by the protease thrombin or factor XA, allowing the protein cloned gene target can be distinguished from the GST fragment using the specified endopeptidase.
The promoter region can be distinguished from any desired gene using vectors that contain the unit of transcription of the reporter, which does not include the promoter region, such as the transcription unit chloramphenicolchloramphenicol (cat) or luciferase, in the direction along the transcription from the restriction site or sites designed to embed a possible promoter fragment, i.e. a fragment, which may include a promoter. It is well known that the embedding containing the promoter fragment in the vector at the restriction site, the RA is put against the course of transcription relative to the cat gene or luciferase, causes the appearance of the CAT or luciferase activity, which can be detected by standard methods of the SAT-analysis or by using luminometry. Suitable for this purpose vectors are well known and readily available. Examples of such vectors are the three vectors RCC-8, -RSM and pGL3, Promega, E1751, registration number in the Genebank u47295). Promoters for expression of polynucleotides of the present invention include not only well-known and easily accessible promoters, but also promoters that can be easily obtained by the method described above, using the analysis of reporter gene.
Among known bacterial promoters suitable for expression of polynucleotides and polypeptides of the present invention, it should be noted promoters of E. coli lacI and lacZ, the promoters, the T3 and T7, T5 promoter tac, the promoter, the lambda PR, PL and promoter trp. known eukaryotic promoters suitable for this purpose, it should be noted very early promoter of CMV (cytomegalovirus), timeintensity the promoter of the herpes virus simple (HSV), early and late promoters of simian virus S-40, the promoters of retroviral LTRS (long terminal repeats), such as the promoter of the rous sarcoma virus ("RSV"), and promoters metallothionein, such as murine promoter metallothionein-I.
Among the systems based on the use of the cells NASA is omogo, the nuclear polyhedrosis virus of Autographa californica NPV (AcNPV) is one of several systems based on the use of insect cells, which can be used as a vector for expression of foreign genes. The viruses are grown in cells of Spodoptera frugiperda. The coding sequence of differentially expressed gene can be cloned individually in essentially areas (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful embedding the coding sequences of differentially expressed gene may result in inactivation of the polyhedrin gene and production of "neutropenia" recombinant virus (i.e., a virus has no protein shell encoded polyhedrin gene). Then such recombinant viruses use to infect cells of Spodoptera frugiperda, in which the expression of gene (see, for example. Smith and others, J. Virol. 46, 1983, 584; Smith, U.S. patent No. 4215051).
In the case of use as host cells of mammalian cells can be applied to numerous expression systems based viruses. In those cases, when the expression vector using the adenovirus of interest coding sequence of differentially expressed gene can Legerova the b complex, controlling the transcription/translation of adenovirus, e.g., the late promoter and tripartite leader sequence. Then this chimeric gene can be integrated into the genome of adenovirus using the methods of recombination in vitro or in vivo. Embedding in essentialyou region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and has the ability to Express the protein differentially expressed gene in infected hosts (see, for example, Logan and Shenk, Proc. Natl. Acad. Sci. USA 81, 1984, cc.3655-3659). For efficient broadcast of embedded coding sequences of differentially expressed gene may also require specific initiation signals. Such signals include the initiating ATG codon and adjacent sequences. In those cases, when in an appropriate expression vector embed full differentially expressed gene, including its own initiator codon and adjacent sequences, may not require the inclusion of additional regulating broadcast signals. However, in cases where embed only part of the coding sequence of differentially expressed gene, it is necessary to introduce exogenous regulatory broadcast signals, which may include the initiation the ith codon ATG. In addition, in order to guarantee the transmission of full insertion, the initiating codon must meet the reading frame of the desired coding sequence. Such exogenous regulatory broadcast signals (Kozak sequence) and initiating codons can have different origins, they can be obtained from natural sources or by synthesis. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancers, transcription terminators, etc. (see Bittner and others, Methods in Enzymol, 153, 1987, cc.516-544).
The procedure of selection of appropriate vectors and promoters for expression in the cell host is a well-known and the methods used for expression vector construction, integration of the vector into the host and expression in the host are the standard for professionals in this field.
Generally, recombinant expression vectors should include the site of initiation of replication, a promoter derived from having a high level of gene expression, which is used to control transcription of the structural sequences located downstream transcription, and breeding marker that allows you to highlight cells containing the vector, after their processing by the vector.
In addition, you can choose the strain of host cells that allows Modulare the AMB expression built-in sequences or modify and processional gene products required in a specific way. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein may be important for the function of the protein. Different cell owners have their characteristic and specific mechanisms for post-translational processing and modification of proteins. To ensure the correct modification and processing of the expressed foreign protein can choose the appropriate cell lines or owners. For this purpose you can use eukaryotic cells-owners who have a cellular mechanism accordingly to carry out the processing of the primary transcript, glycosylation, and phosphorylation of the gene product. These cells are the masters isolated from mammals include but are not limited to Cho, VERO, KSS, HeLa, COS, MDCK, 293, T, WI38, etc.
For long term production of recombinant proteins with high yield it is preferable that the expression was stable. For example, it is possible to construct a cell line, which stably Express a differentially expressed protein. Rather than using expression vectors that contain viral sites of replication initiation, the cells of the host can be transformed with DNA controlled by appropriate control of arousih expression elements (for example, promotor, enhancer sequences, transcription terminators, polyadenylation sites, and so on), and using breeding marker. After embedding alien DNA, engineered cells grown in enriched environments within 1-2 days and then transferred to selective medium. Breeding marker confers resistance recombinant plasmids to the selection and allows stable integration of the plasmid into the chromosome of the cells and grow them getting a small group of cells, which in turn can be cloned and reproduced with obtaining cell lines. This method preferably can be used to construct cell lines that Express differentially expressed protein. Such engineered cell lines may be used, in particular, for screening and identifying compounds having the ability to affect the endogenous activity of the differentially expressed protein. Such stable cell lines can be applied directly or after encapsulation for cell therapy.
You can apply multiple systems selection, including (but not limited to, genes timedancing herpes virus simple (Wigler, etc., Cell 11, 1977, p.23), hypoxanthineguanine (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48, 1962, s) and infosfacilitiesfree (Lowy and others, Cell 22, 1980, C) in cells tk-, hgprt-or aprt-respectively. You can also use system selection based on the selection on the principle of resistance to antimetabolites, for breeding dhfr gene causing resistance to methotrexate (Wigler, etc., Natl. Acad. Sci. USA 77, 1980, s; O'hare and others, Proc. Natl. Acad. Sci. USA 78, 1981, s); gpt, which cause resistance to mycophenolate acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78, 1981, s); neo, which cause resistance to the aminoglycoside G-418 (Colberre-Garapin, etc., J. Mol. Biol. 150, 1981, c.1); and hygro, 1981, causing resistance to hygromycin (Santerre and others, Gene 30, 1984, s).
The use of alternative systems based on the fused protein allows for easy cleaning Undenatured fused proteins expressed in human cell lines (Janknecht and others, Proc. Natl. Acad. Sci. USA 88, 1991, cc.8972-8976). In this system, the gene of interest subcloning in recombinant plasmids on the basis of cowpox virus in such a way that the open reading frame of the gene by broadcast merge with the N-terminal tag consisting of six histidine residues. Extracts of cells infected with recombinant vaccinia virus, bring in columns filled with Ni2+-nitriloacetate-agarose, and labeled with histidine proteins selectively elute using containing imidazole buffers.
The door is correctly expressed protein when it is used as a component in systems analysis, such as the systems described below, you can mark either directly or indirectly, to facilitate detection of a complex formed of differentially expressed protein and the test substance. You can apply any of the many tagging systems, including (but not limited to radioisotopes, such as125I; labeling system using the enzyme that generates a detectable calorimetric signal or emission of light during the processing of a substrate, and a fluorescent label.
If to obtain differentially expressed protein intended for use in such systems analysis using recombinant DNA, it may be desirable to construct fused proteins, which can simplify the labeling, immobilization and/or detection.
Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to any product differentially expressed gene. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single-chain antibodies, Fab fragments and fragments produced using expression libraries Fab.
In another embodiment of the invention in the framework of gene therapy to stimulate fu the work of the cones introduced nucleic acid, containing the sequence encoding the protein RDCVF1 or RDCVF2 or its functional derivative. Gene therapy refers to therapy performed by injecting the patient with a nucleic acid. In this embodiment of the invention nucleic acid leads to the formation encoded by the protein that causes therapeutic effect, stimulating the function of the cones.
According to the present invention can use any of the methods of gene therapy, which is used in the art. Examples of methods are described below.
In a preferred embodiment of the invention therapeutic medicinal product contains nucleic acid Rdcvf1 or Rdcvf2, which is part of an expression vector expressing the protein RDCVF1 or RDCVF2 or its fragment or containing chimeric protein in a suitable host. In particular, such a nucleic acid has a promoter functionally linked to the coding region of Rdcvf1 or Rdcvf2, the promoter is inducible or constitutive and tissue-specific optional. In another specific embodiment of the invention using the nucleic acid molecule in which the coding sequences Rdcvf1 or Rdcvf2 and any other necessary sequences are flanked by regions that enhance g is mological recombination at a desired site in the genome, thereby vnutricherepnuyu the expression of a nucleic acid Rdcvf1 or Rdcvf2 (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86, 1989, cc.8932-8935; Zijlstra and other, Nature 342, 1989, cc.435-438).
Introduction of nucleic acid in the patient's body can be done either directly, in this case, the patient is treated directly nucleic acid or carrying nucleic acid vector, or indirect, in this case, cells are first transformed nucleic acid in vitro and then transplanted them into the patient. These two approaches are called, respectively, the gene therapy in vivo or ex vivo.
According to a particular variant of the invention, the nucleic acid is administered directly in vivo, where its expression with the receipt of the encoded product. This can be accomplished by any of numerous known in this area by, for example, by constructing it as part of the respective carrier nucleic acid expression vector, and introducing him so that he got into the cell, e.g., by infection using defective or attenuated retrovirus, or other viral vectors (adenovirus, adeno-associated virus and lentivirus) (see, for example, U.S. patent No. 4980286 and others mentioned patents), or by direct injection ogole the Neu DNA or by microparticle bombardment (e.g., using a gene gun; firm Biolistic, Dupont), or coating with the use of lipids or located on the cell surface receptors or transfective agents, encapsulation in liposomes, microparticles, or microcapsules, or by introduction through binding with the peptide against which it is known that he has the ability to penetrate into the nucleus by introducing after binding with ligand-sensitive mediated receptor endocytosis (see, for example, US 5166320; 5728399; 5874297; and 6030954, which are all included in the present description by reference in their entirety) (which can be used for directional migration in those types of cells that specifically Express the receptors), etc. In another embodiment of the invention it is possible to form a complex of nucleic acid and ligand, where the ligand contains fusogenic (able to merge) viral peptide possessing the ability to destroy endosome that avoids degradation of the nucleic acid in lysosome. According to another variant embodiment of the invention it is possible to carry out the introduction of nucleic acids in vivo to specific cell uptake and expression by the directed transfer to a specific receptor (see, for example, PCT publication WO 92/06180; WO 9/22635; WO 92/20316; WO 93/14188; and WO 93/20221). In an alternative embodiment, to implement the expression of the nucleic acid can be entered into the cell and be embedded in the DNA of the host cell by homologous recombination (see, for example, US 5413923; 5416260; and 5574205; and Zijlstra, etc., Nature 342, 1989, cc.435-438).
In a specific embodiment of the invention using a viral vector that contains a nucleic acid Rdcvf1 or Rdcvf2. For example, you can apply retroviral vector (see, for example, US 5219740; 5604090; and 5834182). Such retroviral vectors modify to exclude retroviral sequences that are not required for packaging the viral genome and integration into the DNA of the host cell. Nucleic acid Rdcvf1 or Rdcvf2 intended for use in gene therapy, clone in the vector, which provides the introduction of the gene into the patient.
In gene therapy as other viral vectors can also be used adenoviruses. Adenoviruses are especially preferred carriers for the introduction of genes in the respiratory epithelium. Adenoviruses typically affect the respiratory epithelium, causing a weak form of the disease. Other targets delivery systems based on the use of adenoviruses, are the liver, Central nervous system, endothelial cells and muscle tissue. The advantage of adenoviruses is the I, that they are able to infect non-dividing cells. Methods for gene therapy based on the use of adenoviruses as described, for example, in US 5824544; 5868040; 5871722; 5880102; 5882877; 5885808; 5932210; 5981225; 5994106; 5994132; 5994134; 6001557; and 60338843, which are all fully incorporated into the present description by reference.
For gene therapy has also been proposed to use adeno-associated virus (AAV). Adeno-associated viruses are particularly preferred carriers for the introduction of genes into the retina. Methods of obtaining and application of AAV described, for example, in US 5173414; 5252479; 5552311; 5658785; 5763416; 5773289; 5843742; 5869040; 5942496; and 5948675, which are all fully incorporated into the present description by reference.
Another approach for the implementation of gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, mediated by calcium phosphate transfection or infection with a virus. Generally, the method of transfer involves the transfer of breeding marker in cells. The cells are then subjected to selection to highlight those cells that contain and Express the transferred gene. Then these cells are injected into the patient's body directly or after encapsulation.
According to this variant of the invention, the nucleic acid being introduced into the cell prior to the introduction in vivo is received recombinant cells. Such introduction can be performed by any method known in this field, including (but not limited to transfection, electroporation, microinjection, infection with vector-based virus or bacteriophage carrying nucleotide sequences, cell fusion, mediated chromosome gene transfer mediated by microcidal gene transfer, merge spheroplasts etc. According to the present invention for introduction of foreign genes into cells, you can apply numerous techniques known in this field, provided that you will not be disturbed necessary functions of development and physiological functions of the recipient cells. The method should provide for the stable transfer of the nucleic acid into the cell so that the nucleic acid can be expressed by the cell and preferably heritable and expressed her cell is derived.
The obtained recombinant cells can be introduced into a patient by various methods known in this field. In a preferred embodiment of the invention epithelial cells are administered by injection, for example, subcutaneously. In another embodiment of the invention as a skin graft for a patient can be applied recombinant skin cells. Recombinant blood cells (e.g., hematopoietic stole the s cells or precursor cells) are preferably administered intravenously. The number of cells, designed to be inserted depends on the desired action, the condition of the patient, etc. and it can be determined by a specialist in this field.
As the cells in which you can enter the nucleic acid for the purposes of gene therapy, it is possible to use any desired and available cell type, including but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T-lymphocytes, b-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem cells or precursor cells, in particular hematopoietic stem cells or precursor cells such as those derived from bone marrow, blood from the umbilical cord, peripheral blood, liver, embryo, etc.
In a preferred embodiment of the invention, the cell used for gene therapy is autologous to the patient.
In the embodiment of the invention, involving the use of recombinant cells in gene therapy, nucleic acid Rdcvf1 or Rdcvf2 being introduced into the cells so that it could be expressed by the cells or their progeny, and then the recombinant cells are introduced into the body to achieve therapeutic effects in vivo. In concrete the m variant of the invention, the use of stem cells or precursor cells. In principle according to the present invention can use any stem and/or cell precursors, which can be selected and maintained in vitro. Such stem cells include, but are not limited to, hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the intestine, heart muscle cells of the embryo, stem cells of the liver (see, for example WO 94/08598), and neural stem cells (Stemple and Anderson, Cell 71, 1992, cc.973-985).
Epithelial stem cells (ESC) or keratinocytes can be obtained from tissues such as skin and the lining of the intestine, using well known methods (Rheinwald, Meth. Cell Bio. 21A, 1980, s). In stratified epithelial tissues, such as skin, recovery occurs as a result of mitosis of stem cells in the germinal layer, i.e. the layer closest to the basal layer. Stem cells inside the lining of the intestine provide a high recovery rate of this tissue. ESC or keratinocytes derived from skin or mucosa of the intestine of a patient or donor, can be grown in tissue culture (Pittelkow and Scott, Mayo Clinic Proc. 61, 1986, s). If ESC receive from the donor, it can be used as a method of suppressing graft versus host (e.g., radiation, medication, or antibodies to stimulate moderate immunosuppression) (Tropepe and others, Retinal sem cells. Science, 287, 2000, s).
According to this variant embodiment of the invention for hematopoietic stem cells (HSC) can be used in any method that allows the selection, reproduction and the maintenance of HSC in vitro. Methods by which this can occur include (a) the selection and creation of HSC cultures from bone marrow cells isolated from an organism of a future owner or donor, or (b) the application of previously established long-lived cultures of HSC, which can be allogeneic or xenogeneic. Neautrogena HSC is used preferably in combination with a method of suppressing immune responses to transplantation for a future owner/patient. In a specific embodiment, the present invention cells of human bone marrow can be obtained from the rear podsosnogo ridge by needle biopsy (see, for example, Kodo, etc., J. Clin. Invest. 73, 1984, cc.1377-1384). In a preferred embodiment of the invention HSC can be obtained in substantially enriched or substantially pure form. This enrichment can be performed before, during or after prolonged cultivation and can be accomplished using any method known in this field. Long-lived culture of bone marrow cells, you can create and maintain, for example, using a modified method of obtaining a culture cleto is dexterous type (Dexter and others, J. Cell Physiol. 91, 1977, s) or a method of obtaining a culture of Vitakka-white (Witlock and Witte, Proc. Natl. Acad. Sci. USA 79, 1982, cc.3608-3612).
In a specific embodiment of the invention, nucleic acid, intended for the introduction for the purposes of gene therapy, contains the inducible promoter functionally linked to the coding region, allowing the expression of the nucleic acid can be controlled by adjusting the presence or absence of the appropriate inducer of transcription.
In the present description presents methods for producing antibodies that can specifically recognize one of several epitopes of differentially expressed gene. Such antibodies may include, but is not limited to polyclonal antibodies, monoclonal antibodies (MAB), humanized or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab')2-fragments, the fragments obtained through libraries expressed Fab fragments, antiidiotypic (anti-Id) antibodies, and epitopespecific fragments of any of the above antibodies. Such antibodies can be used, for example, to detect a fingerprint, target gene in a biological sample, or alternatively in the method of inhibiting abnormal activity of the target genes. Thus, these antibodies can be used as part of the method is economical treatment of diseases and/or can be used as part of diagnostic techniques, according to which patients can be tested against abnormal levels of Rdcvf1 or Rdcvf2, or the presence of abnormal forms of Rdcvf1 or Rdcvf2, taking samples of tissue fluid and/or the vitreous body by methods known to experts in this field (see Forster RK, Abbott RL, Gelender H., Management of infectious endophthalmitis Ophthalmology 87, 1980, cc.313-319).
For the production of antibodies to differentially expressed gene can give different hosts-animals by injection of differentially expressed protein or part thereof, or by immunization with DNA. Such animals are hosts include, but are not limited to, rabbits, mice and rats, etc. depending on the type of owners to enhance the immunological response, you can apply various adjuvants, including but not limited to, beta-blockers (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyols pluronic, polyanion, peptides, oil emulsions, hemocyanin lymph snails, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as the product of the target genes or E. what about the antigenic functional derivative. To obtain polyclonal antibodies, host animals such as those listed above can give by injection using a product of differentially expressed gene, also supplemented the above-described adjuvants.
Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any method which allows the production of antibody molecules in a continuous cell culture. Such methods include, but are not limited to the method based on hybridoma Kohler and Milstein (see, Nature 256, 1975, cc.495-497; and US 4376110), the method based on hybridoma human b-cells (Kosbor and other Immunology Today 4, 1983, p.72; Cole and others, Proc. Natl. Acad. Sci. USA 80, 1983, 1983, cc.2026-2030) and the method based on hybridoma EBV (Epstein-Barr) (Cole and others, Monoclonal Antibodies And Cancer Therapy, published by Alan R. Liss, Inc., 1985, cc.77-96). Such antibodies can refer to any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD and any subclass. Hybridoma producing a Mat according to the invention can be cultivated in vitro or in vivo. The possibility of obtaining high titers Mat in vivo makes this method more preferred at present.
In addition, you can apply the methods developed to obtain a "chimeric antibodies" (Morrison and others, Proc. Natl. Acad. Sci., 81, 1984, cc.6851-6855; Neuberger and others, Nature, 312, 1984, cc.604-608; Takeda and others, Nature, 314, 1985, cc.452-454) by splicing is and genes from a mouse antibody molecule, having specificity against the corresponding antigen, in combination with genes from a molecule of human antibodies with the appropriate biological activity. A chimeric antibody is a molecule in which different fragments derived from different animal species, for example, those that have variable or hypervariable region derived from a mouse Mat and a constant region of human immunoglobulin.
Alternatively, to obtain single-stranded antibodies to differentially expressed genes can adapt the method described for obtaining single-chain antibodies (US 4946778; Bird, Science 242, 1988, cc.423-426; Huston and others, Proc. Natl. Acad. Sci. USA 85, 1988, cc.5879-5883; and Ward and others, Nature 334, 1989, cc.544-546). Single-chain antibodies can be obtained by linking the fragments of the heavy and light chain Fv region with amino acid bridge, resulting in a single-chain polypeptide.
It is most preferable to obtain antibodies to the polypeptides, fragments, derivatives and functional equivalents specified in the present description, it is possible to apply methods that are suitable for the production of "humanized antibodies". Such methods are described in US 5932448; 5693762; 5693761; 5585089; 5530101; 5910771; 5569825; 5625126; 5633425; 5789650; 5545580; 5661016 and 5770429, which are all fully incorporated in the present description, the quality is TBE reference.
Fragments of antibodies that recognize specific epitopes, can be obtained by the known methods. Such fragments include, but are not limited to, for example, F(ab')2-fragments, which can be obtained by cleavage of the molecule pepsin antibodies, and Fab fragments, which can be obtained by restoring the disulfide bridges of F(ab')2-fragments. Alternatively, you can create a library of expressed Fab fragments (Huse and others, Science, 246, 1989, cc.1275-1281) for quick and easy identification Fab fragments of monoclonal antibodies having the desired specificity.
From the standpoint of ease of detection particularly preferred is a composite analysis, for which there are numerous options, and it should be borne in mind that these fall within the scope of the present invention.
For example, in a typical forward-analysis of unlabeled antibody immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. Then carry out incubation over a period of time sufficient for the formation of a binary complex of antibody-antigen. Then after the formation of the complex add second antibody labeled reporter molecule that is capable of inducing a detectable signal, and incubated during the period of time, sufficient for the formation of a ternary complex of antibody-antigen-labeled antibody. All unreacted material is removed by washing and the presence of the antigen is determined by detection of the signal, or you can quantify antigen by comparing with a control sample containing known amounts of antigen. Ways forward for analysis include the simultaneous analysis in which the sample and the antibody are added simultaneously to the bound antibody, or a reverse analysis, in that we combine the labeled antibody and the sample incubated and added to unlabeled linked antibody. Such methods are well known to specialists in this field and the ability to make small variations is obvious. It should be borne in mind that in the context of the present description the term "sandwich-analysis" includes all options based on the method of using two sites. The only limiting factor for immunoassay according to the present invention is that the unlabeled and labeled antibodies are specific against RdCVF1 or RdCVF2 antibodies.
The most commonly used reporter molecules in this type of analysis are either enzymes or containing fluorophores or radionuclide molecules. In the case of enzyme immunoassay enzyme conjugar the Ute with the second antibody, as a rule, using glutaraldehyde or periodate. It is easy to understand, however, that there is a wide variety of different methods of ligation, which are well known to the person skilled in the art. Commonly used enzymes include, among others, the horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase. Designed for use with specific enzymes, substrates, typically, is chosen so that after hydrolysis by the corresponding enzyme from them was detectable color change. For example, for use in the conjugates, including alkaline phosphatase can be applied para-nitrophenyl; for conjugates, including peroxidase, typically used 1,2-phenylenediamine or toluidine. You can also use not chromogenic substrates and fluorogenic substrates that yield fluorescent product. Then the solution containing the appropriate substrate is added to the ternary complex of antibody-RdCVF1 or RdCVF2-labeled antibody. The substrate reacts with the enzyme linked to the second antibody, resulting in high-quality visible signal, which can then be quantified, usually spectrophotometrically by getting an estimate of the amount Rdcvf1 or Rdcvf2 present in the serum sample.
Alternatively, the flu is rescently connection such as fluorescein and rhodamine, can chemically bind to antibodies without altering their binding capacity. When activated by illumination with light of a specific wavelength labeled fluorochrome antibody absorbs the light energy inducyruya the state of excitation in the molecule, followed by emission of light with a longer wavelength. The issue manifests itself in the form of a characteristic color, which can be visually detected by using a light microscope. Both methods, i.e. the method immunofluorescence assay and ELISA (enzyme-linked immunosorbent assay) is well known in this field and are especially preferred for this method. However, you can also use other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules. The person skilled in the art it is obvious how to alter a procedure to ensure that it meets the desired goals.
The invention relates also to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene encoding the polypeptide selected from the group that includes the polypeptides presented in sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, which is associated with dysfunction, is a diagnostic tool which can be used additionally or using it directly to make the diagnosis of a disease or susceptibility to disease, resulting from insufficient expression, overexpression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene, can be detected at the DNA level using various methods.
Nucleic acids for diagnosis may be obtained from cells of the patient, such as blood, urine, saliva, tissue biopsy or material obtained at autopsy. Genomic DNA can be used to detect directly, or can be subjected to enzymatic amplification using PCR or other amplification methods known in this field. Similarly you can also use RNA or cDNA. Deletions and insertions can be detected by changing the size of the amplified product in comparison with the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled nucleotide sequences. Perfectly matching sequences can be distinguished from the erroneously paired duplexes with cleavage by RNase or by differences in melting temperatures. Differences in DNA sequences can detect changes elektroforeticheskoi mobility of DNA fragments in gels in the presence of denaturing agents (SSCP) or without them, or by direct DNA sequencing (see, for example, yers and others, Science, 230, 1985, cc.230-1242). Sequence variation in the specific provisions can also be assessed using tests of protection against nucleases, for example, protection from RNase and S1, or using chemical cleavage (see Cotton and others, Proc Nati Acad Sci USA, 85, 1985, cc.4397-4401). In another embodiment of the invention for efficient screening of, for example, genetic mutations, it is possible to construct a set of oligonucleotide probes containing the nucleotide sequence of Rdcvf1 or Rdcvf2, or its fragments. Methods based on the technology using a set of well known and are generally applicable and can be used to solve many questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, M. Chee and others, Science, volume 274, cc.610-613, 1996).
Diagnostic analysis is a method of diagnosing or determining the susceptibility to disease by detecting mutations in the gene Rdcvf1 or Rdcvf2 used with the methods described above. In addition, such diseases can be diagnosed by methods comprising determining the abnormal expression of Rdcvf1 or Rdcvf2 in a sample taken from the patient's body: the expression can be measured at the RNA level using any of the well-known in the field of methods for quantitative evaluation of polynucleotides, is Aceh, for example, the amplification of nucleic acids, for example, PCR, RT-PCR, protection from RNase, Northern blotting and other hybridization methods. Methods of analysis that can be applied to determine protein levels, such as the polypeptide of the present invention, in a sample derived from a host are well-known to specialists in this field. Such assay methods include radioimmunoassay analyses, analyses of competitive binding assays by the method of Western blotting and ELISA (enzyme-linked immunosorbent assay).
Thus, another object of the present invention is a diagnostic kit, which includes:
(a) polynucleotide of the present invention, where preferably the nucleotide sequence encoding a polypeptide selected from the group comprising the polypeptide presented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, or a fragment;
(b) a nucleotide sequence complementary to the sequence specified in (a);
(C) a polypeptide of the present invention, preferably the polypeptide or its fragment; or
(d) antibody to a polypeptide of the present invention, preferably to the polypeptide selected from the group comprising polypeptides having the sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 1.
It should be borne in mind that in any of these sets listed in (a), (b), (C) or (d) the elements can contain crucial component. This set can be used for diagnosis of a disease or susceptibility to disease, such as retinitis pigmentosa, age related macular degeneration syndrome Barda, syndrome Bessin-Kornzweig, the disease best, choroidea, atrophy of the cerebral convolutions, congenital Amoros, syndrome Resana, syndrome Stargardt and usher syndrome. The nucleotide sequence of the present invention can also be used to determine the localization on the chromosome. Localization on the chromosome can be determined by PCR using as template DNA obtained from a panel of hybrid cell lines (mouse-hamster). The position of the human gene in the chromosome can be predicted by the position of the mouse gene-based sintenie (McCarthy and others, Genome research, 7, 1997, s). Carry out specific transfer sequence at a particular locus on a single chromosome, including murine or human chromosome, and subjected to hybridization.
Mapping of suitable sequences on the chromosomes of the present invention is an important first step in correlating these sequences with the gene associated with the disease. After after the outermost Carteret in the exact position on the chromosome, the physical position of the sequence on the chromosome can be correlate with genetic map data. Such data can be found, for example, V.McKusick, Mendelian Inheritance in Man (you can get them in the mode of on-line medical library at Johns Hopkins University, Welch (Johns Hopkins University Welch Medical Library, RetNet). Then by analyzing the relationship (co-inheritance of physically adjacent genes) identify the relationship between genes, mapped in the same region of a chromosome, and diseases.
You can also define the differences cDNA or genomic sequences affected and healthy individuals. If the mutation have several or all affected individuals, but there is no one healthy individual, the mutation is likely to be the causal agent of the disease.
Another variant embodiment of the invention relates to the introduction of pharmaceutical compositions in combination with pharmaceutically acceptable carrier to provide any of the above therapeutic action. Such pharmaceutical compositions may contain Rdcvf1 or Rdcvf2, mimetics or agonists. The composition can be entered individually or in combination with at least one additional agent such as a stabilizer, which you can enter in any sterile, biocompatible, pharmace the optical carrier, including (but not limited to, saline, buffered saline, dextrose, and water. Songs you can enter the patient individually or in combination with other agents, drugs or hormones.
Pharmaceutical compositions falling under the scope of the invention, it is possible to enter any of numerous ways, for example by transcleral the introduction possessing the biological reactivity of the protein in choroid and the retina according to the method described by Ambati and others, Investigative Ophthalmology and Visual Science, 41, 2000, s. Composition of ophthalmic drugs intended for local use, including ophthalmic solutions, suspensions and ointments, well known to specialists in this field (see Remington's Pharmaceutical Sciences, 18th ed., Chapter 6, cc.1581-1592, published by Mack Publishing Company, 1990). You can use other routes of administration, including in-vessel injection (which you can do directly in front of the camera or directly into the chamber of the vitreous body), subconjunctival injections and retro-bulbar (in the rear part of the bulb of the eyeball) injection methods and means of obtaining ophthalmic preparations suitable for such routes of administration are well known.
In the context of the present description the term "extraocular" refers to the surface of the eye and the (external) simple is ansto between the eyeball and eyelid.
Examples extraocular areas include REDD century or blind bag, the surface of the conjunctiva and cornea. This area is external to all other tissues of the eye and for access to this area is not required to use invasive procedures. Examples of such extraocular systems include insert and enter the "local" drops, gels or ointments that can be applied for the introduction of therapeutic material in these areas. Extraocular devices, as a rule, can be easily removed, it can make even the patient himself.
The following patents described extraocular system, which is used for administering drugs in extraocular area. Higuchi et al described in US 3981303, 3986510 and 3995635 biodegradable ocular insert containing the drug. It is possible to make the insert having a different shape for retention in a blind bag eyeball, extraocular the space between the eyeball and eyelid. Described a number of conventional biocompatible polymers that can be used for the manufacture of such devices. Such polymers include alginate zinc, poly(lactic acid), poly(vinyl alcohol), poly(anhydrides) and poly(glycolic acid). The patent also described device is covered with a membrane with a reduced permeability for drug environments is tion and having a hollow chamber, containing pharmaceutical composition.
Theeuwes in patent US 4217898 described microporous reservoirs used for the controlled injection of a medicinal product. Such devices are placed extraocular in a blind eye bag. Interest polymeric systems include copolymers of poly(vinyl chloride) and poly(vinyl acetate). Kaufman in patents US 4865846 and 4882150 described the ophthalmic system of administration of medicinal products containing at least one biodegradable material or an oil medium, for introduction into the conjunctival SAC. As suitable systems introduction the patent describes polymer systems, such as polylactide, polyglycolide, polyvinylalcohol and cross-linked collagen.
The advantage presented in this description of the applicability of protein RDCVF1 or RDCVF2 for the treatment of diseases or injuries of the retina also lies in the fact that the ophthalmic composition for topical application comprises an agent that promote penetration or transport of therapeutic agent into the eye. Such agents are known in this field. For example, Ke and others in the patent US 5221696 has described the use of materials that enhance the penetration of ophthalmic drugs across the cornea.
Intraocular systems are such systems that can be applied in any tissue compartment inside, IU the remote control or on the outside of the tissue layers of the eye. Such areas include subconjunctival (under the mucous membrane of the eye adjacent to the eyeball), orbital (behind the eyeball) and the chamber (inside the chambers of the eyeball). Unlike extraocular systems for access to these areas requires an invasive procedure, which is the injection or implantation.
Intraocular device described in the following patents. Wong in patent US 4853224 described microencapsulation medicines for introduction into a chamber of an eye. Polymers, which are used in such a system, include polyesters, and polyethers. Lee in patent US 4863457 described biodegradable device that is surgically implanted intraocular for continuous release of therapeutic agents. The device is intended to be implanted surgically under the conjunctiva (the mucous membrane of the eyeball). Krezancaki in patent US 4188373 described pharmaceutical carrier, which turns into a gel when the temperature of the human body. This device is an aqueous suspension of drug and resin or synthetic cellulose derivatives. Haslam et al described in patents US 4474751 and 4474752 system polymer-drug, which is in a liquid state at room temperature and turns into GE is ü at body temperature. Suitable polymers that can be used in this system include polyoxyethylene, polyoxypropylene. Davis and others described in the patent US 5384333 polymer for the introduction of biodegradable injectable drug, which provides prolonged release of the drug. The composition of the medicinal product represents having pharmaceutical activity of the agent in a biodegradable polymeric matrix, where the polymer matrix is in a solid state at temperatures from 20 to 37°C, and is fluid at temperatures from 38 to 52°C. the Use of the polymer for the introduction of medicines are not limited to soluble or liquid compositions medicines. For example, the polymer can be used as a matrix for stabilization and retention at the injection containing the drug microspheres, liposomes or other particles medicines.
Particularly preferred carrier for intraocular injection is sterile distilled water, with which on the basis of protein RDCVF1 or RDCVF2 prepare sterile isotonic solution, preferably canned. Another ophthalmic preparation may contain the composition of the protein RDCVF1 or RDCVF2 agent, such as injectable Mick is ospery or liposomes which provides a delayed or continuous release of the protein and which is then administered by injection in the form of a depot. Other means suitable for intraocular injection of protein RDCVF1 or RDCVF2 include implantable device for injecting drugs or which contain protein RDCVF1 or RDCVF2.
Ophthalmic preparations of the present invention, in particular, the topical preparations may contain other components, for example, ophthalmically acceptable preservatives, agents for adjusting toychest, co-solvents, wetting agents, complexing agents, sautereau agents, antimicrobial agents, antioxidants, and surfactants that are well known in this field. For example, increase toychest agents include halides of alkali metals, preferably sodium chloride or potassium), mannitol, sorbitol and the like, Preferably add increasing toychest agent in an amount such that the composition is intended for insertion into the eye, was gipotonichnaya or nearly isotonic. Suitable preservatives include, but are not limited to) benzylaniline, thimerosal, finitely alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, etc. as preservative can be applied also peraki the ü hydrogen. Suitable co-solvents include, but are not limited to, glycerin, propylene glycol and polietilenglikol. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include, but are not limited to, esters sorbitan, Polysorbate, such as Polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol etc. as buffers it is possible to apply conventional buffers such as borate, nitrate, phosphate, bicarbonate or Tris-HCl.
The components of the composition are present in concentrations that are acceptable for introduction into extraocular or intra-ocular region. For example, buffers are used to maintain physiological pH or pH of the composition, less than physiological significance, as a rule, the pH value from about 5 to about 8. Additional components of the composition may include materials that provide a prolonged presence in the eye extraocular injected therapeutic agent so as to maximize the local contact with the eye and contribute to the absorption. Suitable materials include polymers or gelling materials which would increase the viscosity of ophthalmic PR is parathas. Especially preferred material as the agent for controlling the rate of release in the eye, which is used in liquid ophthalmic compositions of the medicinal product is chitosan (see patent US 5422116 on the name of the Yen and other). The suitability of the compositions of the present invention for controlled release (e.g., continuous and prolonged introduction) ophthalmic medicinal agent in the eye can be determined by various methods known in this field, for example, as described in the Journal of Controlled Release, 6, 1987, cc.367-373, 1987, as well as their options.
In addition to the active substances such pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, representing excipients and auxiliaries that facilitate processing of the active substances with obtaining drugs that can be used for pharmaceutical purposes. Additional details regarding methods of preparation of compositions and their administration can be found in the latest edition of the Remington''s Pharmaceutical Sciences (published by Mack Publishing Co., Easton, Pennsylvania).
Pharmaceutical compositions for oral administration can be prepared using pharmaceutically acceptable carriers well known in the art, applying them in doses suitable for pearling the introduction. Such carriers enable you to prepare pharmaceutical compositions in the form of tablets, pills, coated tablets, capsules, liquids, gels, syrups, emulsions, suspensions, etc. intended for absorption through the mouth of the patient.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, if necessary, by an optional grinding the resulting mixture and processing the mixture of granules, after adding, if necessary, suitable excipients to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; corn starch, wheat starch, rice starch, potato starch or starch from other plants; cellulose, such as methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose sodium; gums, including gum Arabic and tragacanth gum; and proteins such as gelatin and collagen. If necessary, you can add leavening agents, or solubilizing agents, such as crosslinked polyvinylpyrrolidone, agar, alginic acid or its salt, such as sodium alginate.
You can apply the kernel of pills in combination with a suitable coating, for example, obtained by using koncentrira is the R sugar solutions, which may also contain gum Arabic, talc, polyvinylpyrrolidone, carboloy gelling, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In the coating of tablets or pills, you can add dyes or pigments to identify the product or to specify the amount of active substance, i.e. the doses.
Pharmaceutical preparations which can be used orally include easy to swallow gelatin capsules (push-fit), soft sealed capsules having a coating of glycerine or sorbitol. Capsule type push-fit may contain active ingredients mixed with a filler or binders such as lactose or starches, sizing, such as talc or magnesium stearate, and optionally stabilisers. In soft capsules the active substances can be dissolved or suspended in suitable liquids, such as fatty oils, liquid or liquid polyethylene glycol, containing stabilizers, or without them.
Pharmaceutical compositions suitable for parenteral administration, can be prepared in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, ringer's solution, or buffered saline. Water injecor the administered composition may contain substances, which increase the viscosity of the suspension, such as carboxymethylcellulose sodium, sorbitol, or dextran. Additionally, suspensions of the active substances can be prepared as appropriate oily suspensions for injection. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters, such as etiloleat, or triglycerides, or liposomes. For injection can also be used Nazirova poly aminopolymers. The optional suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds, which allows to obtain drugs in the form of highly concentrated solutions.
In the composition for local or nasal application, you can use the penetrants (substances that promote penetration), suitable for penetration through the concrete barrier. Such penetrants are widely known in this field.
The pharmaceutical compositions of the present invention can be prepared by a method known in this field, for example using conventional mixing, dissolving, granulation, production drops, grinding into powder, emulsifying, encapsulating, capture or freeze-drying.
The pharmaceutical composition can be prepared in the form of a salt which m is should be formed with many acids, including (but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts, typically, have greater solubility in aqueous or other protonic solvents than the corresponding form of a free base. In other cases, the preferred preparation may be a lyophilized powder containing any or all of the following components: 1-50 mm histidine, 0.1 to 2% sucrose, and 2-7% mannitol, at a pH value of from 4.5 to 5.5, which is before applying the join buffer.
After preparation of pharmaceutical compositions they are placed in an appropriate container and provided with a label indicating the conditions of treatment of the condition for which illustrates the application of the composition. For the introduction of Rdcvf1 or Rdcvf2 this label must include the amount, frequency and method of administration.
Pharmaceutical compositions suitable for use according to the invention include compositions in which the active ingredients are contained in amounts effective to achieve the desired goal. The effective dose is in the competence of the specialist in this field.
For any connection, therapeutically effective dose can be estimated initially either by analysis using cell cultures, for example, neoplastic cells, or on models with the use of what Finance animals usually mice, rabbits, dogs, or pigs. Models with animals can also be used to determine the appropriate concentration range and route of administration. Then this information can be used to determine useful doses and routes of administration to humans.
A therapeutically effective dose is a quantity of the active substance, for example Rdcvf1 or Rdcvf2 or its fragment, antibody Rdcvf1, agonists, which leads to a weakening of symptoms or improvement. Therapeutic efficacy and toxicity is possible to define a standard pharmaceutical methods using cell cultures or experimental animals, e.g., defining the ED50(dose having therapeutic efficacy for 50% of the population) and LD50(the dose lethal to 50% of the population). The ratio of doses that characterize the toxic and therapeutic effects is a therapeutic index, and it can be expressed as the ratio LD50/ED50. Preferred are pharmaceutical compositions having high therapeutic indices. Data obtained from analyses of cell culture and animal studies, are used to support dose range, designed for a person. The dosage contained in such compositions, before occhialino is in the range of concentrations in blood, which include ED50and are accompanied by low toxicity or do not have a toxic effect. The dosage varies within this range depending upon the dosage form, the sensitivity of the patient and route of administration.
The exact dosage should be determined by the attending physician taking into account factors related to the patient in need of treatment. The dose and route of administration regulate to maintain sufficient levels of the active fragment or to maintain the desired action. Factors that could be taken into account include the severity of the disease condition, General health of the patient, age, weight and sex of the patient, diet, time and frequency of administration, combination(s) of drugs, reaction sensitivities, and tolerance/response to therapy. Pharmaceutical compositions with prolonged action, you can enter every 3-4 days, every week or once every two weeks depending on half-life and rate of clearance of a particular composition.
Usual amount of doses, depending on the route of administration can vary from 0.1 to 100,000 micrograms, up to a total dose of constituting approximately 1, Instructions regarding how specific doses, methods of administration, bringing in the literature and is generally available to doctors. Specialists in this field can apply compo is icii containing nucleotides, other than compositions containing proteins and their inhibitors. Similarly, the route of administration of polynucleotides or polypeptides must be specific to particular cells, conditions, areas of localization, etc. Pharmaceutical compositions suitable for oral administration of proteins, are described, for example, in US 5008114; 5505962; 5641515; 5681811; 5700486; 5766633; 5792451; 5853748; 5972387; 5976569 and 6051561.
Below the invention is illustrated in the examples, not limiting its scope.
Cleaning General ARN of the healthy retina of mice 5 weeks of age
The cDNA library from the retinas of mice 5-week-old C57BL/6@N designed, following in General the method described Glissin and others, Biochemistry, 13, 1974, cc.2633-2637. In General, the method was as follows: after killing the animals were subjected to enucleation and eyes first were placed in phosphate buffered saline (SFR), supplemented with 0.1% diethylpyrocarbonate (DEPC). The neural retina was immediately cut off (from this tissue preparation was removed pigmented epithelium of the retina). Immediately after each cut tissue homogenized in a freshly prepared 6M grandiflora (GC). United 10 retinas 2.4 ml GC in sterile test tubes with a volume of 4 ml and the fabric was completely destroyed by intensive homogenization for 1 min at room temperature.
Purification of messenger RNA, mRNA) from a healthy retina of mice 5 weeks of age
mRNA was isolated using coated with oligo-dt porous granules (type Oligotex, the company Qiagen) under strict conditions according to the method Kuribayashi, etc, Nucleic Acids Res. Symposium series, 19, 1988, cc.61-64. In General, the method was as follows: 100-150 µg total RNA of mouse retina were mixed with 15 µl coated with oligo-dt beads in buffer for binding [10 mm Tris pH 7.5; 0.3 m NaCl; 0,1M etc; 0.5/vol.% LTOs (sodium dodecyl sulphate)] and incubated for 6 min at 65°C in a beaker in 0,5 l of water, then gradually cooled to room temperature for about 3-4 hours, and then centrifuged at room temperature, highlighting the agarose pellets. They are then washed twice by incubation for 10 min in 0.4 ml of solution (0.1m Tris pH 7.5; 0.1 m NaCl; 1 mm etc; 0.5/vol.% LTOs). Associated RNA (mRNA) was suirable in two stages using 50 ál heated to 70°C water, not containing RNase, besieged by using 10 µl sodium acetate, pH of 5.2 and 0.25 ml of ethanol, and incubated for 12 h at -70°C. mRNA was collected by centrifugation (1 h at 15,000 rpm, then washed twice with 70%ethanol) and resuspendable in 20 μl of water, not containing RNase. The mRNA concentration was measured at 260 nm and the absence of impurities in the form of rRNA was checked by gel electrophoresis in the above-described denaturing conditions.
Synthesis of cDNA
Synthesis of cDNA was carried out according to the method of Okayama and Berg, Mol Cell Biol., 2 1982, cc.161-170. For the synthesis of the first chain made premirovanii using 2.5 µg adaptorname nucleotide for NotI (5'-TGTTACCAATCTGAAGTGGGAGCGGCCGACAA(T)18-3'), and incubated for 2 h with 50 units of reverse transcriptase modified virus murine leukemia (M-MLV) (Superscript II, firm Life Technology) under conditions recommended by the supplier. For synthesis of the second chain, the reaction mixture was incubated for 4 h at 14°C with 4 units of RNase H and 100 units of DNA polymerase I in SS-buffer [40 mm Tris pH 7,2; 85 mm potassium chloride; 4,4 mm magnesium chloride; 3 mm DTT (dithiothreitol); 5 μg/ml bovine serum albumin (BSA)] in a final volume of 0.25 ml EcoRI-adapter (5'-HE AATTCGGCACGAGG 3'-OH/3'-OH GCCGTGCTCC5'-PO4) ligated to both ends of the double-stranded cDNA for 14 h at 16°C using 40 units DNA ligase of phage T4 (firm Promega, Madison, USA) in a total volume of 20 µl in the conditions recommended by the supplier. The products of this reaction was a double-stranded cDNA (dzanc), which had poluchit EcoRI at the 5'-end and poluchit NotI at the 3'-end that allowed you to Orient it in the cloning vector.
Embedding tzcdnk by ligating into pcDNA3
Embedding tzcdnk by ligating in pcDNA3 was carried out according to Maniatis T., Molecular cloning: a laboratory manual, 2nd ed., 1992, using 10 μg of plasmid pcDNA3, Invitrogen), obtained by cleavage with EcoRI and NotI (firm Promega, Medic is h, USA) under conditions recommended by the supplier.
Propagation of recombinant clones
Propagation of recombinant clones was carried out generally according to the method of Birnboim and others, Nucleic Acids Res., 7, 1979, cc.1513-1523. In General, the method was as follows: to obtain pools of 100 primary clones was used by the Protocol transformation of XL1 Gold (firm Strategene), in which were made the following small changes. After incubation in the culture medium of the reaction mixture for transformation was made in the medium containing 20% vol. glycerol and 8% vol. horse serum albumin (LCA, firm Life Technologies). LCA and glycerin to prevent death in the thawing after freezing. The titration was carried out by sowing on agar plates (100 μg/ml ampicillin) increasing the volume of each reaction mixture for transformation in order to calculate the volume, allowing you to get 100 colonies, with uterine reaction solution for transforming stored at -80°C. Recombinant plasmids from the library was purified using at one time 96 pools. To obtain 96 pools, consisting of 100 clones, the calculated amount corresponding to 100 clones were sown on agar and grown for 20 h at 37°C. DNA was purified directly from the colonies, taken from agar plates. The mother liquor of every culture in 23%glycerol and kept at -80°C. DNA PTS is looking through the device Qiawell ultra (firm Qiagen), using the Protocol recommended by the supplier. As a rule, received 10 µg of purified plasmid concentration of each drug was evaluated by measuring the optical density at a wavelength of 260 nm. For the separation of selected pools containing 100 clones, pools containing 10 clones, 50 ál of dilution (1/250000) glycerol stock solution of the original pool were sown on agar plate supplemented with 100 μg/ml ampicillin. After growing for 16 h at 37°C 160 individual colonies were replicated on 16 agar plates (10 colonies per plate) and grown for 16 h at 37°C. each plate were collected from 10 colonies were grown in liquid medium [broth, Luria (LB), 100 μg/ml ampicillin] for 3 h at 37°Smutny solution of these crops in 30%glycerol and kept at -80°C. Plasmid DNA was obtained according to the above-described method. To separate subpool consisting of 10 clones, individual clones 50 ál of dilution (1/250000) glycerol stock solution were sown on agar plate supplemented with 100 μg/ml ampicillin. After growing for 16 h at 37°C, collected 16 single colonies and grown for 16 h in 2 ml LB supplemented with 100 mg/ml ampicillin. The mother solution of these crops in 30%glycerol and kept at -80°C. Plasmid DNA was obtained according to the above method.
Short Tr is speccie cell line Cos-1
Transient transfection of cell line Cos-1 was carried out using the method of Chen and Okayama, High-efficiency transformation of mammalian cells by plasmid DNA, Mol Cell Biol., 7, 1987, cc.2745-2752).
Cell culture of chicken embryo retina
For this purpose, we adapted the Protocol described by Adler and Hatlee, Science, 243, 1989, s. The retina of the chick embryo (6-day embryos in the egg) were subjected to dissociation and cultured in monolayer culture. Under these culture conditions in the absence of signal differentiation 60-80% of the cells are cones. In the claimed invention was obtained in the rabbit polyclonal antibodies to vizinio (marker cones of the retina of chickens, registration number M in Genbank) and checked that the proportion of cones in the above culture was 60-80%. Simple environment used in this model (specific chemical composition of the environment, lack of contact cells), in addition to the simplicity and speed of implementation of the method make this system very preferred for the study of trophic factors involved in the survival of cones. In General, the method consisted of the following: retinal embryos obtained from the reference isolate progenitors, cut after 6 days of development in the egg, the cells were subjected to dissociation and sown with low density (105cells/cm2). For 10 days was evaluated isnes osobnosti cells (60-80% of the cones) using the method of analysis of LIVE/DEAD (the company Molecular probes, Eugene, USA), i.e. analysis, which allows us to estimate the number of live and dead cells. The number of living cells is reduced to 8% from the initial number of cells after culturing for 7 days in having a certain chemical composition of the environment. When carrying out analysis in the presence of conditioned media from COS1 the number of living cells, transfection clones from the library were counted after in vitro culturing for 7 days.
Chickens-ancestors (line 657, red label) for the purpose of this experiment is contained in a separate room at the poultry farm, located 25 km from the laboratory. The resulting natural fertilization of the eggs collected weekly and after incubation was kept in the laboratory at 17°C (which corresponds to their biological zero). Daily 5 eggs were incubated for 24 h at 20°C, and then for 136 h at 37°C With periodic rotation angle of eggs in a special chamber. On the first day of cultivation, the surface of the eggs were washed Mucocit-A, then the shell was broken, chicken embryos transferred in SFR. Verify that the developmental stage of each embryo corresponded to 29-th stage, was carried out by visual comparison in accordance with the method of Hamburger and Hamilton, Essential Development Biology, edited by Stern and Holland, 1951. Selected two embryos and nucleosomal eyes bore is in a medium with arbitrary content of CO 2(firm Life Technologies). The retina was cut out and transferred into the buffer ringer's solution and washed twice. The retina was cut into small pieces and treated for 20 min at 37°C with trypsin solution (0.25% of wt./vol.). The reaction was stopped by adding culture medium (M199, firm Life Technologies), supplemented with 10% inactivated FCS. Cell suspension was treated for several minutes in 25 µl of Gnkazy I (1 mg/ml, the firm Sigma). Then, in order to remove the FCS, the suspension cells were twice washed in culture media having a specific chemical composition (Chemical Defined Culture Media) [CDCM, equal volumes of media DMEM and M199 (firm Life Technologies) and environment AV with additives (5 μg/ml insulin; 5 μg/ml transferrin; 64 nm progesterone; 0.1 mm of putrescine; 5 ng/ml selenium; 3 mm taurine; 2,7 µm citizen-5'-diphosphocholine; 5,2 μm citizen-5'-diphosphocholine, 0.2 ág/ml hydrocortisone; 30 nm 3,3'-5-triiodothyronine-L-thyronine; 1 mm sodium pyruvate), 0.3 μm prostaglandin D2; 0.1 mg/ml linoleic acid]. The concentration of cells stained Trifanova blue, was measured using cell Malassezia and brought up to two concentrations (5,6 and 1.12×105cells/ml), corresponding to the two planting densities (2 and 4×105cells/cm2).
Conditioned medium containing transfection cells Cos-1 were subjected to thawing on ice, and 50 μl was transferred to two 96-well Chernichenko for tissue culture (firm Corning Costar), which was senzibilizirani a solution containing 100 µg/ml poly-L-lysine (firm Sigma) according to the following plan.
The first round of screening:
|18||18||19||19||19td align="left"> 19||P||20||20||20||20|
In this table, the numbers correspond to the numbers pools, consisting of 100 clones, indicates the conditioned medium obtained from cells Cos-1, transfection empty vector (pcDNA3), R denotes the positive control (air-conditioned environment, transfection pcDNA-mouse GDNF).
The second and third cycle screening:
In this table x (second cycle) and in (third cycle) denote the number of pools selected in the first and second cycles, respectively. Number from 01 to 16 indicate the used supply. x(y) denotes the parent pool, which brought 16 pools. With has the same value as that of the first cycle. R modified using pCMVScript-CNTF in the second cycle and then pcDNA-939.09.08 in the third cycle. 0 denotes the cone cells of the retina of chickens in environments that sod is rasih only CDCM. C57 and SN denote air-conditioned environment, obtained from explants of retinas, which were isolated as described above (getting air-conditioned environments from retinal explants of mice), from retinas 5-week-old C57BL/6@N and C3H/He@N, respectively.
50 μl of the cell suspensions corresponding to the two densities (2 and 4×105cells/cm2), was added to each well of two 96-well plates filled with air-conditioned environments, using 8-channel automated pipette (company Biohit) in order to minimize experimental errors. Cells were incubated for 7 days at 37°C in atmosphere containing 5% CO2.
Receiving the conditioned media from explants of mouse retinas
In the second and third cycle of screening used positive controls, which were obtained according to the method adapted Mohand-Saïd and others (1998). Of mice 5C57BL/6@N (wild type) and C3H/He@N (rdl) 5 weeks of age were killed and analizirovali. Cut out two of the retina, and incubated for 24 h at 37°C in atmosphere containing 5% CO21.5 ml CDCM 12-hole tablets. Allocated air-conditioned environment and concentrated 40-fold by ultrafiltration using a device type (company Sartorius, level cutoff 10 kDa). Conditioned medium was frozen in liquid nitrogen and kept in altvaterisch at -20°C until use. On the day of use conditioned medium was subjected to thawing on ice, diluted 10 times in CDCM and sterilized by filtering through a filter with a cell size of 0.22 μm (type Acrodisk 13, the company Gelman Sciences).
Functional analysis, Live/Dead analysis
Functional analysis is based on determining the number of chicken cells of the retina, surviving after incubation in vitro for 7 days. This used kit Live/Dead analysis (company Molecular probes, Eugene, USA), which is based on the use of two fluorogenic dyes (calcein AM and ethidium dimer), staining living and dead cells, respectively. A living cell has a metabolic activity (in this case esterase activity), resulting in the substrate (calcein AM) is converted to its fluorescent product that emits fluorescence at a wavelength of 520 nm. The permeability of the membrane of dead cells is changed, which allows the staining of the DNA in the nucleus using ethidium dimer emitting fluorescence at a wavelength of 635 nm. If the cell is alive, the emission occurs at a wavelength of 520 nm after excitation at a wavelength of 485 nm, if dead, the emission occurs at a wavelength of 635 nm after excitation at a wavelength of 520 nm. Using EPI-fluorescence microscopy can be individually visualize two types of fluorescent cells. After cultivation the project within 7 days in vitro, cells were incubated for 30 min at room temperature in the dark in the presence of 2.7 μm calcein S and 0.3 mm ethidium dimer.
In General, the image acquisition is autofocusing each hole, automated counting cells at two wavelengths of fluorescence and subsequent processing of primary data using specialized software, for example, Metamorph, Universal Imaging Corporation, West Chester, USA), to obtain digitized images of each of the wells. Used inverted microscope (Nikon TE 200)equipped with EPI-fluorescence mercury lamp with two filters for excitation wavelengths of 485 and 520 nm, two filters for wavelengths of emission of 520 and 635 nm, a lens (×10)computer controlled motorized plate (Multicontrol 2000, the company Martzauzer) and CCD camera (camera charge-coupled) (firm Cohu).
To obtain an image of the tablet was placed on a mechanical plate and manually focused on the first hole and got the image in this plane (the z coordinates). Based on the data obtained for the first hole, set the threshold visualization of dead and alive cells. The position of the center of the first hole regulated in white light by combining manually the bottom of the first hole with the bottom of the image on the computer monitor, and then combined the right edge of the first hole on the right side of the image on the computer and recorded two positions. To calculate the position of the center of the first hole and determined the position of the center of each of the wells. During method development, it was found that in the corner of the wells has a few more high density of cells and therefore the angular region were excluded from treatment. It is important that the image of each well was strictly centered in order to avoid any results misleading. When configuring carried out first scan to obtain an image of dead cells. Under these conditions, the density of dead cells is less variable. The program was executed avtookutyvanie by taking images in different focal planes and chose the most striking the right focus. This z-position was memorable and the plate was carried out by programmed movement with respect to the axes x and y, when it received a total of 4 images, which after conversion to a single picture cover 2/3 of the surface of the well. The entire volume of images out of the focal planes, maintained for inspection. Using plates made avtookutyvanie and 4x data collection for each of the wells, from wells A1-A12, then B12-B1, C1-C12, etc. At the end of the plate has moved beyond a tablet to preexponential the last hole (H1). Scan the dead to etoc took 30 minutes The second scan (living cells) was carried out after changing the filter. During this second scan used the z-position of each well recorded while scanning the dead cells. As well as dead cells for each well received 4 images. After completion of the second scan (22 min) transformed images of live and dead cells were preserved in the file, which is automatically assigned a number with date. The number of cells (dead and alive) were calculated automatically in accordance with predefined morphometric parameters (mean values) and displayed on the computer monitor in order to verify the correctness of the experiment. It is important to check daily that the number of living cells is not too large. It was found that when sowing with too high density of cells of the chicken retina live longer apparently due to the fact that they produce their own survival factor. Cells were subjected to screening in the absence of such actions. Before scanning the second tablet (the same experiment was carried out at twice the density of cultured cells) at the end of the file name registration for the first tablet was added to the letter A. Images obtained in each experiment were stored on CD-ROM. Was created Jbeil is swelling, containing more than 250 CD-ROMs.
Counting cells and selection of pools
The number of cells (live and dead) were counted using images obtained in each experiment and stored on CD-ROM. Downloaded the log file for a specific experiment were loaded into a computer calculation was performed in off-line mode) and open it using the software Metamorph. The first stage was opened image files corresponding to 14 holes With (conditioned medium from cells Cos-1, transfection empty vector). After adjusting the threshold image used the command (program) Integrated Morphometric Analysis (Integrated Morphometry Analysis) to measure for these monitoring wells distribution of the premises (number of living cells for each full square) in the range from 10 to 250. The distribution was described by a Gauss curve, with the maximum number of objects corresponded to an isolated cell. Then this is the default value (SV) is used to calculate the magnitude of the square, above which the object should be considered as two cells (the standard threshold value of the object, SOC), using the obtained the inventors empirical functions: SOC=29/20,74 SV. The amount of SOC for each individual tablet is used for counting the number of live cells per tablet. According to the scientists the data is then transferred to an Excel table.
For the first round of screening on the basis of the data for each pool built by the graph of fold difference (increase or decrease quantities of living cells with respect to the average value for 14 control wells) ± standard deviation. In order to eliminate variations caused by the difference of the provisions on the tablet, expected average fold difference individually between 80 holes corresponding to the positions where tested pools and 14 control wells for 200 independent tablets. On average, the observed differences were due only to the difference in the position and number of cells was adjusted taking into account this factor. In order to more rigorously determine intended for breeding pools, build a dependency graph of the amounts of living cells in the form of a fold difference relative to the control, which used all the pools, for which the value is a multiple of the difference did not exceed 1.3, but exceeded the 0.4. Thus, it was believed that all the pools for which fold difference compared with 14 holes, containing an empty vector, were in the range from 0.4 to 1.3, had no action and considered as control. After the correction fold difference relative to the control for the two tablets were perennial and the results were arranged in order of decreasing fold difference. PU is s, situated at the head of this list, were subjected to further validation by visual examination of the graph corresponding to 20 pools, which conducted the experiment (both tablet), and images of live and dead cells in order to avoid screening doubtful pools.
For the second and third cycle in screening tablets for subpool used for more control. To prepare conditioned medium from retinal explants of mice 5 weeks of age. Selected experiments were found positive steps to retinal explants obtained from C57BL/6@N and excluded others. The results were presented in the form of a graph fold difference relative to the 14 monitoring wells for controls conversion is not made. Was perennially fold difference relative to the control for the two tablets and the results for 16 subpool had in order of decreasing fold difference.
Selected cDNA sequenced using T7 primer (5'-GTAATACGACTCACTATAGGGC-3') on capillary sequencing machine (type CEQ2000, the company Beckman Coulter). The DNA sequence was compared with the database using the Basic Local Alignment Search Tool (BLAST).
Identification of Rdcvf2 and human homologues
Using sequence Rdcvf1 (nucleotide sequence encoding the polypeptide presented in SEQ I NO: 2 or SEQ ID NO: 4) and the BLAST program identified homologous mouse and human polypeptides (Fig). Were identified following EST-clone, homologous to mouse RdCVF2 (registration number in GenBank: bc016199): registration number in GenBank: be552141, bi517442, bg707818, bi603812, ai433287, be088414, bg297383, bg297304 (see also Fig). Were identified following EST-clone, homologous to mouse Rdcvf1 (SEQ ID NO: 1): registration number in GenBank: bg299078, ai716631, bg294111, be108041, bg395178 (see also Fig).
The real-time analysis of the expression of Rdcvf1 using RT-PCR
The expression of Rdcvf1 in the retinal cells C57BL/6@N and C3H/He@N 5 weeks of age, and congenic mice SN (+/+ and rd/rd) of the same age were studied using RT-PCR in real time in a transparent cell (firm Roche) using sybergreen-set for PCR (firm Roche). s was obtained by random premirovany using a review of the oligonucleotide (pdN6, firm Amersham), reverse transcriptase M-MLV (superscript II, firm Life Technologies) and total RNA from mouse retina, obtained as described in 1). cDNA was standardized with the use of ubiquitous carrier of glucose-6-phosphate dehydrogenase (G6PDH). 0.2 µl of the synthesized first chain cDNA (equivalent to 10 ng total RNA) is amplified using a 2 μm of oligonucleotides, the sequence of which is presented in SEQ ID NO: 24 and SEQ ID NO: 25, in triplicate in a total volume of 25 ál according to the following program: 30 s at 95°C and 35 cycles carried out in the following serial the major: (1 s at 95°C, 18 s at 55°C, 10 s at 72°C). The results of the analysis (Fig) suggests that the expression of Rdcvf1 decreases after degeneration sticks in rd1 mice (C3H/He@N). With help FROM real-time PCR using RNA obtained from the outer layer of the retina with the use of vibratome for making cuts, it was found that Rdcvf1 is expressed directly by the photoreceptors. Products were checked by agarose gel electrophoresis. Similar results were obtained using another pair specific in respect of Rdcvf1 primers. As a positive control was assessed by the expression of arrestin sticks (registration number M) under the same conditions using primers (5'-CTATTACGTCAAGCCTGTAGCC-3' and 5'-ALTSTADTFEST-3'). Confirmation that Rdcvf1 is a protective factor cones, can be obtained by adding the corresponding number of Rdcvf1 to the retinal Explant mouse lines rd1 (C3H/He@N) 5 weeks of age. The required number can be estimated by a few preliminary experiments by titration of the initial amount. Compared with the corresponding controls after 7 days survival of cones should be increased.
RT-PCR analysis of Rdcvf2
RT-PCR for the expression of Rdcvf2 was performed using primers 5'-GCCAGCGTTTTCTGCCTTTTAC-3' and 5'-AAGCCCTGCCTGCTCTAACATC-3'. The results of the analysis of the evidence of the expression of RdCVF2 depends on sticks and that the expression of Rdcvf2 occurs not only in the retina and other nerve cells also Express Rdcvf2 (Fig), while the expression of Rdcvf1 apparently occurs only in the cells of the retina.
Live-Dead analysis of Rdcvf1 or Rdcvf2
Cells COS-1 was transfectional suitable expression vector carrying Rdcvf1 or Rdcvf2 under control of the inducible promoter. Control cells were transfectional empty vector. Cells were incubated for a certain period of time after induction of expression of Rdcvf1 or Rdcvf2. Then the number of surviving cells, cone cells, which were incubated in conditioned media from cells COS-1, transfection Rdcvf1 or Rdcvf2, and the number of surviving control cells was calculated according to the above-described method. Found that cells expressing the Rdcvf1 or Rdcvf2, are characterized by a significantly higher survival rate than the control cells.
Specific in relation sticks factor
The results of the standard conditions of RT-PCR-real-time analysis of the expression of arrestin sticks (control) and Rdcvf1 in retinal explants of 5-week-old mice C57BL/6@N and C3H/HE@N using primers
SEQ ID NO: 24: 5'-TCTATGTGTCCCAGGACCCTACAG-3'
SEQ ID NO: 25: 5-TTTATGCACAAGTAGTACCAGGACAG-3'
indicate that RdCVF1 is expressed only in the presence of the sticks (you the Eden Popsicle CVF1).
Obtaining polyclonal antibodies
Polyclonal antibodies were obtained in the body of rabbits as a result of injection of rabbit purified fused protein carrying a glutathione-S-transferase (GST) (GST-Rdcvf1), and a fragment of the peptide sequence of the mouse RdCVF1 extending from amino acid at position 11 to the amino acid at position 32 of SEQ ID N02 (At No. 2), and a fragment of a peptide sequence extending from amino acid position 79 to the amino acid at position 96 of SEQ ID N02 (At No. 3). Fused design pGST-Rdcvf1 was obtained by amplification using the oligonucleotides presented in SEQ ID NO: 26 and SEQ ID NO: 27, using pcDNA-Rdcvf1 as a matrix in standard conditions. Open-reading frames (OPC) Rdcvf1 cloned in frame read into pGex2TK (firm Pharmacia) between the restriction sites BamHI and EcoRI and transformed E. coli [BL21 (DE3) pLysS, the company Promega] using the standard method. An individual colony was grown in 3 l of liquid LB medium supplemented with 100 µg/ml ampicillin at 30°C, the protein production was induced by adding 1 µg/ml isopropylthio-b-D-galactoside (IPTG) and continued for 5 h at 30°C. Cells were harvested, literally by exposure to ultrasound, and was purified on glutationreductase according to the standard Protocol. Fused protein was suirable using 10 mm reduced glutathione at room temperature is. Suirvey protein is dialyzed in SFR before injection to rabbits. The purity of the protein was assessed by electrophoresis in polyacrylamide gel. Two rabbits were immunized by intradermal injection in 80 locations with 100 µg of purified GST-Rdcvf1. The serum was collected after 8 weeks.
1. The use of the polypeptide with the amino acid sequence selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, for the preparation of drugs for the treatment of retinal dystrophy.
2. The pharmaceutical composition intended for the treatment of retinal dystrophy and comprising a therapeutically effective amount of a polypeptide selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, and a pharmaceutically acceptable carrier.
3. The use of a nucleic acid that encodes a polypeptide with the amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, for the preparation of drugs for the treatment of retinal dystrophy.
4. The pharmaceutical composition intended for the treatment of retinal dystrophy and comprising a therapeutically effective amount of a nucleic acid selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 13, and a pharmaceutically acceptable carrier.
5. The purified polypeptide having the ability to increase vyjivaemosti retina and including amino acid sequence, presented in SEQ ID NO: 2.
6. Nucleic acid with a nucleotide sequence of SEQ ID NO: 1 and the nucleic acid encodes a polypeptide that increases the survival of cones of the retina.
7. The expression vector including a nucleic acid sequence that encodes a polypeptide with the amino acid sequence SEQ ID NO: 2, which is functionally connected with the sequence controlling transcription, for use in the preparation of a medicine for the treatment of retinal dystrophy.
8. The expression vector according to claim 7, derived from an adenovirus or virus associated with adenovirus.
9. The purified antibody obtained by immunization of an animal with the polypeptide with the amino acid sequence presented in SEQ ID NO: 2, which specifically binds to the specified polypeptide, or a functionally active fragment.
10. The fragment of the antibody according to claim 9, representing the Fab or F(ab')2 fragment.
11. The antibody according to claim 9, representing a polyclonal antibody.
12. The antibody according to claim 9, representing a monoclonal antibody.
SUBSTANCE: invention represents plasmid vector for transfer of DNA, which comprises sequence coding various fragments of oncoprotein p185neu, which are able to induce immune response in respect to tumors, which hyperexpress p185neu. Invention is also related to pharmaceutical composition on the basis of vector for prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.
EFFECT: invention makes it possible to increase efficiency of prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.
10 cl, 14 dwg, 2 tbl, 2 ex
SUBSTANCE: glia cells are extracted, seeded, and cell is cultivated to produce monomolecular layer. Then cells are extracted and washed. Total RNA is extracted from them, reverse transcription is carried out, as well as amplification of produced DNA. Genes expression is assessed under action of tested compound.
EFFECT: invention makes it possible to accelerate screening of compounds, to search for new compounds with highest activity, which are potential neuroprotectors.
4 cl, 4 dwg, 1 tbl, 4 ex
SUBSTANCE: there is described method for preparing a polymorphous region of gene PAR1 which can contain T to C replacement in position 3090 and/or A to C replacement in position 3329 of the polynucleotide sequence of wild type gene (NM_001992) with applying a pair of specific primers, and also the method for observing said region prepared of a DNA-containing biological sample for the presence or absence of the specified replacements. There are offered complete sets of the components application of which provides both amplification of the polymorphous region of gene PAR1 under the invention, and if needed, further analysis for genetic modifications in positions 3090 and/or 3329.
EFFECT: higher accuracy of estimating risk of cardiovascular diseases.
7 cl, 17 dwg, 1 ex
SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.
EFFECT: creation of model cat for research of retroviral infection.
24 cl, 10 dwg, 6 tbl, 8 ex
SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.
EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.
16 cl, 3 tbl, 7 ex
SUBSTANCE: as a result of substitution of part of amino-acid sequence of human protein PRG-4 coded with exon 6 with artificial polypeptide, including 4-15 sequences KEPAPTT or KEPAPTT-like sequences, recombinant proteins are produced with considerably lower number of glycolised repetitions compared to natural lubricin, which at the same time preserve its lubricating properties. It is suggested to use new recombinant proteins in pharmaceutical compositions and in methods of treatment, where natural lubricin is traditionally used.
EFFECT: application of the present invention for medical purposes has certain advantages related to simplification, compared to native protein.
28 cl, 1 dwg, 3 tbl, 9 ex
SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.
EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.
20 cl, 50 dwg, 1 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to production of versions of the Gla domain of human factor VII or human factor VIIa, and can be used in medicine. Amino acid sequence of the FVII or FVIIa version is obtained, which differs on 1 to 15 amino acid residues with amino acid sequence of the human factor VII (hFVII) or human factor VIIa (hFVIIa), in which a negatively charged amino acid residue is introduced by substitution in position 36. Obtained variants of FVII or FVIIa are used in a composition for treating mammals with diseases or disorders, where blood clotting is desirable.
EFFECT: invention allows for producing versions of FVII or FVIIa with high clotting activity and/or high activation of factor X, compared to natural form of hFVIIa.
42 cl, 3 dwg, 5 tbl, 11 ex
SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.
EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.
5 cl, 2 ex, 2 tbl, 4 dwg
SUBSTANCE: thrombin derivative is described that includes chain A and chain B where chain B has an aminoacid sequence whose aminoacids of the series active center in position 205 and histidine in position 43 in the aminoacid sequence of chain B of thrombin are substituted and where: the specified thrombin derivative decomposes thrombin substrate to the extent of 10% or less when interacting with thrombin substrate in 50 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl at the temperature of 37°C for 3 hours and the specified thrombin derivative retains the ability of getting combined with C-terminal hirudin peptide immobilised in gel.
EFFECT: pharmaceutical formula is disclosed that contains the thrombin derivative described.
54 cl, 48 dwg, 2 tbl, 31 ex
SUBSTANCE: invention is related to biotechnology and represents monoclonal antibody or its antigen-binding fragment, containing antigen-binding site, which specifically binds with protein of prostatic stem cells antigen (PSCA). At the same time monoclonal antibody is produced with postfusional cell line, selected from group of postfusional cell lines deposited in American typical cultures collection (A.T.C.C.) under inventory No.PTA-6698, PTA-6699, PTA-6700, PTA-6701, PTA-6702 and PTA-6703. Besides invention is also related to expression vector, which contains polynucleotide coding this antibody, and also to method for analysis for detection of PSCA protein presence in biological sample with application of this antibody. Moreover, invention is related to method for delivery of cytotoxic agent or diagnostic agent to cell, which expresses PSCA protein, with application of this antibody, and also to method for detection of PSCA protein in biological sample with application of above-mentioned antibody.
EFFECT: invention may efficiently be used in active and passive immunisation against cancer diseases.
21 cl, 27 dwg, 11 tbl, 33 ex
SUBSTANCE: invention can be used for production of monoclonal antibodies (MCAs) to heat shock protein 70 (HSP 70). A hybridoma strain is made by immunisation of BALB/c mice with bovine HSP 70 within 78 days. For the third days, splenocytes of immune mice (108 cells) are hybridised with murine myeloma cells P3-X63 Ag/8-653 (107 cells). A fusion agent is polyethylene glycol of molecular weight 4000 (Merk, Germany). The hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma. Hybridoma 6G2 is deposited in the microorganism collections of "ГНТТ ПМБ" under No. H-2. MCA.
EFFECT: produced hybridoma under the invention is more evident to be detected as HSP 70 on the cell surfaces, and change of endocellular HSP 70 level when exposed to the stress factors.
4 dwg, 1 tbl, 6 ex
SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.
EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.
16 cl, 4 dwg, 1 tbl, 13 ex
SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.
EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.
16 cl, 34 dwg, 7 tbl, 20 ex
SUBSTANCE: invention relates to Bcl-2 proteins, fragments thereof, and to application thereof in patients with a malignant tumour. The declared proteins and peptide fragments particularly are applicable in vaccine compositions for treatment of malignant tumour. Besides, the invention concerns the methods of treatment with application of specified compositions. Also, an aspect of the invention is production of T-cells and receptors thereof which are specifically recognise declared proteins and peptide fragments.
EFFECT: higher clinical effectiveness with respect to tumours.
61 cl, 5 ex, 2 tbl, 12 dwg
SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.
EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.
4 cl, 6 dwg, 22 tbl, 19 ex
SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.
EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.
10 cl, 21 dwg, 11 ex
SUBSTANCE: strain A-4A7 is prepared by fusion of mouse myeloma cells of line SP2/0.Agl4 with mouse lymphocytes of line Balb/c immunised by introduction in pads of a purified preparation AMGF (alpha2-microglobulin of fertility) separated from amniotic fluid, and deposited in the transplantable mammal cell culture collection of the Research Institute of Human morphology of the Russian Academy of Medical Science numbered 131/2002. Strain A-4A7 synthesises monoclonal antibodies (MCA) of IgGI class specifically reacting in solid-phase immune-enzyme analysis (IEA) with AMGF isoforms of endometrial, follicular and sperm nature. Activity of the strain: cultural supernatant contains MCA 3-5 mkg/ml, while ascetic fluid contains MCA 2-5 mg/ml. The antibody titre in cultural fluid is 1:500-1:1000, in ascetic fluid up to 1:1×107. A-4A7 bonds various AMGF protein glycoforms produced in male and female reproductive organs. Application of MCA A-4A7 as an immunodiagnosis test systems allows for high-specific and high-sensitive (1 ng/ml) quantitative analysis of various AMGF/glykodeline isoforms in biological liquids.
EFFECT: new compounds are characterised with valuable biological properties.
SUBSTANCE: method for making polyclonal Nogo protein antibodies by protein immunisation of an animal, with at least 6 amino-acid residues of active area Nogo A, free from any other myelin material of central nervous system whereto bound in natural conditions. Said active area covers position 1-171, 260-974, 1163-1178 of the corresponding amino acid sequence of protein Nogo A. There is disclosed separated antiserum based on polyclonal antibodies made under said method. There are disclosed methods for immunisation of a non-human animal to make polyclonal antibodies, as well as method for making monoclonal antibody, and the specified monoclonal antibody.
EFFECT: making active protein Nogo A antibodies to be applied in medicine for neurite growth activation.
26 cl, 18 dwg, 2 tbl, 8 ex
SUBSTANCE: polypeptides include single-domain antibody against vWF, A1 domain of vWF, A1 domain of activated vWF, A3 domain of vWF, gp1b and/or collagen. Invention claims methods of obtaining indicated polypeptides, methods of coating devices applied in medical practice (e.g. in X-ray structural analysis, endoprosthetics) with indicated polypeptides.
EFFECT: obtainment of polypeptides for treatment of diseases requiring modulation of thrombocyte-mediated aggregation.
40 cl, 69 ex, 30 dwg, 32 tbl
SUBSTANCE: invention refers to medicine, veterinary science and agriculture. There is presented exogenous modulator of metabolic and energy interchange processes between a cell and environment representing a polypeptide metabolic process modulator of molecular weight 15 to 10 KDa made of human placenta by chemical modification of placental biobased products with reactions of chlorination, oxidation, hydrolysis, and containing covalently bound chlorine atoms with nitrogen atoms of peptide bond in amount 0.5 to 40% of weight of an end product. There is also offered method for making said modulator.
EFFECT: correction and regulation of metabolic processes to ensure maximum viability of human body, animals and plants.
7 cl, 8 tbl, 10 ex