Peptide ded and its application for identification and/or purification of recombinant proteins, vector pded

FIELD: medicine.

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
Alagcu gcc gca gcgalanine
Argaga agg cgu cgc cga eggarginine
Asnaau aacasparin
Aspgau gacaspartic acid
Cysugu ugccysteine
Glncaa cagglutamine
Glugaa gagglutamic acid
Glyggu ggc gga gggglycine
Hiscau cachistidine
Ileau auc aua isoleucine
Leuuua uug cuu cue cua cugleucine
Lysaaa aaglysine
Metaugmethionine
Pheuuu uucphenylalanine
Proccu ccc cca ccgProline
Seragu agc ucu ucc uca ucgserine
Thracu ACC ACA acgthreonine
trmuaa uag ugathe stop codon
Trpuggtryptophan
Tyruau uactyrosine
Valguu guc gua gugvaline

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.

Examples

Example 1

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.

Example 2

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.

Example 3

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,').

Example 4

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).

Example 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).

Example 6

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.



 

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11 cl, 21 dwg, 9 tbl, 17 ex

FIELD: agriculture.

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

FIELD: medicine.

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

FIELD: medicine.

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

FIELD: medicine.

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

FIELD: biotechnologies.

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

FIELD: medicine.

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

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