Method of obtaining system of directed delivery of protein molecules (oncolytic proteins) into tumour cells based on activated lymphocytes
SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.
EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.
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The invention relates to the field of medicine and biotechnology.
Modern methods of cancer therapy based on the application of an integrated approach using a combination of funds focused on certain features of the biological properties of tumor cells. With the deepening of knowledge about tumor cells and determine the most universal mechanisms that distinguish cancer cells from normal, it becomes possible to develop more effective, specific and safe for the body biological therapy. In contrast to the lack of specific existing chemotherapeutic agents, biological therapies are designed to use natural activity and mechanisms to identify and kill cancer cells.
Currently more and more attention of researchers is attracted by the prospects of using approaches to therapy of malignant tumors, based on the use of biomolecules - proteins with selective inhibitory or even toxic effects against tumor cells, while not exerting similar effects on normal cells. Such approaches include the use of specially designed antibodies against ofwholesale epitopes that inhibit tumor growth or by blocking the re�of atarov, necessary for active proliferation of the tumor, either by direct cytotoxic effect of the toxin is covalently linked to the antibody. Another group of protein molecules, with no less potential use in therapy, oncolytic are protein molecules which when injected into the tumor cell, initiating a cascade of Pro-apoptotic signals, dependent on pathological signaling pathways characteristic of transformed cells, but not normal.
The main advantage of protein molecules compared to the traditionally used cytotoxic chemical compounds is that the mechanism of their action is aimed at fundamental features of cancer cells, which provides exceptionally high selectivity of their action, in combination with low toxicity for the body and no side effects. Due to this, a strong cumulative effect of several drugs on the tumor can be achieved without complications, usually observed in cancer therapy.
In recent years more and more works describe the inactivation of tumor suppressor p53 as one of the main causes of cancer. As you know, p53 is a key component responsible for launching of protective signaling pathways in response to DNA damage in mammalian cells. �adaplene damage in the genetic apparatus of the cell leads to activation of p53 protein, resulting in a stopping of the cell cycle and DNA replication, with strong damage occurs programmed cell death or apoptosis. Thus the mechanism that can prevent and remove potentially oncogenic cells, and mutation or loss of p53 gene leads to the accumulation of a multitude of DNA damage, cell degeneration and, eventually, to cancer. About 50% of all malignant tumors detected mutation namely p53 gene. Therefore, of great scientific and applied importance are studies devoted to the search for alternative, p53-independent pathways of apoptosis. Recently it was found that such a mechanism has apoptosis induced by Apoptin.
Apoptin (product VP3 gene of the genome of the virus of infectious anemia CSD) - Proline-rich protein with a molecular mass of 13.6 kDa, consisting of 121 amino acid residues.
Greater interest is the ability apoptin to selectively induce apoptosis of transformed cells of birds and mammals, while not affecting normal, untransformed cells. Based on the obtained data, we can speak about 70 different tumor lines that are sensitive to apoptin. Apoptin region has nuclear localization, which is located in the C-terminal region of the protein between 80-121 amino acid residues, and the area of nuclear�exports, located in the Ν-terminal region, between 33 and 46 amino acid residues. Many studies have shown that nuclear localization apoptin in tumor cells is required for induction of apoptosis, whereas in normal cells the protein is localized mainly in the cytoplasm, where it is unstable and rapidly degraded.
As already mentioned, VP3 can cause the death of cancer cells while not affecting normal. Such selectivity may explain the Nucleo-cytoplasmic transport. Experimental data showed that nuclear export apoptin in normal cells is identified by a unique signal sequence (nuclear export sequence) interacts with exportin CRM1, exercising, thus, the permanent withdrawal of protein from the nucleus. In cancer cells the reverse transfer does not occur, due to phosphorylation 108 threonine (Thr 108) in the nuclear export sequence, which blocks the interaction with CRM1. Thus, feofilivka Thr-108, as it seems, may be one of the key events required for the accumulation apoptin in the nucleus and apoptin-induced apoptosis. Further, more detailed study of this phenomenon showed that the phosphorylation apoptin is not a prerequisite of its tumor-specific on�of aplane in the kernel. Thus, replacement 107 and 108 of threonine for alanine residues, and further validation of the subcellular localization of the mutant apoptin in MCF-7 cells and normal cells did not reveal any changes. This protein still accumulated in the nuclei of tumor cells and subsequently lead to apoptosis. This indicates the existence of other mechanisms of accumulation apoptin in the nuclei of tumor cells.
Medical application of oncolytic proteins, including apoptin, while complicated by the low efficiency of the used methods of delivering biological agents in the areas of the tumor. Another challenge is to deliver the protein directly to tumor cells, because unlike small chemical molecules, proteins lack the ability to penetrate through the plasma membrane. A significant limitation is the immunogenicity of proteins and the rapid formation of antibodies, which, over time, can completely neutralize the activity of oncolytic protein. In order oncolytic proteins become effective anticancer therapy, we need to find ways to effectively address these problems: to determine modifications of protein structure, necessary for its smooth passage through the plasma membrane and to develop a fundamentally new means of targeted delivery of such therapeutic use�quarter of agents to tumor cells. These delivery methods have, on the one hand, to protect the protein from the action of antibodies, and to bring the release of the active protein to the location of tumor cells.
Currently there are attempts to adapt apoptin as the most promising oncolytic protein for clinical use.
Having a high selectivity against tumor cells, apoptin has good prospects for application in cancer therapy. To ensure delivery apoptin in tumor cells, you must either enter into these cells a genetic construct expressing apoptin, or to modify the structure apoptin, giving it the ability to penetrate into tumor cells through the plasma membrane. The first method of delivery used in cancer therapy using oncolytic viruses, while the second method of delivery allows you to use apoptin a variety of ways: traditionally, introducing synthetic recombinant apoptin, and using biological means of delivery.
Medical application prospects apoptin and oncolytic proteins, in General, depend upon the development of the means of delivery of recombinant proteins in the tumor. Injecting the synthesized protein is not n�will create a local high concentration in the tumor and bears the risk of immunization the patient to input a squirrel or development of allergic reactions. To administer an injection directly into the tumor is not always possible, in addition, this injection may cause a number of complications. The best option the use of oncolytic proteins would be the presence in the tumor cells producing these proteins. In this case, the impact would be long, and the concentration of the drug is maximal in the region of the tumor. To achieve this purpose, commonly used recombinant viral vectors are collected on the basis of lentivirus or adenovirus.
Vectors based lentivirus described, for example, Ye Feng, etc., Construction of three recombinant lentiviruses containing apoptin driven by survivin promoter of different lengths and comparison of their tumor suppressing efficency in vitro, Journal of Third Military Medical University, 2012-01. Lentiviral vectors will allow you to write and to Express the constitutive recombinant apoptin in tumor cells and surrounding tissues. This expression will be longer, hazards of immunization against the virus almost never occurs because he is not able to replicate. However, the nature of integration of viral DNA into the genome carries the risk of damage and, as a consequence, additional malignancy of the tumor.
Known patent RU 2252255 describing recombinant adenoviral vector containing apoptin for the treatment of cancer, which are obtained by cotransfection in a helper cell line 911 adaptor plasmids�p ID.AMb-VP3 (in the case of expression of protein VP3) or plasmids pMAb-VP2 (in the case of VP2 protein expression and plasmid DNA JM17. Plasmids pAMb-VP3 gene are apoptin in orientation 5'-3', the expression of which is under the control of adenovirus late chief promoter. Plasmid DNA JM17 contains the complete DNA of adenovirus with the exception of region E1 and E3. Plasmids pMAb-VP2 gene are apoptin, with 2 point mutations within the coding region.
Vector delivery of genes capable of inducing apoptosis in a cell comprising the DNA fragment encoding the protein having adapteroptions activity, and apoptosis is induced in cancer cells and to a lesser extent or not at all in normal untransformed diploid/non-cancerous cells, wherein the gene delivery vector is independent of the infective vector and represents the adenovirus.
A significant disadvantage of this method is gradually emerging immunization of patient-specific adenoviral strain.
Known patent RU 2492238 C1, which relates to recombinant plasmid DNA pGEM-Puro-DS-Apo, carrying the synthetic gene apoptin size 363 BP with a specific nucleotide sequence, flanked by fragments of the genome of vaccinia virus has a size 6298 D., a molecular weight of 4.19 MDA, and recombinant strains of vaccinia virus producing apoptin anemia virus of chickens. Using this method will be n�to blogatize also gradually emerging immunization of the patient.
Described drawbacks necessitate the development of a new, non-viral, method of delivery and expression of recombinant proteins in tumors
Known patent US 5656465 concerning ways to deliver genes in vivo. Describes the use of non-cumulative viral vectors having a low replicative efficiency for inserting a gene into a cell such as a lymphocyte or a tumor cell is a privileged system for transforming such cells for use in somatic cell therapy or gene therapy. Genes are inserted into the target cell, in particular in the target cell, representing the lymphocytes infiltrating the tumor tissue. It is noted that the interest are genes encoding TNF, TGF-α, TGF-β, hemoglobin, interleukin-1 and the like, as well as biologically active mutiny of these proteins. Not mentioned the possibility of targeted delivery apoptin.
The object of the present invention is to provide a new means of targeted delivery apoptin to tumor cells, which allows, on the one hand, to protect the protein from the action of antibodies, and to bring the release of the active protein to the location of tumor cells.
Technical result: increased ability apoptin to penetrate into tumor cells and provide oncolytic effect in respect of� all of the tumor cells. This therapy can be used in combination with other anticancer drugs that will reduce the duration of treatment and reduce side effects.
The problem is solved by a method for producing the targeted delivery system apoptin in tumor tissue. Method harakteryzuyetsya that modify apoptin by fusion of secretory signal lactotransferrin and transductional signal (ST-CTP-apoptin), get a lentiviral construct expressing ST-CTP-apoptin specified construct is used to produce recombinant lentiviral particles defective in replication and load-bearing modified apoptin, which are then injected into autotransplantation lymphocytes infiltrating in the tumor tissue.
As a means of delivery and the producer of oncolytic protein proposed autotransplantation lymphocytes infiltrating in the tumor tissue (TIL, tumor infiltrating lymphocytes). Lymphocytes can be obtained during surgical removal of the tumor or in the process of obtaining the biopsy. Can also be used isolated from peripheral blood lymphocytes, activated by cultivation in the presence of interleukin-2. Such cultivation enhances the tropism of lymphocytes to the tumor, and, after entering back into the bloodstream, a significant portion of lymphocytes localized in tumor tissue.<> The main difference of the present invention is the use onkologicheskih lymphocytes as a carrier and the producer apoptin - is new and not described in the literature. This approach will allow to combine oncolytic effect apoptin with the antitumor effect of cell and immunotherapy of malignant tumors. Unlike the known solutions in that it describes the constructs reintegriruet into the genome of the vector, while the stated lentiviral construct is integrated.
To solve the problem were developed signal peptide ensuring secretion of the expressed oncolytic protein from cells and subsequent penetration into the surrounding cells by protein domain that provides penetration through the plasma membrane. In the invention used property of cytotoxic lymphocytes to infiltrate and accumulate in the tumor. Expression of secreted oncolytic lymphocyte protein would release active therapeutic molecules directly at the location of the cancer cells and reduce the probability of neutralization of the active protein antibodies.
The invention is demonstrated below by examples.
Example 1. Determination of the optimal variant of recombinant apoptin have�its maximum oncolytic activity
In cDNA apoptin PCR integrated domain cytoplasmic transduction (P) and secretory signal, and the resulting sequence is cloned in the lentiviral vector. Created a series of constructs containing different variants of the P-domain (option 4, a fragment of the TAT protein of HIV (PTD domain) and 3 synthetic peptide), and variants of the ST-peptide (5 options, 3 of which are synthetic, derived on the basis of the predictions of the hmm-algorithm for determining the effectiveness transducing peptide, and 2 options are taken from natural secreted proteins, cystatin C and lactotransferrin). Series obtained constructs introduced into cells-producers, and one of them made the selection of the most effective option. This is filtered by the environment in which producers were cultivated, processed cell cancer cell lines SAOS2 and RKO, and comparative evaluation of cytotoxicity of each variant protein. This approach allows us to determine the functional activity of the protein in conditions similar to real. As control structures, as well as visual evaluation of the efficiency of secretion and capture of the modified protein, the resulting set of strains, secreting fluorescent protein, fused with a test PAGE - and ST-domains.
Has created a series of genetically engineered structures, designed to Express�Yu different options oncolytic protein apoptin and red fluorescent protein tagRFP. Were created constructs expressing wild apoptin (wt-apoptin), apoptin, merged with secretion signals (ST-apoptin), obtained from proteins lactoferrin, cystatin C, as well as three synthetic ST-signals obtained on the basis of the predictions of the hmm-algorithm for determining the efficiency of the secretory peptide. Also the resulting construct expressing apoptin, merged with a strong cytoplasmic transduction is one option, which is a fragment of TAT protein of HIV (PTD domain), and three synthetic peptide. In addition to constructs expressing apoptin fused separately with signals secretion and cytoplasmic transduction (5 and 4 items, respectively), were obtained expressone vectors containing all versions apoptin, and merged with secretory and transductional signal at the same time.
All apoptin-expressing constructs were introduced by the method of lentiviral transfection in culture tumor cell lines SAOS2 and RKO, and comparative evaluation of cytotoxicity of each variant protein. To do this, the obtained recombinant lentiviruses, after concentration with polyethylene glycol was determined the titer (by RT-PCR in real time), and then, after infection of cell cultures with an equal number of viral particles was determined, in which case the cytotoxicity Mac�Kalina. In fact, various options apoptin showed a cytotoxicity of less than "wild" apoptin (which was expected), but some options, including apoptin fused with the secretory signal lactotransferrin and transductional signal PTD showed an almost unchanged level of cytotoxicity.
In parallel with these experiments, cell lines SAOS2 and RKO were transfected by tagRFP-containing structures. After the selection and sorting, in which 90-95% of the cells in the culture became tagRFP-expressing cells were mixed in a ratio of 1/1 with nitrostilbene SAOS2 and RKO, respectively. After co-culturing for 24 hours, comparative fluorescence of two populations of co-cultured cells was changed on the cytometer. On the basis of the gain characteristic tagRFP fluorescence in nitrostilbene the clack, conclusions were drawn about the effectiveness of a couple-secretory and transducing peptides. The experiments showed that the greatest efficiency of the tested variants has a couple of secretory signal lactotransferrin and transductores PTD signal.
The sequence apoptin with secretory and transductive peptides SEQ ID NO 1 shown in Fig. 1. Example expressing constructs containing apoptin with a signal peptide, �of redstapler in Fig. 2.
Thus, it was determined the most effective structure through which onkologicheskie proteins can be introduced into tumor cells - ST-CTP-apoptin. The expression of ST-CTP-apoptin in a small subset of tumor cells, or cells of the tissue adjacent to it, leading to long-term presence of high concentrations apoptin in the intercellular space of the tumor.
Example 2. The cultivation and activation of lymphocytes in model animals, lines Nude thymus of mice
The homozygous Nude thymus of mice was inoculated with the tumor, in parallel, in heterozygous animals of the same line from peripheral blood were isolated mononuclear cell fraction. Lymphocytes were transducible the lentiviral expression tagRFP, and subjected to cultivation with different activating conditions (phytohemagglutinin, IL-2, IL-4, GM-CSF, lectins). After the operating time of a sufficient number of activated lymphocytes, they were introduced to Nude thymus mice with grafted tumors. Then, the removed tumor was analyzed on a flow cytometer, for the purpose of determining the amount of the contained fluorescent lymphocytes.
Example 3. A comparative study of the antitumor efficacy
Lentiviral construct expressing ST-CTP-apoptin, was introduced in lymphocytes in parallel with the control lentiviral construct. TC�tki were subjected to identical conditions of cultivation and inoculated into animals with tumors. Antitumor efficacy of ST-CTP-apoptin assessed in vivo by measurement of the dynamics of tumor development in mice that received apoptin-expressing and control lymphocytes expressing fluorescent protein. Introduction lymphocytes expressing apoptin leads to significantly higher efficiency of this method against a panel of human tumor cells, cultured on Nude thymus mice.
A method of obtaining a system of targeted delivery apoptin in tumor tissue, comprising a lentiviral construct expressing a modified apoptin, characterized in that the modified apoptin is apoptin fused with the secretory signal lactotransferrin and transductional signal (ST-CTP-apoptin) and having the sequence SEQ ID NO. 1 shown in Fig. 1, get a lentiviral construct expressing ST-CTP-apoptin with the arrangement of the elements shown on Fig. 2, followed by production of recombinant lentiviral particles defective in replication and load-bearing modified apoptin, which are then injected into T-lymphocytes (TILs) obtained during surgical removal of the tumor or in the process of obtaining a biopsy, which have the ability to penetrate into tumor tissue, with subsequent autotransplantation of these lymphocytes to ocosandomeprazole.
SUBSTANCE: method comprises amplification of fragments of genes EGFR, KRAS and BRAF using LNA-blocking multiplex "nested" PCR in which the template for amplification is used as DNA tumour sample. The single-stranded fluorescently labelled PCR product is obtained. The biochip for identification of somatic mutations in the genes EGFR, KRAS and BRAF is obtained. Hybridisation of fluorescently labelled PCR-product in the biochip is carried out. The results of hybridisation on the biochip are recorded and interpreted.
EFFECT: invention enables to carry out analysis in a short period of time and with low material costs.
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SUBSTANCE: invention refers to medicine and aims at diagnosing vaginitis in females of a reproductive age. Expression levels of mRNA of TNF, IL18, GATA3 and TLR4 genes are measured in vaginal smears. Expression levels of mRNA of TLR4/GATA3 and TNF/IL18 genes are related, and the derived threshold cycles are used to predict a probability of vaginitis by formula. If P≤57% for nonpregnant females or P≤68.5% for pregnant females, the absence of vaginitis is stated. If P≥57% for nonpregnant females or P≥68.5% for pregnant females, vaginitis is diagnosed.
EFFECT: invention provides the effective diagnosis of vaginitis in the females of a reproductive age.
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SUBSTANCE: group of inventions relates to the field of biotechnology, in particular to micro-array based genomic selection (MGS). A device contains: (a) at least one reaction zone, containing a micro-array, one or more units of temperature control and regulation for the control and regulation of the temperature inside the reaction zone; (b) at least one non-reaction zone, containing one or more units of temperature control and regulation for the control and regulation of the temperature inside the non-reaction zone, which is connected with the reaction zone with the possibility of liquid transfer; and (c) at least one means for transfer, capable of generating and/or regulating a liquid flow between the said reaction zone (a) and the said non-reaction zone (b).
EFFECT: device can be used for the specific selection of nucleic acids, with an increase of binding and fluorescence intensity.
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SUBSTANCE: invention refers to a method of detection of Mycobacterium tuberculosis isolates resistant to pyrazinamide by detection of mutations in pncA gene, associated with evolving resistance to pyrazinamide, by PCR in real time mode using HRM analysis. It involves amplification in a mixture of equal amounts of tested DNA and wild type DNA using primers: Pnc18U: 5'-TACGCTCCGGTGTAGGCAC-3' and Pnc15R: 5'-GAAGCGGCGGACTACCATC-3', formation of heteroduplexes due to simultaneous co-amplification of wild type DNA and test DNA, where the first PCR stage includes concentration equalisation by quantitative PCR using the same pair of primers (Pnc18U/Pnc15R) with calibration curve generation and use of regression equation.
EFFECT: reliable and fast detection of mutations in pncA gene associated with evolving resistance to pyrazinamide, due to high specificity and sensitivity.
SUBSTANCE: invention relates to field of biochemistry, in particular to set of oligonucleotide probes for diagnostics of human population, living on RF territory, for hereditary monogenic diseases, by detection of mutations and/or polymorphisms. Also claimed are method of obtaining DNA-microchip, set for research of population of people, as well as method of detection of associated with monogenic diseases mutations and/or polymorphisms, in which claimed set of oligonucleotide probes is applied.
EFFECT: invention makes it possible to increase self-descriptiveness, accuracy and reduce research term.
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SUBSTANCE: method comprises performing PCR with electrophoretic detection of results, the transfer of amplification product on the gel, and assessment of the reaction. At that the study of each sample is carried out in two reactions using primers. In the first reaction the bacterium Pasteurella multocida is detected and the serogroups A and D are genotyped, in the second reaction the serogroups B, E and F are genotyped. The method enables to identify and genotype the strains and isolates of bacterium Pasteurella multocida of five serogroups - A, B, D, E, and F in pure or mixed cultures, as well as directly in samples of biological material from animals. The method may be used in veterinary microbiology for diagnostics of pasteurellosis in farm animals.
EFFECT: increase in the accuracy of diagnostics.
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SUBSTANCE: invention relates to a set of oligonucleotide primers and probes for the identification and subtyping of a bacterial DNA of Pasteurella multocida of serotypes A, B, D, E, F by the PCR method in real time. The invention can be used in genetic engineering and veterinary practice for the detection of a genetic material (DNA) of the bacteria P. multocida of the said serotypes.
EFFECT: improvement of the method.
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SUBSTANCE: invention refers to biotechnology, namely to a method for identifying tuberculosis mycobacteria of cluster Beijing B0/W148. A polymerase chain reaction is conducted for amplification of M. tuberculosis DNA fragments with selected primers P1, P2 and P3, sequences of which are presented by SEQ ID NO 1, 2, 3, respectively in the amplification mode of 98°C - 10 minutes, 30 cycles: 98°C - 30 sec, 64°C - 30 sec, 72°C - 50 sec, 72°C - 10 min, 4°C. The amplification products are separated by electrophoresis in 2% agarose gel. If the fragment of 1,021 base pairs is produced, the strain of tuberculosis mycobacteria of cluster Beijing B0/W148 is detected.
EFFECT: presented invention enables fast and high-specificity identification of tuberculosis mycobacteria of cluster Beijing B0/W148.
SUBSTANCE: invention relates to biotechnology, namely to a method of detecting a nucleic acid target-sequence from DNA or a mixture of nucleic acids with the application of a target signal generating primer (TSG-primer). The method includes hybridisation of the nucleic acid target-sequence with the TSG-primer, which contains a hybridisable nucleotide sequence, complementary to the nucleic acid target-sequence, and a reporter molecule and a quencher molecule. The TSG-primer does not represent a loop-hairpin structure. In case if the TSG-primer does not hybridise with the nucleic acid target-sequence, the reporter molecule and the quencher molecule are located close to each other spatially without forming loop-hairpin structures, which makes it possible for the quencher molecule to quench the signal from the reporter molecule. In case the primer hybridises with the nucleic acid target-sequence, the reporter molecule and the quencher molecule are separated spatially, making it not possible for the quencher molecule to quench the signal from the reporter molecule, which results in the generation and detection of the signal indicating the presence of the nucleic acid target-sequence. Bringing the product in contact with a matrix polymerase of nucleic acids under conditions of primer elongation in such a way that the reaction of 3′-elongation is induced on 3′-end of the TSG-primer. Detection of the signal, indicating the presence of the nucleic acid target-sequence.
EFFECT: claimed invention makes it possible to detect the nucleic acid target-sequence with high sensitivity and specificity.
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SUBSTANCE: probes included in the set have a structure of a "pin" with fluorophore and fluorescence extinguisher at the ends and have complementarity to products of an amplification reaction with primers. The developed set of hybridisation probes can be used for the fluorescent detection of the results of typing the actinobacillus mallei by a method of amplification of differentiating fragments of a genome in the implementation of the epidemiological surveillance of the glanderous infection.
EFFECT: possibility of simultaneous analysis of nine DFR-loci.
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SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.
EFFECT: described is a method of the target product and culture medium preparation.
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FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.
EFFECT: present invention can find further application in cancer therapy.
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SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.
EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.
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SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.
EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.
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FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.
EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.
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SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.
EFFECT: invention can find further application in diagnosing and treating cancer.
43 cl, 98 dwg, 20 tbl, 26 ex
SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.
EFFECT: invention enables to increase the expression level of recombinant human erythropoietin.
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SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.
EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.
23 cl, 74 dwg, 17 tbl, 33 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.
EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.
25 cl, 24 dwg, 8 tbl, 17 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. Presented are antibodies targeting integrin α2β1 containing humanised anti-integrin alpha-2 (α2) antibodies, as well as a method of treating by the integrin α2 antibodies. The humanised integrin α2 antibodies comprise a variable region of a light chain domain, a constant human light chain domain and a variable constant heavy chain domain of human IgG1, which exhibit the altered effector function. The variable constant heavy chain domain of human IgG1 comprises an S324N substitution. The invention can be used in medicine.
EFFECT: antibodies exhibit complement-dependent cytotoxicity, improved antibody-dependent cell-mediated cytotoxicity and improved CDC and ADCC.
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SUBSTANCE: invention represents a culture medium for the micropropagation of calciphilic plants in culture in vitro, comprising vitamins and amino acids dissolved in distilled water, on Murashige and Skoog, sucrose in an amount of 20,000 mg/l, agar-agar in an amount of 8,000 mg/l, and also growth regulators 6-benzylaminopurine, gibberellic acid, and 3-indolyl acetic acid, the mineral composition Woody Plant Medium, kinetin, at that the growth regulators are taken in the following concentration, mg/l: 6-benzylaminopurine 0.1-0.3, kinetin 0.9-1.1, gibberellic acid 0.9-1.1, 3-indolyl acetic acid 0.4-0.6.
EFFECT: invention enables to increase the propagation factor by activating the axillary meristems without compromising on the quality of regenerants.