Vβ-dβ-jβ oligonucleotide of t-cell receptor, pair primers, oligonucleotide probe, method for detection of mbp83-99vβ13.1 clone pf t-cells, espressing lgraglty motive of t-cell receptor, kit, method for treatment of autoimmune decease and method for monitoring thereof

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to T-cell receptor sequence being detected in patients with extended sclerosis and is useful in diagnosis and therapy. Oligonucleotide including sequence which represents or is derived from 5'-CTAGGGCGGGCGGGACTCACCTAC-3' or nucleotide sequence being fully complementary thereto. Oligonucleotide together with nuclear acid including nearly 15-30 oligonucleotides, which doesn't comprise oligonucleotide sequence and presents in region from Vβ to Jβ of Vβ13.1 gene in T-cell Vβ13.1-subgroup, wherein oligonucleotide and nuclear acid sequences don't present in the same chain of pair sequences of Vβ13.1 gene, is used in Vβ13.1 gene part amplification. In method for detection of LGRAGLTY motive, which is present in T-cell receptors of T-cell Vβ13.1-subgroup, oligonucleotide is used in combination with labeling particle. Once LGRAGLTY motive is detected, development monitoring and treatment are carried out by removing of LGRAGLTY motive-containing peptide.

EFFECT: simplified methods for detection of LGRAGLTY motive in T-cell receptors and treatment of patients with extended sclerosis.

21 cl, 7 dwg, 3 tbl, 3 ex

 

Background of invention

According to the grant room NS36140 received from the National institutes of health (National Institute of Health) the government of the United States may be entitled to the present invention.

1. The technical field to which the invention relates.

The present invention generally relates to the field of treatment of autoimmune diseases such as multiple sclerosis (MS). More specifically it refers to the sequence of the T-cell receptor, found in some sufferers of MS patients, and to the method of its detection.

2. Description of the prior art

In humans and other mammals on the surface of T cells detected T-cell receptors. T-cell receptors contain αand β-chain, and β-chain consists of the following areas (listed from N-Terminus to the C-end): Vβ-Dβ-Jβ-Cβ. In vivo T-cell receptors have different Vβ-Dβ-Jβ-field.

When the antigen is presented to T cells by antigen-presenting cells (APC), T-cell receptor variable regions (including Vβ-Dβ-Jβ), which has the ability to recognize the antigen binds to the antigen on the APC. After that T-cells bearing T-cell receptor, undergoes activation (clonal expansion).

The pathogenesis of some autoimm is the R diseases, apparently, associated with autoimmune T-cell responses to antigens, which are normally present in the body. An example of such a disease is multiple sclerosis (MS), which usually occur T-cell responses to myelin antigens, in particular on the basic protein of myelin (MBP). Found that MBP-reactive T cells are activated in vivo, and this occurs when higher compared with the control frequency of occurrence of precursors in the blood and cerebrospinal fluid in patients MS patients. These MBP-reactive T cells produce Th1-cytokines, such as IL-2, TNF and γ-interferon. These Th1-cytokines facilitate the migration of inflammatory cells into the Central nervous system and exacerbate the damaging myelin inflammatory response in MS.

In the treatment of MS can be used a variety of regulatory mechanisms. One of them is vaccination with the use of one or more related membrane peptides from extracellular domains, which reduces the number of T-cells. Vandenbark in patent US 5614192 offers treatment of autoimmune diseases using the immunogenic peptides of the T-cell receptor consisting of 15-30 amino acids that include at least a portion of the secondary hypervariable segment (CDR2) T-cell receptor. In the process of simultaneous considered the I the patent application US in the name of Zhang (60/099102) the proposed treatment of autoimmune diseases using the immunogenic peptides of the T-cell receptor in combination with immunogenic marker peptides T-cell activation.

One of the possible ways to improve the effectiveness of vaccination with peptide T-cell receptor is to identify what General motives (if any) present in T-cell receptors in patients suffering from autoimmune disease such as MS. If such motives are detected, the patient can be vaccinated peptides identical to these motives, in order to facilitate treatment.

Thus, there is a need to identify amino acid sequences common motifs present in T-cell receptors in patients suffering from autoimmune disease. It is also important to have the ability to easily detect such motifs in a patient using a conventional method such as PCR. In addition, it is desirable to apply the peptides identical to the identified motives for the treatment of a patient suffering from an autoimmune disease.

The present invention describes such a common motif found in T-cell receptor Vβ13.1-subgroup of T cells, i.e. the "LGRAGLTY-motif", which has the amino acid sequence Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3), and the method of its simple detection by PCR. This motif is found in some T-cell receptors of those T cells that recognize amino acids 83-99 MBP (hereinafter designated as "MUR-99"). The motif in the context of a specific Vβ131-subgroups of T cells may be further designated as "Vβ 13.1-LGRAGLTY". Identical to the motif peptides can be used for vaccination of patients with the aim of treating or preventing autoimmune diseases is associated with the motif Vβ13.1-LGRAGLT. One such autoimmune diseases is MS.

Summary of the invention

One of the embodiments of the present invention is an oligonucleotide consisting of about 15-30 nucleotides that includes at least 10 consecutive nucleotides of SEQ ID NO:1 or the sequence complementary to it or derived from it. Even more preferred is an oligonucleotide consisting of about 15-30 nucleotides, which includes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of about 15-30 nucleotides, which includes the nucleotide sequence of SEQ ID NO:1 or the sequence complementary to it.

In some additional embodiments, the oligonucleotide can be used for amplification or detection of the nucleotide sequence identified in T-cell motif Vβ13.1-LGRA GLTY. In a subgroup of embodiments of the oligonucleotide used in a pair of primers, and primer pair consists of or is derived from:

and parvovirinae, which is an oligonucleotide consisting of about 15-30 nucleotides, which contains at least 10 consecutive nucleotides of SEQ ID NO:1 or the sequence complementary to her; and

(b) a second primer which is an oligonucleotide consisting of about 15-30 nucleotides, which does not include the sequence specified in (a), and this second Primera sequence can be found in a region extending from Vβ to Jβ gene Vβ13.1 (SEQ ID NO:2) in the T-cell receptor T-cells, and the sequence specified in (a) and (b), are not present on the same chain gene T-cell the receptor.

Preferably, the first primer is an oligonucleotide consisting of about 15-30 nucleotides, which includes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of about 15-30 nucleotides, which includes the nucleotide sequence of SEQ ID NO:1 or the sequence complementary to it.

In another subgroup of embodiments of the oligonucleotide used as an oligonucleotide probe and the oligonucleotide probe includes:

(a) oligonucleotide consisting of about 15-30 nucleotides, which include the AET at least 10 consecutive nucleotides of SEQ ID NO:1 or the sequence complementary to her; and

(b) particle labeling.

Preferably the oligonucleotide consists of about 15-30 nucleotides and includes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of about 15-30 nucleotides, which contains the nucleotide sequence of SEQ ID NO:1 or the sequence complementary to her. Particle labeling preferably selected from32R or digoxigenin.

According to another variant implementation of the present invention relates to a method for detection of clone PSR-99 Vβ13.1-subgroup of T-cells expressing the motive LGRAGLTY, which provides:

(I) obtaining a sample of nucleic acid from the clone PSR-99 Vβ13.1-subgroups of T cells;

(II) bringing the sample nucleic acid into contact with a pair of primers that are selected or derived from:

(a) a first primer which is an oligonucleotide consisting of about 15-30 nucleotides, which contains at least 10 consecutive nucleotides of SEQ ID NO:1 or the sequence complementary to it or derived from it; and

(b) a second primer which is an oligonucleotide consisting of about 15-30 nucleotides, which does not include the sequence is, specified in (a), and is present in the area extending from Vβ to Jβ T-cell gene Vβ13.1 (SEQ ID NO:2),

moreover, the sequence specified in (a) and (b), are not present on the same chain gene Vβ13.1; and

(III) detecting the presence of nucleic acid encoding the motif LGRAGLTY.

Preferably, the first primer is an oligonucleotide consisting of about 15-30 nucleotides, which contains 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of about 15-30 nucleotides, which contains

the nucleotide sequence of SEQ ID NO:1 or the sequence complementary to it.

According to another variant implementation of the present invention relates to a method of treatment of autoimmune diseases, including:

(a) isolation of clone PSR-99 Vβ13.1-subgroups of T cells from the human body;

(b) identifying the presence of a nucleic acid that encodes a motive LGRAGLTY, using the above method; and if the nucleic acid is detected, then

(C) introduction to the human peptide Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3).

According to another variant implementation of the present invention relates to a method of monitoring an autoimmune disease, OEM home button Flex cable is travelmenu:

(a) isolation of clone PSR-99 Vβ13.1-subgroups of T cells from the human body;

(b) identifying the presence of a nucleic acid that encodes a motive LGRAGLTY, using the above method; and if the nucleic acid is detected, then

(C) quantification of nucleic acids.

Brief description of drawings

The following drawings form part of the present description and are included to further explanations of some of the objects of the present invention. The invention will become better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments.

Figure 1 shows the experimental procedure cloning and sequencing PCR products isolated from mononuclear cells of peripheral blood (RVMS). cDNA obtained from samples RVMS, were subjected to amplification using primers 5'Vβ13.1 and primer 3'Jβ 4 samples RVMS, positive in relation to the expression of motive LGRAGLTY, and was built by ligating into the TA-cloning vector pCR2.1 and transformed E. coli them. Plasmid DNA was subjected to screening by PCR using primers M13 and LGRAGLTY-specific primer. Positive plasmids, which revealed a noticeable PCR amplification, sequenced in relation to the availability of sequences VβDβJβwith Vβ 13.1-primer.

Figure 2 presents the scheme of the reactivity of the two T-cell clones MUR-99 in respect of peptide analogues with the replacement of alanine. Two pairs of T-cell clones MUR-99, which are characterized by the same rearrangement of the gene Vβ13.1 (MS7-E2.6 and MS27-C3.1) and the same sequence Vα-Jα-interface (MS7-E2.6 and MS27-C3.1), was evaluated from the viewpoint of reactivity against a panel replaced by alanine peptides in the experiments on the incorporation of [3H]-thymidine. As a source of antigen-presenting cells used line of mouse fibroblasts expressing DRB1*1501. Proliferative responses of clones for each of the peptide analogs were evaluated after 72 h and the result is presented as the number of CPM (pulses/min). Enclosed in the rectangle (shaded) data are consistent with the reduced proliferation of T-cell clones by more than 50% in response to the impact of peptide analogues.

Figure 3 presents data on cross-specificity CDR3-oligonucleotides with respect to source and unrelated T-cell clones. The set of oligonucleotides, specific VDJ-region of T-cell receptor (TCR), evaluated from the point of view of their specificity in the detection of known DNA sequences of target present in the original T-cell clones MUR-99, as well as an unrelated T-cell clone PSR-99, obtained from the same or different individuals. Was carried out by PCR reaction using DR3-specific oligonucleotides as primers for synthesis during transcription (direct primer)and 3'-β-primer as a primer for synthesis of progress against transcription (reverse primer). Solid rectangles indicated positive results of determination of the DNA sequences present in the original T-cell clones or T cell (s) clone(s)support(s) the same CDR3 sequence. All primers were also evaluated for their ability to bind with DNA products randomly selected T-cell clones, which carried an unrelated CDR3 sequences (shaded rectangles).

Figure 4 shows the detection of a sequence of target DNA, complementary motif Vβ13.1-LGRAGLTY, in randomly selected samples RVMS taken from patients suffering from MS. cDNA obtained from samples RVMS taken from randomly selected patients suffering from MS (n=48), is first amplified by PCR with reverse transcriptase (RT-PCR)using 5'-Vβ13.1-specific primer and 3'-β-primer. Amplificatoare PCR products were then subjected to hybridization with labeled using digoxigenin oligonucleotide is first probe, specific motive LGRAGLTY. The original clone PSR-99 (MS7-E2.6) and unrelated T-cell clone (MS32-B9.8) were used as positive and negative controls, respectively. MS-7 MS-27 was a source samples RVMS of clone MS7-E2.6 (MS-7 in table 1) and clone MS27-C3.1 (MS-27 in the table). Asterisks shows positive expression of DRB1*1501.

Figure 5 shows the identification of motif Vβ13.1-LGRAGLTY in randomly selected samples RVMS taken from healthy people. Types RVMS taken from 20 healthy individuals (NS), were analyzed in the same way as presented in the description of figure 4. The original clone (MS7-E2.6) and unrelated T-cell clone (MS32-B9.8) were used as positive and negative controls, respectively. Asterisks shows positive expression of DRB1*1501.

Figure 6 shows the semi-quantitative comparison of the expression of motive LGRAGLTY in samples RVMS taken from suffering from MS patients and healthy people. The expression of motif Vβ13.1-LGRAGLTY analyzed using semi-quantitative PCR in comparison with the expression Withβ in each cDNA obtained from RSMS suffering from MS patients and healthy people. The relative expression level was calculated from the following equation: (the expression of the motive LGRAGLTY/expression of Cβ) × 100%.

7 shows the identification of motif Vβ13.1-LGRAGLTY in the short-lived T-Claix the full line MUR-99, obtained from patients suffering from MS. From the blood of 5 suffering from MS patients using synthetic peptide MBP 83-99 received a panel of independent short-lived T-cell lines MUR-99. For all of these T-cell lines was confirmed by specific reactivity against peptide MUR-99 (number of pulses per minute (CPM) in response to the processing line MUR-99/number of pulses per minute in the control > 5). The products of cDNA amplified by PCR using 5'-Vβ13.1-specific primer and 3'-β-primer PCR. Amplificatoare PCR products are then hybridized with labeled using digoxigenin oligonucleotide probe corresponding to the motif Vβ13.1-LGRAGLTY, using the method of southern blotting. cDNA products obtained from the original clone PSR-99 (MS7-E2.6) and unrelated T-cell clone (MS32-B9.8), were used as positive and negative controls, respectively.

Description of the preferred embodiments of the invention

With the aim of better understanding the inventions listed below are some concepts.

"PCR" means polymerase chain reaction, for example, as it is generally described in US patent No. 4683202 (issued July 28, 1987 in the name of Mullins), which is incorporated into this description by reference. PCR is a method of amplification, whereby selected oligonu leotide or primers hybridized with nucleic acids-matrices in the presence of polymersomes agent (such as polymerase) and four nucleotidase, and from primers receive the products lengthening. Then these products are denatured and used as matrices in a cyclic reaction in which amplificates a significant number of used copies of nucleic acids, which facilitates their subsequent detection. Numerous PCR methods, and they can be used according to the method according to the invention.

"Primer" means an oligonucleotide probe of natural origin or synthetic, which has the ability to initiate DNA synthesis, complementary to specific DNA sequences on a molecule-matrix.

"Obtained from/extracted from" in the context of the concept of "primer(s) or probe(s), receipt(s) or inferred(s)" indicates that the primer or probe is not limited to the specified one(s) in nucleotide sequence(s), but also includes(ut) variations specified(s) nucleotide(s) sequence(s), including nucleotide additions, deletions or substitutions to the extent that variations of the specified(s) sequence(s) retains the ability to act as primers in the detection of DNA T-cell receptor, which encodes the sequence Vβ13.1-LGRAGLTY, i.e. Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO:3).

"Immunogenic", if this term is used to describe the peptide indicates that the peptide has the ability inducion the th immune response, mediated or T-cell or antibody, or both factors. "Antigen" means that the peptide can be detected in free form antibodies and MHC molecules in the case of antigen specific T cells.

"Associated with immune system disease" means a disease in which the pathogenesis of the disease involved the immune system. Subgroup associated with immune system diseases are autoimmune diseases. Autoimmune diseases in the context of the present description include, but are not limited to) rheumatoid arthritis, myasthenia severe paralysis, multiple sclerosis, systemic lupus erythematosus, autoimmune thyroiditis (Hashimoto's thyroiditis), graves ' disease, inflammatory bowel disease, autoimmune uveoretinitis, polymyositis and certain types of diabetes. On the basis of the present description, the person skilled in the art can easily determine other autoimmune diseases that can be treated using compositions and methods of the present invention. "Mediated T-cell disease" means a disease arising in the body as a result of the recognition of T-cell peptides that are present normally in the body.

The term "treat" or "treatment" refers to the protection of an animal from a disease that includes prevention, inhibition (suppression) or suppression of Zabol the cation. Disease prevention includes the introduction of the compositions of the present invention to an animal to disease. The inhibition of the disease includes the introduction of the compositions of the present invention to an animal after the occurrence of the disease, but before the appearance of clinical symptoms. The suppression of the disease includes the introduction of the compositions of the present invention to an animal after the occurrence of clinical symptoms of the disease. It is well known that in medicine is not always clear when in the process of disease must be entered composition of the present invention.

One of the objects of the invention is a pair of primers comprising a sequence derived from:

(a) a first primer which is an oligonucleotide consisting of about 15-30 nucleotides, which contains at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleotide sequence complementary to her; and

(b) a second primer, which represents an oligonucleotide consisting of about 15-30 nucleotides, which does not include the sequence specified in (a), and which is present in the field from Vβ to Jβ gene T-cell receptor Vβ13.1-subgroup of T-cells, and the sequence specified in (a) and (b), are not present on the same chain gene of the T-cell receptor.

Preferably the first primer is an oligonucleotide, consisting of about 15-30 nucleotides, which includes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of 15 to 30 nucleotides, which includes the nucleotide sequence of SEQ ID NO:1 or the sequence complementary to it.

The primers according to the invention create for amplification of a fragment of the gene encoding T-cell receptor from human T-cell subgroups Vβ13.1, where the fragment includes amino acid motif Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO:3). Gene from Vβ13.1-subgroup of T cells, encodes a T-cell receptor, which contains a motif LGRAGLTY was placed in the gene Bank under the registration number AF117132. The sequence of the gene Vβ13.1-subgroup of T cells, which encodes a T-cell receptor, including the motive LGRAGLTY presented in the present description as SEQ ID NO:2. According to the method according to the invention, the fragment length of approximately 400 base pairs of the gene of the T-cell receptor Vβ13.1-subgroup of T-cells amplified with two primers, the first primer is located in the CDR3 region, and the second primer is Fromβ-region. Vβ-Dβ-Jβ-region gene of the T-cell receptor must be between CDR3 andβ-areas inclusive. According to a preferred variant the NTU implementation of the primers are described above pair of primers.

The primers according to the invention also include oligonucleotides derived from the primers (a) - (b). The sequence is considered derived from the primers (a) or (b)if it represents or contains almost the same sequence as the sequence of one of the primers, and retains the ability to polling annealing to approximately the same CDR3 - orβ-areas Vβ-Dβ-Jβ-region gene of the T-cell receptor Vβ13.1-subgroup of T cells, as described above. More specifically, the primer may differ from the primer (a) or (b) the length or type of nucleic acid in one or several positions in the sequence, if it preserves the selectivity with respect to the identified areas Vβ-Dβ-Jβ-region gene of the T-cell receptor Vβ13.1-subgroups of T cells. For example, the primer can be an oligonucleotide comprising at least 15 nucleotides, and these 15 nucleotides identical to the set of 15 consecutive nucleic acids, selected or derived from the sequence of the primers (a)-(b). The primer may be any oligonucleotide consisting of about 30 nucleotides or less, which includes a segment having a sequence selected or derived from any of the primers (a)-(b). The number of nucleotides in the primer due is to be large enough to maintain selectivity and small enough to remain effective, or functional ability in the synthesis using primers in PCR-based. Primer can have variations, including nucleotide deletions, additions or substitutions to the extent that variations of the sequences of the primers (a)-(b) retains the ability to act as primers in the detection process Vβ13.1-LGRAGLTY.

Method detection Vβ13.1-LGRAGLTY according to the invention is based on the use of a pair of the above-described primers in the process of identifying the presence of any motive Vβ13.1-LGRAGLTY in the sample. The sample to be checked in relation to the presence of Vβ13.1-LGRAGLTY, is a nucleic acid, preferably DNA. DNA can be a genomic DNA, cDNA, DNA, pre-amplified by PCR, or any other form DNA. The sample can be allocated, directly or indirectly, from any tissue of the animal or human body, which expresses genes β-chain T-cell receptor. Preferred tissue of the body are mononuclear cells of peripheral blood (RVMS). If the sample is a genomic DNA, it can be selected directly from the tissues of the body. If the sample is a cDNA, it can be selected indirectly via reverse transcription of mRNA directly isolated from the tissues of the body. If the sample is a DNA, pre-amplified by PCR, it is may be selected indirectly by amplification of genomic DNA, cDNA or any other form of DNA.

According to a preferred variant implementation of part of the gene of the T-cell receptor Vβ13.1-subgroup of T cells, i.e. the part containing a sequence that encodes a motive LGRAGLTY, amplified to increase the possibility of detecting the presence of Vβ13.1-LGRAGLTY (5'-CTAGGGCGGGCGGGACTCACCTAC-3' (SEQ ID NO:1). Amplification can be carried out using PCR with the use of any specific PCR methods or equipment, which provides a sensitive, selective and rapid amplification of the sample.

For example, PCR amplification can be carried out according to the process in which to prepare the reaction mixture containing the following ingredients: 5 ál of 10 × PCR buffer II (100 mm Tris-HCl, pH 8.3, 500 mm KCl), 3 ál of 25 mm MgCl2, 1 ál of 10 mm mixture dNTP, of 0.3 µl Taq polymerase (5 units/μl) (type AmpliTaq Gold, the company Perkin Elmer, Norwalk, Connecticut), 30 pmole primer and 30 pmoles primer B. In light of the present description, the person skilled in the art can choose the appropriate primers a and B for PCR amplification of the gene of the T-cell receptor Vβ13.1-subgroups of T cells. The above mixture can be used for amplification, 1 ál of DNA sample. Further, in the context of the present description to be DNA amplification can be designated as "matrix".

After addition of sample DNA to enter the described PCR reaction mixture can be carried out with the following amplification profile: 1 min at 95° C (denaturation); 20 s at 56°C (annealing or denaturate) and 40 s at 72°C (elongation), only using 35 cycles.

When performing PCR matrix can be subjected to thermal denaturation and annealing of two oligonucleotide primers. Oligonucleotides limit the scope of the nucleotide sequence, subject to amplification. In the reaction mixture include heat-resistant DNA polymerase. Polymerase extends the primers, denaturirovannyj to complementary DNA by adding complementary nucleotides. Preferred polymerases have the following characteristics: they must maintain stability at temperatures comprising at least 95°With, be processevent (the number of repeated acts of catalysis) 50-60 and have a speed of elongation greater than 50 nucleotides per minute.

In a normal reaction PCR amplification used about 40 PCR cycles. However, some PCR reactions can be carried out using only 15-20 cycles or when using a large number of cycles, up to 50. Each cycle includes a step of melting, in which the matrix is heated to a temperature above about 95°C.

The temperature was then PCR is reduced, allowing annealing of the primers to the matrix. At this stage of the annealing temperature of the reaction is maintained at the level of about 55° With up to 72°for about 20 C., depending on the specificity of the reaction, this period may be lengthened or shortened.

Then the temperature increase PCR, which allows the maximum elongation of the primers using the polymerase. At the stage of elongating the reaction temperature is maintained at about 70°C to 75°for about 40 C., depending on the specificity of the reaction can be used in more high or low temperature and/or the interval may be lengthened or shortened.

In addition, before starting the first cycle of the reaction mixture may be subjected to an initial denaturation for 5 min to 15 min Similarly, after completion of the final cycle, the reaction mixture may be subjected to a final elongation for about 5 min-10 min

Amplification can be carried out using a two-step PCR. When using this technique, the first reaction of PCR amplification carried out in order to amplify the first area, which includes interest area and longer. Then carry out the second reaction PCR amplification using the first region as the template to amplify interest area. If any of the primers used in the first PCR, can use the SJ in the second PCR, the second PCR is called "semi-closed". If none of the primers used in the first PCR, cannot be used in the second PCR, the second PCR is called "closed".

According to a preferred variant implementation of the method according to the present invention motif Vβ13.1-LGRAGLTY amplified using a two-step PCR. In the first PCR, the sample is amplified using a first primer, which centurybut to Vβ-region gene of the T-cell receptor, and a second primer, which centurybut up Withβ-region gene of the T-cell receptor, using the above reaction mixture and the reaction profile. The first PCR allows to amplify the first area of approximately 600 base pairs and carry out the extension from Vβ through the joint Vβ-Dβ-Jβ toβ. The second PCR is "closed" or "semi-closed"; a portion of the first area amplificates using a pair of primers (a)-(b). The second PCR allows to amplify interest area.

After amplification in the amplification product to detect any DNA encoding the Vβ13.1-LGRAGLTY in the sample. This detection can be carried out using a variety of methods. For example, aliquot amplification product can be made to gel electrophoresis, to which are connected an electric field to separate DNA molecules by size. According to a friend of the method employed in an aliquot of the amplification product to make gel painted SYBR green, ethidium bromide, or other substance which can bind with DNA and emit a signal that can be detected. For example, ethidium bromide binds to DNA and emits visible light when irradiated with ultraviolet light. In another embodiment, the dried gel may contain radio - or chemically-labeled oligonucleotide (hereinafter in the context of the present description may be designated as "oligonucleotide probe"), the complementary parts of the sequence amplified matrix, which upon exposure of the gel film are autoradiogram.

According to the following alternative implementation of the present invention relates to oligonucleotide probe, including

(a) oligonucleotide consisting of about 15-30 nucleotides that includes at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleotide sequence complementary to her; and

(b) particle labeling.

Preferably oligonucleotide "(a)" consists of about 15-30 nucleotides and includes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of about 15-30 nucleotides, which contains the nucleotide sequence of SEQ ID NO:1 or the placenta is the sequence, complementary to it.

Preferably particles for labeling preferably selected from32P or digoxigenin.

Typical radioactivedecay oligonucleotide that can be used to detect the amplification products obtained using primers of the present invention, is obtained from Vβ-Dβ-Jβ-region. If Vβ13.1-LGRA GLTY-region amplificateur using the above-described two-stage "semi-closed" PCR, which apply primer corresponding to a sequence that encodes a motive LGRAGLTY, you can use any oligonucleotide consisting of about 10 or more nucleotides, and preferably from about 18 or more nucleotides complementary part of any chain amplified Vβ13.1-LGRAGLTY - region. More preferably as a probe can be used oligonucleotide 5'-CTAGGGCGGGCGGGACTCACCTAC-3' (SEQ ID NO:1) or complementary him nucleotide sequence.

The present invention also includes a test set, which includes the first primer (a), consisting of about 15-30 nucleotides that includes at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleotide sequence complementary to it.

According to one of the preferred embodiments the test set which also includes a second primer (b), where the second primer is a nucleotide sequence consisting of about 15-30 nucleotides, which does not include the sequence (a) and which is present in the field from Vβ to Jβ gene Vβ13.1 T-cell receptor in T-cells, and the sequence (a) and (b) are not present on the same chain gene of the T-cell receptor.

More preferably, the first primer is an oligonucleotide consisting of about 15-30 nucleotides, which includes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides SEQ ID NO:1 or the sequence complementary to her. Most preferred is an oligonucleotide consisting of about 15-30 nucleotides, which includes the nucleotide sequence of SEQ ID NO:1 or the sequence complementary to it.

In this embodiment, the test set further includes at least one reagent suitable for amplification of DNA motif Vβ13.1-LGRA GLTY using PCR, as described above.

Examples of reagents that can be included in the set are (but not limited to, buffers, dezoksinukleozidtrifosfaty, thermostable DNA polymerase, such as Taq polymerase, DNA Vβ13.1-LGRAGLTY as a positive control and DNA, which is not related to Vβ13:1-LGRAGLTY as negative con is playing. Other reagents which can also be included in the test set, well-known experts in this field.

According to another preferred variant implementation of the test set further includes a particle labeling. Preferably the particle labeling is a32R or digoxigenin.

The present invention also relates to a method of treatment of autoimmune diseases. This disease is one of the diseases in which at least some patients in the Vβ13.1-subgroup of T cells detected including motive LGRAGLTY T-cell receptors. Patients may present other types of T cells and/or Vβ13.1-subgroups of T cells lacking T-cell receptors, including the motive LGRAGLTY.

A method for the treatment of autoimmune diseases includes:

(a) obtaining a clone PSR-99 Vβ13.1-subgroups of T cells from the human body;

(b) identifying the presence of T-cells a nucleic acid that encodes a motive LGRAGLTY, using the above method; and if the nucleic acid is detected

(C) introducing a peptide Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3) person.

Autoimmune disease can represent any autoimmune disease in which T-cell receptor bearing the motif LGRAGLTY found on T-cells under the group Vβ 13.1. Autoimmune diseases falling under the scope of the present invention, include, but are not limited to) rheumatoid arthritis, myasthenia severe paralysis, multiple sclerosis, systemic lupus erythematosus, autoimmune thyroiditis (Hashimoto's thyroiditis), graves ' disease, inflammatory bowel disease, autoimmune uveoretinitis, polymyositis and certain types of diabetes. Preferred autoimmune disease is multiple sclerosis (MS).

If the nucleic acid encoding the motif LGRAGLTY identified above described methods, the autoimmune disease can be treated by introduction of a peptide comprising Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3). The peptide may be introduced individually or in combination with marker peptide T-cell activation. Preferably the peptide is administered in combination with a marker peptide T-cell activation, as described by Zhang, patent application US 60/099102, which is incorporated into this description by reference. Introduction peptide may cause immunogenic reactions in which the patient must antibodies and T-cell receptors that recognize and bind to the motif LGRAGLTY T-cell receptors found on the surface of the Vβ13.1-subgroups of T cells.

Because Vβ13.1-LGRAGLTY may be present as in patients with MS and in healthy people, the e suffering from this disease, it is likely that the peptide Gly Arg Ala Gly Leu Thr Tight (SEQ ID NO: 3) can be entered as suffering from MS, and healthy people.

In an alternative embodiment, if the nucleic acid encoding the motif LGRAGLTY identified by the methods described above may be carried out monitoring of autoimmune disease by quantification of nucleic acids. The greater the amount of nucleic acid present in the sample, such as RVS, the have more Vβ13.1-subgroups of T cells and a higher probability of serious symptoms autoimmune disease. In addition, depending on the time elapsed between the appearance of increased levels of Vβ13.1-subgroups of T cells and symptoms, your doctor may be able to receive a treatment regimen designed to minimize the severity of symptoms and/or treatment of a disease before symptoms appear.

The following examples are included to illustrate preferred embodiments of the invention. Professionals in this field should be obvious that described in examples of the methods are representative of the techniques described with the purpose of realization in practice of the invention and therefore may be considered as part of the preferred ways of its practical implementation. However special the Stam in this area in light of the above descriptions it should be clear in specific embodiments, the implementation can be done is described the many changes that still provide the same or similar results without deviating from the essence and scope of the invention.

Examples

Example 1

The DNA sequence Vβ-Dβ-Jβ-the T-cell receptor and sequence motifs found in MUR-99-specific clones of T cells obtained from various suffering from MS patients

A panel of 20 independent CD4+clones of T cells obtained from 7 suffering from MS patients. All clones of T-cells recognize peptide 83-99 basic protein of myelin (MVR-99) in the context of HLA-DR2, which are identified using transfection DRB1*1501 mouse fibroblasts (L-cells) as antigen presenting cells. Clones of T cells was assessed in relation to rurangirwa V-TCR gene by PCR with reverse transcriptase (RT-PCR), using Vα- Vβ-specific oligonucleotide primers, and then enforcing sequencing in terms of areas Vα-Jα- Vβ-Dβ-Jβ-joints. Area series joints are shown in tables 1 and 2.

Table 1 summarizes the results of the analysis pane of 20 independent MUR-99-specific clones of T cells, characterized from the point of view of the frequency of occurrence of a gene Vβ using PCR with reverse transcriptase is by using a panel of oligonucleotide primers, specific collection Vαgenes (unique sequence of the applied primers is underlined in the sequence of DNA corresponding to each clone). Amino acid sequence "Vα"-, "n", "Jα"and "α"-areas of each clone identified in table 1 as follows: n-region nepodarkom, "Vα" - "Jα"sequences in bold on their respective sides ″n"sequences and "Withα"-sequence is shown in normal font without the underscore. Amplificatoare PCR products hybridized with labeled using digoxigenin Withα-region cDNA probes and then analyzed in relation to the DNA sequence.

Table 2 summarizes the results of the analysis pane of 20 independent MUR-99-specific clones of T cells. The clones were characterized from the point of view of the frequency of occurrence of a gene vβ using PCR with reverse transcriptases using a set of oligonucleotide primers specific for 26 representatives of the family Vβgenes (sequence-specific primers for each clone highlighted in the corresponding DNA sequence). "Vβ"-, "D", "Jβ"and "β"-region of each clone are shown in table 2 as follows: "In"-the region emphasized, "Vβ" - "Jβ"sequences in bold on testwuide the parties "n"sequence, and the rest of the sequence, i.e. Withβ"listed in normal font (not underlined and not in bold). Amplificatoare PCR products hybridized with labeled using digoxigenin Withβ cDNA probes, and then analyzed in relation to the DNA sequence.

Although rearrangement Vα and Vβ vary between individual T-cell clones MUR-99, many of these independent T-cell clones obtained from a specific individual, are identical to Vα- Vβ-chains with the same sequence regions Vα-J-α and Vβ-Dβ-Jβ-joints. These results are consistent with earlier data clonal expansion in vivo MVR-99-specific T cells in individual patients suffering from MS (Vandevyver 1995, Wucherpfenning 1994).

It is important to note that as can be seen from tables 1 and 2, independent T-cell clone (clone E obtained from a patient (MS-1), have the same Vβ13.1 and Vα17 with three of the four T-cell clones (clones C, D7.16 and F3.4)received the C of another patient (MS-2). Vβ13.1 these T-cell clones had identical DNA sequence with a sequence region Vβ-Dβ-Jβ-interface.

Example 2

Vβ-Dβ-Jβ-specific oligonucleotide primers with high specificity and sensitivity in the detection of the corresponding DNA sequences present in the original T-cell clones MUR-99, and RVMS containing the original T-cell clones MUR-99

Synthesized a set of 14 oligonucleotide primers that corresponded to the DNA sequences in regions Vβ-Dβ-Jβ-joints independent of T-cell clones MUR-99, and then evaluated for their specificity using RT-PCR. The DNA sequence of these oligonucleotide primers are shown in table 3.

Table 3
The DNA sequence of a Vβ-Dβ-Jβ-specific oligonucleotide primers
T-cell cloneThe DNA sequenceSEQ ID NO
MS1-E3.1AGCAGCCAAGATCGTTTTTGGSEQ ID NO:68
MS1-E2.6CTAGGGCGGGCGGGACTCACCTACSEQ ID NO:69
MS2-C3.1CTAGGGCGGGCGGGACTCACCTACSEQ ID NO:70
MS2-D4.4   
MS3-F5.12TACTCGATTAGGGGACAGGGTAACSEQ ID NO:71
MS3-B9.8  
MS4-D9.3CAAGATCGGGTTGCGCCASEQ ID NO:72
MS4-B9.1ACCCGGCAAGGACCTCAAGAGACCSEQ ID NO:73
MS5-D2.7AGCTTAGGACAGGGGGCTSEQ ID NO:74
MS5-D1.3  
MS6-D8.1GCCAGCCGGGACAGGTCCSEQ ID NO:75
MS6-DL2GAGTAGATTGGTACGGGASEQ ID NO:76
MS7-C.26  
Branch adapter ms8-C7.2TACATCTGAAGTGCTATAGACSEQ ID NO: 77

These Vβ-Dβ-Jβ-specific primers exclusively related to DNA sequences present in the original T-cell clones MUR-99 and are not associated with sequences obtained from unrelated MUR-99 T-cell clones (figure 2), suggesting their specificity in relation to the original DNA sequence Vβ-Dβ-Jβ. The only exception that was discovered is a clone MS1-E2.6 and clone MS2-C3.1, in which the same primer associated with the DNA sequence Vβ-Dβ-Jβ-joint found in both T-cell clones.

With regard to specific is t Vβ -Dβ-Jβ-oligonucleotide primers and the high sensitivity of PCR-based detection systems, the question was raised about the possibility of using this based on the two-stage PCR system using 5'-Vβ-primers and Vβ-Dβ-Jβ-the specific oligonucleotide primers for detection of corresponding Vβ-Dβ-Jβsequences of DNA present in samples of peripheral mononuclear cells blood (RVMS), which received the original T-cell clones MUR-99. The results of two independent experiments gave positive results from the point of view of detection Vβ-Dβ-Jβsequences in the initial samples RVMS. Thus, these data suggest that PCR-based detection system, in which Vβ-Dβ-Jβsequence serves as a fingerprint, is a specific and sensitive detection of T-cell clones MUR-99 present in mononuclear cells of peripheral blood, by sounding identical DNA sequences.

Example 3

Detection of total Vβ-Dβ-JβDNA sequences in samples RVS obtained from various suffering from MS patients and healthy people

It was further analyzed whether the DNA sequence corresponding to the regions Vβ-Dβ-Jβ -joint T-cell clones MUR-99, to be found in RVS, randomly drawn from the group suffering from MS patients and healthy people. Used the same system PCR amplification using primers specific for the respective Vβ-families (first PCR)and primers specific to Vβ-Dβ-Jβsequences (in the second "half-closed" PCR). This system was combined with analysis by the method of southern blotting using hybridization appropriate Vβ-Dβ-Jβ-probes. Taking into account the specific requirements of the detection system based on two-stage PCR, and Vβ-Dβ-Jβ-specificity of primers and probes to identify DNA sequences must originate from a specific Vβ-chains of the TCR and to represent a sequence or identical or similar interest Vβ-Dβ-Jβsequences.

The results indicate that only one Vp-Dp-Jp-oligonucleotide primer (MS1-E2.6, Vβ13.1-LGRAGLTY) allows to detect complementary DNA sequence Vβ13.1 TCR 15 of 48 (31%) of the samples RVS obtained from various suffering from MS patients. Thus, these results indicate the presence of those suffering from MS patients T-cell clone PSR-99, which expresses the motif Vβ13.1-LGRAGLTY. In a similar experiment is analnyj conditions the same primer was allowed to reveal the corresponding DNA sequence in 5 of 20 (25%) of the samples RVMS, obtained from healthy people. Thirteen other Vβ-Dβ-Jβ-primers did not allow to identify any signal sequence using the same panel of samples RVMS. The results were reproduced in three independent experiments. Identical DNA products, amplificatoare using primer I come from T cells expressing Vβ13.1, because for amplification in the first PCR was used Vβ13.1-specific primer.

In addition, the detected sequence Vβ13.1-LGRAGLTY also amplified in 13 of 24 (54%) Korotkova T-cell lines MBP8S-99 obtained from 5 suffering from MS patients (MS-35, MS36 and MS39), for samples RUMS which established the presence of a Vβ13.1-LGRAGLTY sequences. These results confirmed that the DNA sequence Vβ13.1-LGRAGLTY detected in samples RVS obtained from discriminating MBP8S-99 T cells. This result also suggests that T-cell clones MBP8S-99, expressing the sequence Vβ13.1-LGRAGLTY include the entire or almost the totality MBP83-99 T-cell lines, detected some suffering from MS patients.

The joint detection system based on PCR and DNA hybridization, in which Vβ-Dβ-Jβsequences were used as fingerprint, is effective in the instrument for the detection of antigen specific T cells by detecting identical sequences Vβ -Dβ-Jβ-joint. High specificity and sensitivity of the detection system allows the identification of specific Vβ-Dβ-Jβsequences of T-cells in peripheral blood. In the present study first demonstrated that the overall Vβ13.1-subgroup of T cells that recognize immunodominant peptide MBP 83-99 and equally Express identical to Vβ-Dβ-Jβ-the sequence is present in approximately 30% of people with MS patients. This conclusion is made on the basis described in the present description serial experiments. First among independent of T-cell clones MUR-99 obtained from various suffering from MS patients were found to have identical DNA sequence (Vβ13.1-LGRAGLTY). In the second stage, the sequence was identified in the cDNA products amplified from Vβ13.1 TCR samples RVS obtained from various suffering from MS patients. In the third phase, the DNA sequence was detected in Korotkova independent T-cell lines MUR-99 obtained from samples RUMS for which it is proved that they contain a sequence Vβ13.1-LGRAGLTY. T-cell lines MUR-99, expressing the sequence Vβ13.1-LGRAGLTY probably include the entire or almost the totality of T-cell lines MUR-99 was detected in some M suffering from patients. Finally, the presence of the sequence Vβ13.1-LGRAGLTY in samples RWMS was confirmed by cloning of recombinant DNA and direct DNA sequencing.

In addition, it is not surprising that T-cell lines MUR-99, expressing the General sequence Vβ13.1-LGRAGLTY, are also present in some healthy people. The following studies found that MBP-reactive T cells, including T cells that recognize immunodominant peptide 83-99, also found in some healthy individuals (Zhang 1994, Ota 1990). However, there is a functional difference consists in the fact that these T cells are activated in vivo and clonal expansion in patients MS patients in contrast to healthy individuals (Zhang 1994).

These MUR-99-clones Vβ13.1-subgroups of T cells bearing the General sequence Vβ-Dβ-Jβmay constitute a major fraction of T-cell clones MUR-99 was detected in some sufferers of MS patients. The probability of this is confirmed by the fact that the sequence Vβ13.1-LGRAGLTY was present in 40% Korotkova T-cell lines MUR-99 obtained from suffering from MS patients after two cycles of stimulation.

Identified a common sequence Vβ-Dβ-Jβ can be used as a specific marker to quantify the system PCR-based detection to identify MUR-99-subgroups of T cells in the blood and cerebral spinal fluid in a large group suffering from MS patients to monitor clonal expansion in vivo and activity in vivo, potentially associated with disease. This method may have an advantage compared with conventional assays using cell cultures as in vitro selection and expansion MBR-reactive T cells is often hampered by various inhibiting factors inherent in cell culture. This is consistent with current estimates that the frequency of MBP-reactive T cells were unexpectedly high in patients MS patients when applied directly to the analysis of ex vivo for the quantitative determination of MBP-reactive T cells (Hafler is the last author in JEM 1997).

In addition, it was found that synthetic peptides corresponding to the TCR, induce anti-idiotypical T-cell responses to MBP-reactive T cells in patients MS patients (Chou and others, J.I.). Thus TCR-peptide containing the common CDR3 sequence, can have a big opportunity in relation to the manifestation of the ability antiidiotypic T cells to suppress specific subgroup of MBP-reactive T cells in the group of patients whose T-cell clones MUR-99 are the General sequence of CDR3 motif. Immunization with common CDR3 peptide may have an advantage compared to the CDR2-peptides or dependent of the individual CDR3-peptide as a potential treatment for sufferers of MS patients (Vandenbark 1996).

Swete of the present description, all described and claimed in the claims compositions and methods can be carried out without complicated experiments. Although the compositions and methods according to the invention is described in preferred embodiments, the experts in this field should be obvious that various changes may be made in the compositions and/or methods and in the steps or in the sequence of stages described above without deviating from the concept, nature and scope of the invention. In particular, it should be apparent that certain agents that are similar both chemically and physiologically, can be used for replacement are presented in the description of the agents, if it achieves the same or similar results. All such substitutions and modifications are clearly experts in this field and are subject to the nature, scope and concept of the invention, which are presented below in the claims.

1. Oligonucleotide for detecting auto monogo disease, consisting of about 15-30 nucleotides, including at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid fully complementary to it.

2. The oligonucleotide according to claim 1, comprising at least 15 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid fully complementary to it.

3. The oligonucleotide according to claim 1, comprising the sequence of SEQ ID NO:1 or a nucleic acid fully complementary to it.

4. A pair of primers, including

(a) a first primer consisting of about 15-30 nucleotides, which contains at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid complementary to it; and

(b) a second primer consisting of about 15-30 nucleotides, which does not include the sequence specified in (a), and is located in the region from Vβ to Jβ gene Vβ13.1 in T-cell receptor T-cells, and the sequence of the first and second primers are not present on the same chain gene of the T-cell receptor.

5. Primer according to claim 4, where the sequence of the gene Vβ13.1 represents SEQ ID NO:2.

6. Oligonucleotide probe, including

(a) oligonucleotide consisting of about 10-30 nucleotides that includes at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid fully complementary to her; and

(b) a particle labeling.

7. Oligonucleotide probe according to claim 6, where the particle for tagging choose from32R or digoxigenin.

8. The method for detecting clone PSR-99 Vβ13.1 T-cells expressing the motive LGRAGLTY T-cell receptor, which provides

(a) obtaining a sample of nucleic acid from the clone PSR-99 Vβ13.1 T cells;

(b) bringing into contact of the sample nucleic acid with a pair of primers that are selected or derived from (I) a first oligonucleotide comprising from about 15 to 30 nucleotides that has at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid complementary to it, and (II) a second oligonucleotide consisting of about 15-30 nucleotides, which does not include the sequence of the first oligonucleotide is present in the field from Vβ to Jβ gene Vβ13.1 T-cell receptor T-cells, moreover, the sequence of the first and second oligonucleotides are present on the same chain gene of the T-cell receptor; and

(C) identifying the presence of a nucleic acid that encodes a motive LGRAGLTY.

9. The method of claim 8, where the sequence of the gene Vβ13.1 represents SEQ ID NO:2.

10. The method of claim 8, where the fragment of the sample nucleic acid is amplified using polymerase chain reaction (PCR).

11. The method according to claim 10, where the stud is I detecting includes sensing using oligonucleotide probe, which includes

(a) an oligonucleotide probe comprising the sequence of SEQ ID NO:1 or a nucleic acid complementary to it; and

(b) a particle labeling.

12. The method according to claim 10, where the phase detection includes the autoradiograph.

13. The test set designed to detect Vβ13.1 T cells, which includes a first oligonucleotide consisting of about 15-30 nucleotides, where the first oligonucleotide comprises at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid fully complementary to it.

14. The test set according to item 13, further comprising a second oligonucleotide consisting of about 15 and 30 nucleotides, which does not include the sequence of the first oligonucleotide and which is present in the field from Vβ to Jβ gene Vβ13.1 T-cell receptor T-cells, and the sequence of the first and second oligonucleotides are present on the same chain gene of the T-cell receptor.

15. The test set at 14, where the sequence of the gene Vβ13.1 represents SEQ ID NO:2.

16. The test set according to item 13, further comprising a particle labeling, where the particle for tagging choose from32P or digoxigenin.

17. A method of treating an autoimmune disease in humans, which detected the presence of CL is on MUR-99 Vβ 13.1 T cells, including the DNA sequence SEQ ID NO:1, and this method includes the introduction of the human peptide Leu Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO:3).

18. The method according to 17, where the sequence of the gene Vβ13.1 represents SEQ ID NO:2.

19. The method according to 17, where the stage of introducing further includes the introduction of a marker peptide T-cell activation.

20. Method of monitoring an autoimmune disease involving

(A) isolation of clone PSR-99 Vβ13.1 T-cells from the human body;

(B) detecting the presence of nucleic acid encoding the motif LGRAGLTY, by (I) selection of the sample of nucleic acid from the clone PSR-99 Vβ13.1 T cells; (II) bringing the sample nucleic acid into contact with a pair of primers that are selected or derived from (a) a first oligonucleotide comprising from about 15 to 30 nucleotides that has at least 10 consecutive nucleotides of SEQ ID NO:1 or a nucleic acid complementary to it; and (b) a second oligonucleotide consisting of about 15-30 nucleotides, which does not includes the sequence of the first oligonucleotide is present in the field from Vβ to Jβ gene Vβ13.1 T-cell receptor T-cells, and the sequence of the first and second oligonucleotides are present on the same chain gene of the T-cell receptor; and

(C) detecting the presence of nucleic acids, the coding motif LGRAGLTY; and, if the nucleic acid is detected, then

(C) quantification of nucleic acids.

21. The method according to claim 20, where the sequence of the gene Vβ13.1 represents SEQ ID NO:2.



 

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2 cl, 3 dwg, 4 ex

FIELD: biotechnology, genetic engineering, pharmaceutical industry.

SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.

EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.

2 cl, 3 dwg, 4 ex

FIELD: biotechnology, genetic engineering, pharmaceutical industry.

SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.

EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.

2 cl, 3 dwg, 4 ex

FIELD: biotechnology, in particular prephenate dehydrotase-chorismatmutase and DNA fragment encoding the same.

SUBSTANCE: prephenate dehydrotase-chorismatmutase is isolated from Methylophilus methylotropus and may contain replacements, deletions, inserts, or incorporations of one or more amino acids. Said enzyme plays an important role in L-phenylalanine biosynthesis. Method of present invention makes it possible to improve L-phenylalanine production due to increased activity of enzymes involving in L-phenylalanine biosynthesis pathway.

EFFECT: improved L-phenylalanine production.

2 cl, 2 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: raw soft roe should be reduced, treated at certain temperature with 0.2%-acetic acid solution, dehydrated with alcohol, dried to obtain granules, granules should be extracted with sulfuric acid solution at three stages, proteins should be precipitated out of extract by adding triple volume of ethanol, residue should be dissolved, solution should be treated with barium hydroxide up to pH being 7.0-7.5 followed by addition of ammonia up to pH being 10.5-11.5 to separate the residue due to decanting and centrifuging. Solution obtained after decanting and centrifugate should be treated with kieselguhr at heating up to 80 C and filtered upon a Nutch filter, one should apply filtrate through cationite in "H+"-form, eluate should be concentrated due to vacuum evaporation or by applying baromembranous technique, then one should precipitate the product out of eluate with triple volume of alcohol due to a 5-fold reprecipitation along with centrifuging and drying the residue to remove residual moisture and dry ready-to-use product at 55-65 C for 24 h. The present innovation enables to increase the degree of purification of protamine sulfate due to purifying against accompanying foreign proteins.

EFFECT: increased anti-heparin activity.

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