Method of human recombinant lactoferrin obtaining
FIELD: medicine; biotechnologies.
SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.
EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.
The invention relates to biotechnology, in particular to the use of eukaryotes as hosts for adenoviral vectors, and can be used to produce biologically active recombinant lactoferrin (hereafter LF) of a person.
LF - stranded metallovedeniye glycoprotein. A large number of LF is expressed by epithelial cells of the mammary gland, lacrimal, salivary glands. LF is also included in the composition of bile and pancreatic fluid. It is well known that this natural protein has a number of medicinal properties, including bactericidal and bacteriostatic activity, participates in the regulation of humoral and cellular immunological responses, anti-inflammatory and other processes. In this regard, the main task is to obtain LF as the main ingredient for the industrial manufacture of the product.
There are two main directions in obtaining LF.
The first selection from natural sources. Human LF obtained from donor breast milk (n-t of the Russian Federation No. 1709606). However, the disadvantages of the known method of obtaining LF, including the use of donor human milk as a source of LF are the limited availability of raw materials and the necessity of strict control on the presence of infectious agents.
The second is the synthesis of recom is anantnag proteins in prokaryotic and eukaryotic systems. For this purpose, widely used cells of bacteria, yeast, plants, mammals and insects. Recombinant LF Express in various prokaryotic and eukaryotic organisms, including aspergillus (US Pat. No 6.080.559), horned cattle (US Pat. No 5.919.913), rice, corn, yeast Sacharomcyes (US Pat. No 6.228.614) and Pichia pastoris (US Pat. No 6.455.687, 6.277.817, 6.066.469). Each of these methods of synthesis of recombinant proteins has a number of disadvantages, namely:
in prokaryotes, as well as in the cells of many eukaryotes (yeast, insects, plants), except vertebrates, the correct post-translational modification and installation (folding) of the synthesized protein of human rights does not occur at all or is performed incorrectly, which significantly reduces its biological activity;
- receiving recombinant human protein in the culture of cells of vertebrate animals or through their transgenes is a high-cost technology.
Among bioreactors cell-based vertebrates can be classified into three types: cell culture, somatic transgenes and genetic transgenes. As genetic transgenes use different classes of vertebrates, mainly birds and mammals. Bird cages unlike mammalian cells have the advantage that they do not contain viruses, pathoge the data for a person, and prions which could serve as a safe source of recombinant proteins for medical purposes. However, the production of proteins in cell cultures of birds currently in use is limited, due to problems with the delivery of genetic material.
A method of obtaining recombinant human proteins from protein chicken eggs obtained from transgenic chicken, in which the gene is by lentiviral vector was incorporated gene corresponding recombinant protein (U.S. Pat. No 6730822). The use of transgenic birds is related to the duration of the implementation period (up to 24 weeks), high costs, not only for obtaining transgenic herds, but also its contents.
Closest to the claimed invention by a combination of traits is a method for LF, including the transformation of eukaryotic cells with a vector containing a nucleic acid sequence encoding a variant of LF, the cultivation of transformed eukaryotic cells in a suitable nutrient medium before the formation of LF and its secretion (U.S. Pat. No 6111081). However, the known method does not allow to achieve technical results to be obtained from use of the claimed invention, for the following reasons:
1. Requires additional cleaning and testing the resulting p is eparate recombinant LF in the presence of impurities and toxic substances, in particular mycotoxins, which are products of fungi. Allocated originally the Aspergillus aflatoxin potential carcinogens - have a toxic effect on the liver of mammals, birds, fish.
2. The impossibility of using growth medium containing recombinant LF for any purpose without the cleanup phase, whereas allatoona liquid chicken embryo containing recombinant LF, can be used as a dietary Supplement without the cleanup phase.
3. The resulting recombinant LF biological properties is not identical to a protein derived from a natural source.
The claimed invention is directed to solving the problem of preparative production of biologically active recombinant human LF.
Use in clinical practice, the proposed method allows to achieve several technical and economic results:
- the identity of the protein obtained in accordance with the inventive method, biological properties, including biological activity, protein derived from a natural source, through proper post-transcriptional modification and folding of proteins;
- the security of the resulting product due to the fact that it does not contain human pathogens, infectious agents and other dangerous is x impurities;
- short term organization of production up to 10 weeks.
- low cost.
These technical and economic results by carrying out the invention are achieved due to the fact that in the same way as in the known method of recombinant human LF is obtained by injection of viral vector, carrying in their genome a gene human LF, in eukaryotic cell system, cultivation and subsequent isolation of recombinant protein.
The feature of the proposed method lies in the fact that, as a viral vector using recombinant adenovirus birds or mammals, as eukaryotic cellular systems use a chicken egg with the embryo, the cultivation is carried out by incubation at 37 ° °C for 60-90 hours.
The essence of the invention consists in the introduction in allantoin cavity chicken eggs with embryo recombinant adenoviral vector carrying the genome expressing the cassette with the gene LF cheloveka subsequent expression of the gene of this protein and its accumulation preparative quantities allantoine fluid of chicken embryos. Chicken egg with embryo infected with recombinant adenovirus, plays the role of a natural bioreactor for production of recombinant LF.
Compared to known you upomyanutyi systems in the expression of the preparation of recombinant proteins in allantoine liquid eggs with embryo has a number of features and advantages. Found that in the cells of birds occurring glycosylation and folding of proteins, similar to those in mammalian cells. As a consequence, the synthesized recombinant human protein has all the properties of natural protein derived from milk.
On the other hand, in the case of use in the pharmaceutical industry of the claimed invention, it is possible to achieve high economic effect. On the one hand, the cost of extraction and purification of the target protein from allantoine fluid of chicken embryos is comparable to the cost of extraction and purification from other systems. On the other hand, the content of the embryos does not require expensive equipment, and agricultural production scale source products guarantee the possibility of industrial production of recombinant drug. The result is greatly reduced cost of production raw material.
The method is as follows.
Gene human LF is obtained by amplification for DNA synthesized by RT-PCR (Reverse Tpanscrip'tion System "Invitrogene" No. 12236-014, USA), matrix RNA isolated from the genome of human cells using TRIZOL ("Invitrogene" No. 15596-018, USA). The DNA fragment carrying the gene for human LF, clone into plasmid vector pGEM-T Easy (Promega No. A). Cloning is carried out according to the Protocol attached to Kinabalu for cloning PCR products pGEM-T Easy. Received legirovannoi mixture transform cells of E. coli DH5α ClRb method. Transformed cells are selected on agar LB medium with antibiotic ampicillin (40 µg/ml). Plasmid DNA secrete the method of alkaline lysis, analyze using restricted EcoRI, Ncol (No. ER0271, ER0571 "Fermentas", Lithuania) and selected clones carrying the plasmid of the expected size (5316 BP). The primary structure of the cloned fragment in the resulting plasmid pGEM-Lf confirm restriction mapping by restriction endonucleases Bgll (No. ER0071 "Fermentas", Lithuania) and sequencing by the method of Sanger.
For the preparation of recombinant human adenovirus 5-th serotype Ad5-Lf, containing the gene for human LF, produce the cloning of human LF (Lf) from the plasmid pGEM-Lf, hydrolyzed according to the NotI site in the commercial vector pShuttle-CMV, which is set to "AdEasy Adenoviral vector system", on the website for restrictase NotI under the control of the CMV-promoter and polyadenylation signal. The presence of LF gene in the resulting Shuttle vector pShuttle-CMV/Lf confirm restriction fragments length polymorphism analysis using endonuclease EcoRI, EcoRV and PCR. The preparation of recombinant adenovirus Ad5-Lf carried out according to the methodology "AdEasy Adenoviral vector system" (Stratagene Cat. No 240009). The presence of the gene in human LF in the genome of the recombinant adenovirus Ad5-Lf confirmed by PCR.
To obtain recombin nnogo adenovirus birds CELO-Lf, containing the gene for human LF, produce the cloning of human LF (Lf) from the plasmid pGEM-Lf, hydrolyzed according to the NotI site in the Shuttle plasmid vector according to the website for restrictase ESO under the control of the CMV-promoter and polyadenylation signal. This Shuttle plasmid vector is part of a system for the preparation of recombinant adenovirus birds CELO according to the method described by M. Cotten (Michou, A.I., Lehrmann H., Saltik, M., Cotten, M., Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors, Journal of Virology, February, 1999, p.1399-1410, Vol.73, No. 2), according to which then retrieves recombinant adenovirus birds CELO-Lf. The presence of the gene in human LF in the genome of the recombinant adenovirus CELO-Lf confirmed by PCR.
Next, determine the titer of recombinant adenoviruses Ad5-Lf and CELO-Lf method belascoaran on the culture of 293 cells (cells of embryonic human kidney) and LMH (Leghorn male hepatoma cells hepatoma cock the Livorno).
The CELO virus-Lf or Ad5-Lf infect 9-10-day SPF chicken eggs with embryos. As a result of earlier experiments found that the infection of the embryo at this stage of development is the most effective accumulation of recombinant protein in allantoine fluid. The infection produced through a hole with a diameter of 1-2 mm in the eggshell from the side of the air chamber, injecting a needle of the syringe 1.5-2.0 SMOD shell to injection of infectious material under chorioallantoic membrane in the allantois of the embryo.
Chicken embryos infect recombinant adenovirus CELO-Lf or Ad5-Lf in doses of 1×106-1×107The BATTLE/the embryo and 1×107-1×109The FIGHT/embryo, respectively. The optimality of this dose was established in previously performed experiments where it was shown that the dose reduction leads to lower product yield (recombinant protein)and increasing the dose to the premature death of a chicken embryo, so that the product yield is also reduced.
Infected chicken embryos incubated at a temperature of 37°C for 60-90 hours. It is established,that during this period of incubation in allantoine fluid accumulate the maximum amount of recombinant human LF.
Control the level of expression of recombinant human LF is carried out by sampling aliquot (0.5 - 1 ml) allantoine fluid through a hole in the eggshell, a small percentage of infected embryos during the whole period of incubation. The level of expression of recombinant human LF in the selected aliquot allantoine fluid produced by ELISA. After completion of the incubation period in allantoine fluid levels of recombinant protein human LF account for 0.9±0.07 mg/embryo during infection with a virus CELO-Lf and 0.2±0.08 mg/embryo during infection with a virus Ad5-Lf.
On abuses stage produces the selection and purification of recombinant human LF of allantoine fluid by the method of affinity chromatography sorbent antibodies to human lactoferrin (At) - sepharose 4 C. the Yield is 64±4% and 60±3% if using recombinant adenovirus birds CELO-Lf and human Ad5-Lf, respectively.
Of the selected recombinant human LF examined for its biological (antioxidant) activity in vitro and in vivo. These methods are based on the ability of recombinant LF to inhibit lipid peroxidation (LPO) in the liver of mice. The analysis of the biological activity (in vitro and in vivo) recombinant protein obtained in accordance with the inventive method, showed his identity natural human LF isolated from donor breast milk.
The inventive method has significant advantages and meets the criteria of patentability.
A method of obtaining a recombinant human lactoferrin, by introducing a viral vector, carrying in their genome a gene of human lactoferrin in eukaryotic cells, culturing and subsequent secretion of the recombinant protein, characterized in that as a viral vector using recombinant adenovirus birds or mammals, as eukaryotic cells use 9-10-day egg with an embryo that infect recombinant adenovirus CELO-Lf or Ad5-Lf by injection into the embryo allantois in doses of 1×106-1×107 The BATTLE/the embryo or 11×07-1×109The FIGHT/embryo, respectively, the cultivation is carried out by incubation at 37 ° °for 70-75 hours
FIELD: medicine, biotechnologies.
SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.
EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.
20 cl, 2 dwg, 8 ex
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to biotechnology. Method includes addition to fermentation broth or homogenate from E. coli of efficient quantity of ethacridinlactate solution for sedimentation of contamination from host-cells in conditions, when greater part of polypeptide remains dissolved, and isolation of heterological polypeptide from broth or homogenate.
EFFECT: simplification of target polypeptide purification and obtaining it with high degree purity.
23 cl, 15 dwg, 3 tbl
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.
EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.
2 cl, 7 dwg
SUBSTANCE: invention concerns biotechnology, specifically production of new polypeptides regulating carbohydrate metabolism, and can be used in medicine. New polypeptides reacting as both GLP-1 receptor agonists and glucagon receptor antagonists. Polypeptides and coding nucleic acids are used as components of pharmaceutical compositions for treatment of diabetes type 2 and metabolism disorders.
EFFECT: production of compounds providing effective glucose homeostasis for patients, suffering from carbohydrate metabolism disorders.
18 cl, 1 dwg, 4 tbl, 20 ex
SUBSTANCE: invention relates to genetic engineering, namely, to obtaining inhibitors of TGF-β1 and can be used in medicine. Obtained peptides are capable of binding with transforming growth factor TGF-β1 and are potential inhibitors of biological activity of TGF-β1, binding with this cytokine directly. Peptides are obtained by recombinant method using transformed host-cell, by cultivating host-cell in conditions which ensure production of the said peptide, and its separation. The invention allows for efficiently treatment of diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.
EFFECT: possibility to efficiently treat diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.
12 cl, 6 dwg, 4 tbl, 4 ex
FIELD: technological processes; pharmacology.
SUBSTANCE: antagonist of human interleukine-1 receptor is prepared with the help of recombinant strain E. coli that contains plasmid, which provides production of this protein. For this purpose the strain is cultivated. Then from bacterial cells the target product is separated with application of three-stage chromatography and concentration on hydrophobic sorbent. At that the first and the third stage of mentioned chromatographic purification is performed on cation-exchanging resin, and in the second stage of chromatography the anion-exchanging resin is used.
EFFECT: application of invention allows to prepare antagonist of human receptor of high purity.
7 cl, 1 tbl, 4 ex
FIELD: technological processes; medicine.
SUBSTANCE: invention is related to preparation of recombinant analogues of human gamma-interferon and may be used in medicine for prophylactics and treatment of oncological diseases, neoplasms and inflammatory processes of humans. Highly pure genetically engineered analogue of human gamma-interferon - deltaferon with molecular mass of 16.2 kilo Daltons is produced by microbiological synthesis with further chromatographic purification on "KM"-sepharose - cationic-exchange sorbent with high linear speed of flow and suitable for preparative loads, at different pH values. On the basis of deltaferon, which contains by data of polyacrylamide gel-sodium dodecyl sulfate electrophoresis at least 98% of main substance in the form of monomer that possesses antiproliferative and anti-inflammatory activities inherent in gamma-interferon, medical product is prepared, which also includes low molecular polymer filler-stabiliser (reopolyglukine or polyvinylpyrrolidone) and salt buffer system with pH 7.0-7.1.
EFFECT: highly active and stable preparation of deltaferon is produced.
2 cl, 6 dwg, 1 tbl, 3 ex
FIELD: technological processes.
SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.
EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.
7 cl, 20 dwg, 1 tbl
SUBSTANCE: invention concerns glycoforms of VII factor and compositions of VII factor, characterized by modified configurations on basis of asparaginic oligosaccharide chains. In addition invention includes detection method applied for polypeptide glycoforms of VII factor, receiving method and disease treatment method as well.
EFFECT: identification of biologically active forms of recombinant VII factor.
53 cl, 5ex
FIELD: biotechnology, molecular biology, proteins.
SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.
EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.
24 cl, 21 tbl, 32 ex
FIELD: biology, gene engineering.
SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.
EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.
13 cl, 12 dwg,12 ex
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to field of biotechnology and concerns obtaining factor VII protein by method of recombinant DNA. Recombinant plasmid DNA was constructed for expression of blood clotting factor VII in mammalian cells, which is product of ligating of fragment of cDNA of human factor VII gene, flanked by sites of restrictases Xhol and BamHI recognising, with large XhoI/BglII fragment of vector pEFZeo, including genes of resistance to ampicillin and zeocin. As result of BHK cell transformation with new recombinant plasmid, cell line BHK/F7 was obtained, which produces recombinant protein of factor VII with output of up to 40 mkg/ml.
EFFECT: obtaining cell line producing recombinant protein of factor VII.
2 cl, 4 dwg, 4 ex
SUBSTANCE: invention relates to field of biotechnology, namely to genetic engineering. New interferon-binding proteins, which modulate activity of different interferon-α subtypes, as well as interferon-β activity, are obtained. Described is cloning of DNA fragment, which codes interferon-α/β, binding protein IFNAB-BPI, and expression of obtained DNA fragment in host-cells both with formation of respective polypeptide and in form of fused proteins. Practical application of obtained proteins as components of pharmaceutical compositions for inhibiting activity of IFN-α or IFN-β is suggested. Invention can be applied in medicine for inhibiting undesirable impact of IFN-α or IFN-β.
EFFECT: obtaining new interferon-binding proteins, which can find application in medicine for inhibiting undesirable impact of IFN-α or IFN-β.
13 cl, 10 dwg, 6 tbl, 17 ex
SUBSTANCE: invention relates to the field of genetic engineering, namely, to obtaining new proteins, stimulators of MAP-kinases and can be used in medicine. New G-protein-connected receptor polypeptide, which is capable of stimulating MAP-kinases is obtained. By means of expressing vector, which contains sequence of DNA coding new polypeptide, animal host-cell is transformed, cultivated in conditions suitable for expression and thus polypeptide is obtained. Medication is obtained on the basis of new polypeptide or nucleic acid that codes it. The invention allows treating tumor diseases, hypoxia, disorders of cardiovascular, nervous and immune systems.
EFFECT: possibility to treat tumor diseases, hypoxia, disorders of cardiovascular, nervous and immune systems.
14 cl, 66 dwg, 15 ex
SUBSTANCE: according to the invention, the recombinant albumen with anti-cancer effect is selected out of the group including 1) albumen with amino acid sequence presented in SEQ ID N0:2 indicated in the sequence list; 2) albumen with amino acid sequence homologous to the sequence presented in SEQ ID NO:2 by more than 90% and featuring the same effect as the albumen 1); 3) albumen obtained by replacement, deletion or addition of one or more residua to the amino acid sequence presented in SEQ ID NO:2 and featuring the same effect as the albumen 1). The invention also claims a gene coding the described recombinant albumen and its variants, expression vector containing the said gene, and cell line containing the said gene and intended for expression of the recombinant albumen. The recombinant albumen can be applied in obtaining cancer treatment medication. A medication including the aforesaid recombinant albumen as active component has strong selective effect on tumour cells and does not cause apoptosis of normal tissue cells.
EFFECT: developed substance for obtaining cancer treating medication.
14 cl, 16 dwg, 7 tbl, 3 ex
FIELD: parasitology; medicine.
SUBSTANCE: fr316 fragment of mitochondrial gene ND1 with a given nucleotide sequence presented in SEQ ID 1 is obtained from DNA of Opisthorchis felineus.
EFFECT: detection of opistorchosis agents and differential identification of DNA of Opisthorchiidae family species.
SUBSTANCE: invention relates to genetic engineering area and can be applied to medicine in diagnostics of several diseases, associated with metabolic syndrome. For determination of polymorphism Thr312Ala α-fibrinogen (FGA) gene the DNA from investigated sample is isolated and subject to amplification by polymerase chain reaction using allele-specific primers. For prevailing allele FGA T identification reverse primer 5'-TCC-CAG-AGT-TCC-AGC-TTC-CAG-T-3' is used; for rare allele FGA A identification reverse primer 5'-CCC-AGA-GTT-CCA-GCT-TCC-AGC-3' is used; as a direct primer in both cases serves the sequence 5'-TGT-CGA-GGG-TCA-TGC-AGT-AGG-G-3'. The invention makes it possible to carry out high-accuracy, simple, and low-cost analysis of fibrinogen polymorphism, suitable for clinico-diagnostic laboratories.
EFFECT: possibility to carry out high-accuracy, simple, and low-cost analysis of fibrinogen polymorphism, suitable for clinico-diagnostic laboratories.
2 dwg, 1 tbl
SUBSTANCE: invention refers to genetic engineering and biotechnology and can be used for labelling of proteins, cells and organisms. Substitutes added to nucleic acid Aequorea coerulescens, coding colourless GFP-like protein acGFP is considered as means of production of nucleic acid coding fluorescent protein. Expression cassette containing this nucleic acid under regulatory elements control provides in-cell biosynthesis of fluorescent protein. This genetic structure is used for production of transgenic organism and cell producing fluorescent protein, coded by specified nucleic acid. This fluorescent protein is applied in methods for labelling of cells, intracellular structure and protein, as well as for promotor transcriptional activity registration.
EFFECT: invention application ensures widen range of labelling means used in biochemistry, molecular biology and medical diagnostics.
11 cl, 34 dwg, 2 ex
SUBSTANCE: polynucleotide is obtained, coding chromo- or fluorescen mutant wild type DsRed (SEQ ID N0:2), where chromo- or fluorescent mutant contains a substitute in the amino acid position 42 SEQ ID N0:2, and optionally one or more substitutes in the amino acid positions, chosen from a group, consisting of 4, 2, 5, 6, 21, 41, 44, 117, 217, 145. Using the polynucleotide in the vector, the host cells which express chromo- or fluorescent mutant DsRed are transformed. The invention allows for obtaining chromo- or fluorescent polypeptide DsRed, which matures faster than wild type DsRed.
EFFECT: increased effectiveness.
26 cl, 10 dwg, 2 tbl, 4 ex
FIELD: molecular genetics.
SUBSTANCE: method for determining the polymorphism of the PRKAG3 gene - meat productivity marker in pigs, is invented.
EFFECT: invention is applicable in molecular genetics for diagnosing alleles which can be used in animal selection.
5 dwg, 1 ex
SUBSTANCE: invention relates to method for production of porphyrinopeptides satisfying the formula I , wherein R1 and R2 independently from one another represent amino acids or peptides comprising 2-15 of amino acid residues, wherein α-carboxylic groups of amino acids or peptides may be modified by C1-C8-alkyl ester and side functional groups of amino acids or peptides may be protected; in particular R1 is ArgOMe; R2 is -OH (III); R1 is LeuHisOMe; R2 is -OH (IV); R1 is LeuLeuValPheOMe; R2 is -OH (V); porphyrin carboxylic group may be modified by methyl or other C1-C9-ester or pharmaceutically acceptable salt; Y- represents Cl-; Me represents Zn, Cu, Fe, Mn. Claimed method includes activation of porphyrin carboxylic group with N-oxy-5-norbornene-2,3-dicarboxyimede in molar ratio of 1:1 in presence of N,N'-dicyclohexylcarbodiinide; or with diphenylphosphorylazide (DPPA) in equimolar ratio of porphyrin/DPPA in presence of base. Then porphyrin with activated carboxylic group is brought into reaction with amino component (amino acid or peptide) in form of mineral acid salt, which is neutralized with base. Also disclosed are methods for application of compounds (I) as nucleotic agents.
EFFECT: new nucleotic agents.
4 cl, 7 ex, 1 tbl