Peptide ded and its application for identification and/or purification of recombinant proteins, vector pded
SUBSTANCE: there is prepared identification peptide with amino acid sequence: Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. The prepared peptide is applied to identify, purify and recover the recombinant proteins containing it. For making the recombinant proteins with a polypeptide label, an expression vector pDED is applied. Said vector contains a nucleotide sequence coding amino acid sequence of the identification protein.
EFFECT: invention allows extending range of the methods to identify the recombinant proteins.
14 cl, 6 dwg, 1 tbl, 6 ex
The technical field of the present invention
The invention mainly relates to the field of molecular biology. In particular, the present invention relates to new peptide applicable for the identification, purification or selection containing recombinant proteins. In addition, the present invention relates to a nucleotide sequence that encodes the specified peptide tag. The present invention also relates to a vector to clone containing the nucleotide sequence encoding a recombinant protein of interest, fused with preservation of one reading frame with a nucleotide sequence that encodes a specified new peptide.
The prior art of the present invention
In the laboratory, in particular molecular biology, research is often necessary detection of recombinant proteins. For this purpose, usually using specific antibodies. However, due to the high variability of the structure of proteins such an approach to detection requires obtaining specific antibodies for each protein.
The alternative is to use a peptide epitope tag, which is introduced into the recombinant protein and antibodies specific recognizing the label for detection of recombinant proteins containing this label. The label included the CE to recognize its antibodies also allows affinity purification of the containing recombinant proteins.
As a label often use short peptides. The use of short amino acid sequences allows to minimize the effect of the presence of extraneous amino acids on the properties of the recombinant protein. The size of such epitope tags may vary from a few amino acid residues up to tens of amino acid residues. The label can be located on the N end of the recombinant protein (for example, the label FLAGTM, 8 amino acid residues, Sigma-Aldrich, Inc., catalogue number F3290; Norr TR et al. (1988) Bio/Technology 6:1204-10; Chubet R and Brizzard (1996) BioTechniques 20:136-141; Hernan R et al. (2000) BioTechniques 28:789-93), and From-end (for example, the label goes, 144 amino acid residue; Krivi GG et al. (1985) J. Biol. Chem. 260:10263-7), however, it is possible placement of the label on the 3'-end. In addition, the label can be embedded in nikocevic the provisions of the amino acid sequence of the recombinant protein.
To obtain recombinant proteins labeled using plasmid or other vectors, which contain the coding tag nucleotide sequence. Cloning in such vectors, the cDNA of interest protein with preservation of one reading frame of the protein and the label allows to Express the recombinant protein tagged with. Generally, for these purposes, expressing in various systems (e.g., in cells of E. coli or mammalian used is to eat a variety of controlling the expression of auxiliary sequences) using different vectors. The alternative is the introduction of a nucleotide sequence that encodes a tag into cDNA using oligonucleotides with a polymerase chain reaction with subsequent cloning of the product of polymerase chain reaction in the desired vector for production of recombinant proteins.
The closest technical solution, which is the basis of the present invention, it is possible to produce a label FLAGTM.
Brief description of drawings
Figure 1. Given the nucleotide sequence of the region of polylinker vector pDED (figa) and for comparison, a similar region of the vector pBluescript SKII(+) (figb). Shows a frame read lacZ cDNA, which is not broken down as a result of the insertion of the nucleotide sequence that encodes a peptide DED. For vector pDED nucleotide sequence complementary to the sequence that encodes a peptide identification DED dedicated lowercase.
Figure 2. A fragment of the protein sequence Rabatin-5-gamma mouse, comprising the sequence identical to the sequence of the peptide DED, and a fragment of a protein sequence Rabatin-5, in which the sequence identical to the sequence of the peptide DED, is divided into two parts 43 amino acid residues. Sequence identical to the sequence of the peptide DED in the protein Rabatin-5-gamma, and FR is Genty sequence, the identical sequence of peptide DED in the protein Rabatin-5, shown in bold.
Figure 3. Western blot analysis with monoclonal mouse antibodies specifically recognizing the peptide DED (a) and polyclonal anti-FLAG antibodies rabbit (Sigma) (B) lysates of cell line COS-7 (1), lysates of cell line COS-7, producing a protein Rabatin-5 with C-terminal FLAG-epitope (2), and lysates of cell line COS-7, producing a protein Rabatin-5-gamma C-terminal FLAG-epitope (3). Proteins were separated in 7.5% of the LTO-polyacrylamide gel. Detection of primary antibodies mouse was performed using conjugated with horseradish peroxidase antibodies against mouse IgG (GE Healthcare, UK) and ECL+ (GE Healthcare), the detection of the primary antibodies rabbit was performed using conjugated with horseradish peroxidase antibodies against rabbit immunoglobulins (GE Healthcare) and ECL+(GE Healthcare). The left shows the locations of the markers of molecular weights of proteins in kDa. Endogenous produced by the cell line COS-7 protein Rabatin-5-gamma marked by the arrow.
Figure 4. The results of the immunocytochemical detection of green fluorescent protein chimeras with Rebaptism-5-gamma (a-a') and Rabatin-5 (b-B')produced by the cell line KSS-21, using a monoclonal antibody specifically recognizing the peptide DED, and green fluorescent protein chimeras with Rebaptism-5-gamma using monoclonal is NITEL, specifically recognizes the peptide DED that were previously preincubator with a 10-fold molar excess of peptide DED ( -'). Shows the fluorescence of green fluorescent protein (a, B, C) and fluorescence secondary antibodies associated with immobilized antibodies to peptide DED (A', B', A').
Figure 5. Western blot analysis with anti-FLAG antibodies rabbit (a) and monoclonal mouse antibodies to the peptide DED (B) proteins purified using antibodies to the peptide DED immobilized on Protein G-Sepharose from lysates of cell line COS-7, producing a protein Rabatin-5-gamma mouse (1) or protein Rabatin-5 mouse (2) with C-terminal FLAG-epitope. Proteins were separated in 7.5% of the LTO-polyacrylamide gel. Detection of primary antibodies mouse was performed using conjugated with horseradish peroxidase antibodies against mouse IgG and the ECL system+, the detection of the primary antibodies rabbit was performed using conjugated with horseradish peroxidase antibodies against rabbit immunoglobulins and system ECL+. The position of the heavy and light chains of immunoglobulins are marked with asterisks, the position of the protein Rabatin-5-gamma C-terminal FLAG-epitope identified by an arrow.
6. Detection of protein hVEGF-165 C-terminal identification peptide DED in conditioned medium of cell line Cho, transfected with a plasmid pVEGF-DED. Aliquots of conditioned media of cell lines Cho, transpirirovat is related to the plasmid pVEGF-DED (1) and nitrostilbene cell line Cho (2), and besieged of these proteins (3 and 4, respectively), were separated in 12.5% of the LTO-polyacrylamide gel, proteins were transferred to polyvinyl chloride membrane and performed Western blot analysis using monoclonal mouse antibodies specifically recognizing an identification peptide (A) or polyclonal antibodies goat to protein hVEGF-165 (Sigma) (B). Detection of primary monoclonal mouse antibodies was performed using conjugated with horseradish peroxidase antibodies against mouse IgG and the ECL system+, the detection of the primary antibodies goat was performed using conjugated with horseradish peroxidase antibodies against immunoglobulins goats and system ECL+. The left shows the locations of the markers of molecular weights of proteins in kDa.
The disclosure of the present invention
There are several necessary conditions for the creation of an effective system of detection and treatment on the basis of hybrid polypeptides. The first basic requirement to add a marker sequence is the lack of impact marks on native laying protein to which this label is attached. Secondly, the marker peptide sequence should be hydrophilic and expressed essentially on the surface of the protein, so that it can reliably interact with its ligand (e.g. antibody). In addition, it is desirable to use m the TCI allows for affinity purification of relatively simple and inexpensive technique. Finally, it is also preferred that the peptide tag, if necessary, could be easily removed with the receipt of a native protein (Einhauer A. and Jungbauer, A. (2001) J. Biochem. Biophys. Methods 49:455-465).
In accordance with the present invention the peptide tag of the recombinant protein is a peptide DED, characterized by the amino acid sequence Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln.
Two contained in the peptide DED high hydrophilic Tripeptide determine the overall high value of hydrophilicity-end of the peptide by well-known scales (Norr TR and Woods, K.R. (1981) Proc. Natl. Acad. Sci. USA 78:3824-8; Kyte, J. and Doolittle R.F. (1982) J. Mol. Biol. 157(6):105-142). It was shown that when the three-dimensional packing of protein such hydrophilic sequences occupy a position on the surface of the protein molecule, which determines, among other things, high antigenicity hybrid molecules (Norr TR (1986) J. Immunol. Methods 88:1-18).
The Oligopeptide according to the present invention can be encoded by a large variety of molecules of nucleic acid, which is the result of well-known in the art, the phenomenon of degeneracy of the genetic code. The essence of the phenomenon is that any amino acids (except tryptophan and methionine), which is part of the natural peptides, can be encoded by more than one triplet nucleotide codon (see table). All of these viroid is by encoding the molecules of nucleic acids fall under the scope of the present invention.
One of the possible nucleic acid molecules encoding the peptide DED according to the present invention, is a 5'- ggcccggctc cccagcctga cgaggatctg aagaggcaa-3' (SEQ ID NO:1).
|Universal table encoding amino acids|
|Ala||gcu gcc gca gcg||alanine|
|Arg||aga agg cgu cgc cga egg||arginine|
|Asp||gau gac||aspartic acid|
|Glu||gaa gag||glutamic acid|
|Gly||ggu ggc gga ggg||glycine|
|Ile||au auc aua||isoleucine|
|Leu||uua uug cuu cue cua cug||leucine|
|Pro||ccu ccc cca ccg||Proline|
|Ser||agu agc ucu ucc uca ucg||serine|
|Thr||acu ACC ACA acg||threonine|
|trm||uaa uag uga||the stop codon|
|Val||guu guc gua gug||valine|
In accordance with the present invention an auxiliary vector to obtain nucleotide sequences encoding the recombinant protein of interest with a peptide tag, is the vector of pDED. Vector pDED allows by simply cloning it cDNA protein of interest with preservation of one reading frame to obtain a chimeric cDNA encoding a recombinant protein tagged with. This cDNA can be periglomerular in any desired vector for production of recombinant protein. There is no need to use a set of vectors encoding tag for different systems of expression, as well as to synthesize oligonucleotides and use of polymerase chain reaction for the introduction of the label in the composition of the recombinant protein.
The advantage of plasmids pDED is that cloning it cDNA may be accompanied by the use of so-called "blue-white" selection, convenient tool selection of cell colonies containing the cloned cDNA. Selection is based on the fact that the sites of recognition of restricts for cloning in the vector are within the sequence that encodes the enzyme β-galactosidase. If the plasmid does not contain the insert in E. coli under certain conditions, is produced by β-galactosidase, which can convert chromogenic substrate (X-gal), which leads to the blue color of the colonies. When inserting reading frame cDNA of β-galactosidase is broken, the enzyme is not produced, and the colony will not turn blue and remain white in color. During Cloner is of this allows for the color of colonies to determine in cells which contain the desired plasmid containing the insert. Vector pDED-based vector to clone pBluescript II SK(+), which allows the use of blue-white selection.
So, the vector is designed for cloning cDNA of interest with the aim of obtaining cDNA encoding the recombinant protein tagged with Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. While the nucleotide sequence contained in the vector pDED and koderma label, do not shift the reading frame of the β-galactosidase and does not affect its activity. This allows cloning into the vector pDED cDNA of interest to conduct blue-white selection that permits easy identification of colonies with plasmids containing the cDNA insert.
Design vector pDED
The nucleotide sequence of the fragment of the vector pDED to obtain cDNA encoding proteins with peptide identification DED and allowing color blue and white selection of E. coli cells to identify clones containing plasmids with cDNA encoding the recombinant protein identification peptide. Vector pDED obtained by embedding the nucleotide sequence of SEQ ID NO:1 that encodes a peptide DED, supplemented at the 5'-end nucleotide sequence ggagga that encodes a dipeptide glycine, and flanked by fragments of sites usnavy what I restriction endonucleases XbaI at the 5'end and NotI at the 3'-end, in the vector pBluescript SKII(+) (Stratagene, USA) between the recognition sites of restriction endonucleases XbaI and NotI. This preserves the open reading frame of the gene lacZ encoding β-galactosidase, which allows, as in the original vector pBluescript SKII(+), to carry out the identification of recombinant DNA containing the insert in polylinker, on the basis of the color blue-and-white breeding colonies of E.coli; cDNA encoding a protein identification by peptide DED received vector pDED, may be cut from a vector according to any present site in polylinker between sites of recognition between the XbaI and kpni restriction sites at 5'-end and any site between NotI and SacI 3'-end and inserted in any desired vector for production of recombinant protein identification by peptide DED on it-the end (see Example 5). Given the nucleotide sequence of the region of polylinker vector pDED (figa) and for comparison, a similar region of the vector pBluescript SKII(+) (figb). The structure of the vector pDED detail illustrated in figure 1.
Demonstration of specific detection of protein, having in its composition identification peptide DED, the method of Western blot analysis
To demonstrate the specific detection of proteins containing a sequence identical to the sequence of the peptide DED, method Western blot analysis was used protein Rabatin-5-gamma mouse (consistently the th cDNA GeneBank ACC. No. AF248289), which contains inside a polypeptide sequence identical to the sequence of the peptide DED, and protein Rabatin-5 mouse cDNA sequence GeneBank ACC. No. D86066), which contains inside a polypeptide sequence identical to the sequence of the peptide DED, the first 7 amino acid residues which is separated from the next 6 amino acid residues 43 amino acid residues (figure 2). Proteins Rabatin-5-gamma and Rabatin-5 were produced in the cell line COS-7 with C-terminal FLAG-epitope for monitoring the presence of proteins, which cell line COS-7 were transtorno transliterowany plasmid vectors for the expression of the corresponding proteins. 48 hours after transfection cells were subjected to lysis in a buffer for drawing on LTO-polyacryamide gel and aliquots of the lysates were analyzed using Western blotting with anti-FLAG antibodies (Sigma, USA) or monoclonal antibodies specifically recognizing the peptide DED. Anti-FLAG antibody revealed a protein Rabatin-5 and Rabatin-5-gamma with FLAG-epitopes in the lysates of transfected, but not in the lysate nitrostilbene cells (figb). This monoclonal antibody specifically recognizing the peptide DED, confident were detected only protein Rabatin-5-gamma, but not protein Rabatin-5, in lysates of transfected cells (figa). Insignificant the th signal in the lysates of cells, producing Rabatin-5 due to the presence of endogenous protein Rabatin-5-gamma in cell line COS-7, what allows us to conclude the presence of a signal of similar intensity in the lysates nitrostilbene cells.
To demonstrate the feasibility of specific detection of protein, having in its composition identification peptide DED, immunocytochemical methods
Used for the analysis of cell lines KSS-21, transtorno producing as a result of transfection with the appropriate plasmid proteins Rabatin-5-gamma or Rabatin-5, expressed as chimeras with green fluorescent protein N-ends. The cells were fixed with methanol at -20°C and after fixation were stained with monoclonal mouse antibodies specifically recognizing the peptide DED, and their subsequent detection by antibodies to mouse IgG labeled with fluorophore AlexaFluor546 (Invitrogen, USA). Cells producing protein chimeras Rabatin-5-gamma or Rabatin-5 with green fluorescent protein, identified by the green glow figa, B, C). Color monoclonal antibodies to peptide DED were identified by the red glow figa', B', A'). Monoclonal antibodies to the peptide DED able to identify only a Chimera of green fluorescent protein with Rebaptism-5-gamma (figa And'), but not with Rebaptism-5 (figb, B'). The specificity of staining antitetanic the peptide DED by a complete lack of staining with antibody Chimera protein Rabatin-5-gamma green fluorescent protein by incubation of the antibody before detection with a 10-fold molar excess of synthetic peptide DED (FIGU,').
Demonstration of protein purification, having in its composition, the peptide DED, using a monoclonal antibody specifically recognizing the peptide DED
Cell line COS-7, producing a protein Rabatin-5-gamma mouse, which contains inside a polypeptide sequence identical to the sequence of the peptide DED, or protein Rabatin-5 mouse, which contains inside a polypeptide sequence identical to the sequence of the peptide DED, the first 7 amino acid residue which is separated from the next 6 amino acid residues 43 amino acid residues with a C-terminal FLAG-epitope (described in Example 2) was used for preparation of lysates. For this, cells were literally in Tris-buffered saline containing 1% Triton X-100, 0.5% of Nonidet the P-40 and protease inhibitors, and after centrifugation, the lysates were incubated with Protein G-Separate (GE Healthcare) with immobilized therein a monoclonal antibody specifically recognizing the peptide DED, for 3 hours at 4°C. After incubation Sepharose washed 3 times with cold Tris-saline buffer was added to the buffer for application to the LTO-polyacrylamide gel and boiled 5 minutes for elution of proteins and subsequent analysis by the method of Western blotting with polyclonal anti-FLAG antibodies rabbit or monoclonal mouse antibodies to the peptide DED. In the result of the analysis was established, that while both protein - Rabutin-5-gamma and Rabatin-5 with C-terminal FLAG-epitope was present in the lysates (figb), only protein Rabatin-5-gamma, but not Rabatin-5 was associated with Protein G-Separate with immobilized antibodies to peptide DED (figure 5).
Using vector pDED to obtain a plasmid for the production of protein identification by peptide on the C-end
The cDNA fragment growth factor vascular endothelial-165 person (hVEGF-165), containing 16 base pairs 5'-noncoding region and the entire open reading frame was amplified in polymerase chain reccie with primers 5'-agctgaattcgggcctccgaaacc-3' and 5'-taatctagaccgcctcggcttgtcacat-3', which introduced a unique recognition sites of restriction endonucleases EcoRI and XbaI at the 5'- and 3'-ends of the cDNA, respectively, as well as the codon translation termination TGA replaced by TST, encoding a serine residue. Website position recognition restriction endonuclease XbaI at the 3'end of the cDNA was chosen so that the reading frame cDNA hVEGF-165 peptide DED in the vector pDED match. The resulting amplification product was treated with restriction endonucleases EcoRI and XbaI and Legerova in vector pDED processed by the same enzymes. After transformation into cells of E. coli strain XL1Blue MRF' (Stratagene) were inoculated on selective medium containing IPTG and X-gal. Colonies of E. coli containing CL is nerosannow in vector pDED cDNA hVEGF-165, were identified by the white color, and the presence of the insert in the plasmid was confirmed by restriction analysis of isolated plasmid DNA. Then cDNA encoding hVEGF-165 C-konzum identification peptide DED, was subcloned sites EcoRI and NotI in the vector for expression of proteins in eukaryotic cells (plasmid pVEGF-DED).
Products hVEGF protein-165 identification peptide DED located on the C-end, and the demonstration of its identification with monoclonal antibodies specifically recognizing the identification peptide DED
Protein hVEGF-165 is a secretory protein and its secretion is caused by the presence of a signal peptide at the N end of the precursor protein. For production of the protein hVEGF-165-DED into the culture medium of the cell line Cho were transliterowany the plasmid pVEGF-DED. 24 hours after transfection the medium was replaced with medium containing serum (serum-free medium). Cells were incubated in serum-free medium for 48 hours, after which the medium was collected and filtered through a membrane with a pore diameter of 0.22 μm to remove cells and debris. Proteins from the resulting conditioned medium was concentrated by precipitation with methanol. To the aliquot of medium was added to 3 volumes of methanol, and after incubation overnight at -20°C. proteins were precipitated by centrifugation and dissolved in the booth is d for application to the LTO-polyacrylamide gel. Aliquots of conditioned medium and precipitated from her proteins were analyzed using Western blot analysis with a monoclonal antibody that specifically recognizes the peptide identification DED. For control used certified environment and precipitated proteins from it nitrostilbene cell line Cho. Antibodies to peptide DED has detected a protein with a molecular weight of approximately 25 kDa, which corresponds to the molecular weight of the protein hVEGF-165 produced by eukaryotic cells, only in the conditioned medium of cells transfected with plasmid pVEGF-DED and precipitated proteins from it, but not nitrostilbene cells (figa), indicating that production of the protein containing the peptide identification. To confirm that the protein containing the identification peptide is indeed hVEGF-165, the same samples were analyzed using Western blot analysis with antibodies specifically recognizing the protein of hVEGF-165. When using antibodies against protein hVEGF-165 was revealed the same type of coloring, and antibodies to the peptide DED (figb), suggesting that the protein containing the peptide identification DED, is a protein hVEGF-165.
1. Identification peptide characterized by the amino acid sequence Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln.
2. Nucleic acid encoding the amino acid posledovatelno the ü identification of the peptide according to claim 1.
3. The use of the peptide according to claim 1 for the identification of recombinant proteins.
4. The use according to claim 3, whereby the peptide is C-end N-end of the recombinant protein.
5. The use according to claim 3, whereby the peptide is a part of the recombinant protein in any position other than the C-end N-end.
6. The use according to claim 3, and identification of recombinant proteins by Western blotting.
7. The use according to claim 3, and identification of recombinant proteins is carried out immunocytochemical methods.
8. The use of the peptide according to claim 1 for the purification of recombinant proteins.
9. The use of claim 8, where the peptide is C-end N-end of the recombinant protein.
10. The use of claim 8, whereby the peptide is a part of the recombinant protein in any position other than the C-end N-end.
11. The use of claim 8, and purification of recombinant proteins is carried out using the method of Western blotting.
12. The use of claim 8, and purification of recombinant proteins is carried out using immunocytochemical methods.
13. The use of claim 8, and when the purification of the recombinant proteins, monoclonal antibodies immobilized on the stationary phase.
14. Expressing the vector pDED-based vector pBluescript SKII(+) to obtain a recombinant protein comprising the peptide according to claim 1, p is item the specified vector contains the nucleotide sequence according to claim 2, supplemented nucleotide sequence ggagga that encodes a dipeptide of glycine, at the 5'-end and flanked by restriction sites of the vector pBluescript SKII(+) bI at the 5'end and NotI at the 3'-end.
SUBSTANCE: invention refers to genetic engineering and can be used in medicine. The mucosal vaccine contains effective amount of hybrid protein consisting of oncoprotein E7 of human papilloma virus fused with heat-shock protein of mycobacteria Hsp70, chitosan related to hybrid protein 1:0.1-10 and additives pharmaceutically acceptable manufacturing of suppositories. The mucosal vaccine is used in therapy of the diseases associated with human papilloma virus.
EFFECT: possibility for multiple improvement of clinical effectiveness of diseases associated with human papilloma virus, considerable reduction of treatment cost in comparison with common techniques of treating cervical carcinoma and "РПК"; elimination of injection by-effects undesirable and extremely dangerous for the patent's life, eg anaphylactic shock, owing to local application; simplification of medical process - the patient can receive medical treatment out of clinic by independent introduction of the preparation.
6 cl, 4 dwg, 2 tbl, 10 ex
SUBSTANCE: invention is related to preparation of protein, binding tumour necrosis factor (TNF), and may be used in medicine. Strain-producer of baculovirus BvG2RIgG is created with the help of recombinant plasmid DNA pFastBac-G2R-IgG with size of 6444 p.n. and molecular mass 4.18 mDa, which bears fragment of smallpox virus genome of strain India-1967, which codes protein that binds TNF, and fragment of human genome, which codes fragment of heavy chain of human antibody G. Produced strain produces soluble chimeric protein, which consists of smallpoz virus protein, which binds TNF, and fragment of heavy chain of human antibody G.
EFFECT: wider spectrum of new generation preparations intended for treatment of human diseases related to hyperproduction of tumour necrosis factor.
2 cl, 3 dwg, 1 tbl, 8 ex
SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.
EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.
16 cl, 3 tbl, 7 ex
SUBSTANCE: invention refers to biotechnology and can be used for screening of compounds with properties of agonists or antagonists of leptin receptor. There is disclosed method for detecting leptin receptor definition that provides contact of a candidate compound and cells expressing after contransfection with two expressing vectors, respectively, two fused proteins, one of which consists of a short isoform of leptin receptor (OBRs) and an energy donor protein (preferentially luciferase), and the second - of OBRs and an energy acceptor protein (preferentially GFP or its mutant form); measurement of energy transfer (BRET) between the fused proteins; comparison of its value to the relevant indicator measured in the same system, however without tested compound, and estimated result where higher energy transfer indicates binding of the candidate compound-candidate with leptin receptor. Prospective application of the invention is related to development of the preparations for prevention or treatment of disease wherein leptin or its receptor are involved.
EFFECT: development of effective method for detecting leptin receptor ligands.
4 cl, 15 dwg, 3 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.
EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.
3 cl, 1 tbl, 5 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.
EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.
41 cl, 4 dwg, 17 ex
SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.
EFFECT: improved pharmacokinetic properties of erythropoietin.
23 cl, 14 ex, 6 tbl, 11 dwg
SUBSTANCE: there is provided fused protein, including immunoglobulin chain and the molecule IL-7 or its fragment, displaying activity typical to IL-7, that is modified in comparison with IL-7 of wild type, where modification in IL-7 represents amino-acid residues in positions 70 and 91, that are glycated, and amino-acid residue in the position 116 is nonglycated. There is proposed pharmaceutical composition, containing described protein. There is provided the method fro production of described fused protein, including: host cell transformation by DNA, coding specified fused protein IL-7, host cell culture and collection of fused protein IL-7.
EFFECT: invention can be applied for treatment of disorders, accompanied by immune deficiencies.
27 cl, 16 dwg, 1 tbl, 10 ex
SUBSTANCE: according to the invention there is provided protein fused with albumine comprising two or more tandem-oriented polypeptides GLP-1 or its fragments containing (7-36 A8G)), nucleic acid molecules coding albumine proteins according to the investigation as well as vectors containing the specified nucleic acids, host cells transformed by vectors comprising the specified nucleic acids, methods of albumine protein preparation according to the invention and their application methods. Moreover, the present invention refers to pharmaceutical composition containing fused proteins of albumine, treatment of diseases, disorders and conditions using fused proteins of albumine according to the invention.
EFFECT: improved therapeutic polypeptide stability.
34 cl, 81 ex, 11 dwg
SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.
EFFECT: enlarging the application range of medicine.
SUBSTANCE: invention concerns biotechnology, specifically to oligomer compound of length 10-30 nucleic bases, and can be used in medicine. The oligomer compound represents a target-binding domain complementary to a segment of 10-18 sequential nucleotides of the region including base position 1459 (5') to 1476 (3') human mRNA Bcl-2 (HUMBcl2A (approach number M13994) in database GenBank), except maximum one inaccurate coupling with specified site of human mRNA Bcl-2 where said target-binding domain is described by formula: 5' - [(DNA/RNA)0-1-(LNA/LNA*)2-7-(DNA/RNA/LNA*)4-14-(LNA/LNA*)2-7-(DNA/RNA)0-1]-3'.
EFFECT: oligomer compound effectively reducing expression of human gene Bcl-2.
25 cl, 20 dwg, 3 tbl, 16 ex
SUBSTANCE: nucleic acid coding self-activated stability protein contains a limited site of NBS-LRR-gene extended from 5'-end of coding region of NBS-LRR-gene towards 5'-3' prior to NBS-domain, and NBS-LRR-gene is not TIR-NBS-LRR-gene.
EFFECT: plants containing self-activated stability protein gain pathogen resistance.
19 cl, 24 dwg, 3 tbl
SUBSTANCE: this invention is related to the field of biotechnology and may be used in production of various protein products with the help of recombinant DNA technology. New sequences of DNA are defined and generated, which are related to matrix attachment region, which are characterised by ability to improve producing of protein in eukaryotic cells.
EFFECT: methods are suggested for transfection of eukaryotic master cells, including new method of multiple transfection, based on use of active sequenes of DNA MAR according to invention and providing for substantial increase of recombinant protein expression level compared to similar cells transfected by traditional methods.
11 cl, 21 dwg, 9 tbl, 17 ex
SUBSTANCE: double-null line-fertility restorer Brassica napus is obtained for the cytoplasmic male sterility (CMS) Ogura, which is a radish introgression carrying a gene-fertility restorer Rfo cut out of the radish allele Pgi-2 and exchanged with gene Pgi-2 from Brassica oleracea characterised by female fertility, a good transport level of Rfo and a high vegetative capacity. In order to characterise the obtained line-fertility restorer a combination of markers PGIol, PGIUNT, PGIint, BolJon and CP418 is used.
EFFECT: line is distinguished by a good agricultural quality.
7 cl, 24 dwg, 3 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: expression of endogeneous reserve proteins in plant seeds is suppressed through transformation of plant cells using a genetic construct, which contains a seed-specific promoter, functionally linked with a DNA sequence which codes transcription factors of endogenous genes or part of these factors, including their different combinations. The transcribed target of the said sequence is capable of suppressing, slowing down or in some other way, lowering expression of reserve proteins of seeds in a plant cell. Further, this or another plant cell is transformed by the construct, which contains the same seed-specific promoter, functionally linked with the DNA sequence which codes the heterologous polypeptide of concern.
EFFECT: in a plant regenerated from the said cell, expression of endogenous mRNA is reduced, which enables accumulation of heterologous polypeptide in that plant.
25 cl, 5 dwg, 7 ex
SUBSTANCE: there is provided DNA that codes protein able to transform a compound of formula (II) specified in description of invention into a compound of formula (III) specified in description of invention with an electron transport system containing an electron donor. Protein is able to metabolise herbicides.
EFFECT: introduction of DNA to plants with an expression of the specified protein provides herbicide resistance thereto.
26 cl, 66 dwg, 35 tbl, 75 ex
SUBSTANCE: method involves deactivation of definite VGC2 DNA sequence of Salmonella typhimurium positioned between ydhE and pykF genes or its part containing at least 50 nucleotides, or the DNA version of at least 85% identity, representing VGC2 DNA of any microbe out of Salmonella aberdeen, Salmonella gallinarum, Salmonella cubana and Salmonella typhi.
EFFECT: obtainment of microbe with reduced adaptability to specific environmental conditions.
6 cl, 12 dwg, 8 ex
SUBSTANCE: according to the invention there is provided method of person's treatment in case of risk or made diagnosis of autoimmune disease or transplantate rejection, including antagonist GITR injection into the person, where antagonist prevents binding GITRL with GITR on excitatory T-cells. There is provided method of cell population proliferation inhibition, containing excitatory T-cells, including injection of antagonist GITR into the cell population, where antagonist prevents binding GITRL with GITR on excitatory T-cells. There is proposed the method of cell population suppression, containing excitatory T-cells, in the presence of CD4+ CD25+ suppressor cells, including injection of antagonist GITR into the cell population, where antagonist GITR prevents binding GITRL with GITR on excitatory T-cells. There is provided pharmaceutical composition, containing antagonist GITR and pharmaceutically acceptable carrier for autoimmune disorder treatment, inflammatory diseases and transplantate rejection, where antagonist GITR prevents binding GITRL with GITR on excitatory T-cells.
EFFECT: invention can be applied for treatment of disorders, appearing in the result of disarranged immune reactions.
16 cl, 33 dwg, 3 tbl, 16 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: invention relates to biotechnology and concerns protein of protease NS3/4A HCV or its biologically active fragment, which contains succession, in which residue of amino acid, corresponding to amino acid 156 of protease NS3/4A HCV of wild type, is not the residue of alanine and, not obligatorily, residue of amino acid, which corresponds to aminocacid 168 of protease NS3/4A HCV of wild type, is not asparaginic acid. Invention also relates to polynucleotide, coding said protein, as well as application of said protein in detection of HCV presence in biological sample, as well as in method of determination if the infection, mediated by HCV, in patient is resistant to medication against HCV, in estimation method, if the infected by HCV patient has lower sensitivity or susceptibility to VX-950, as well as in method of candidate examination for inhibitor or potential inhibitor of HCV.
EFFECT: expansion of arsenal of medications for HCV treatment.
68 cl, 17 dwg, 6 tbl, 13 ex
SUBSTANCE: invention relates to biotechnology and represents method of obtaining L-amino acid using bacteria of genus Escherichia, bacterium being modified in such way that activity of alkoholdehydrogenase, coded by gene adhE, is enhanced in said bacterium.
EFFECT: invention allows to obtain L-amino acids with high degree of efficiency.
19 cl, 5 dwg, 4 tbl, 20 ex
SUBSTANCE: present invention refers to medicine and concerns methods and compositions for gastroenteric upset therapy. Substance of the invention involves purified polypeptide of 14 amino acids for gastroenteric upset and condition therapy (including, e.g. gastroenteric motor disturbance, functional gastroenteric disease, gastroesophageal reflux disease (GERD), Crohn's disease, ulcerative colitis, inflammatory bowel disease. Additionally the invention involves method of guanylate cyclase activity improvement with using peptide specified above.
EFFECT: advantage of invention consists in extended application.
9 cl, 6 ex, 10 dwg