Vegf binding v9 nanoantibody and method of obtaining said antibody, v9-coding nucleotide sequence and vector containing said sequence, endothelial cell proliferation inhibition method

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention describes a nucleotide sequence which codes the V9 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V9 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V9 nanoantibody, as well as use of the V9 nanoantibody for qualitative and quantitative determination of VEGF in a sample. Use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

EFFECT: invention discloses a monomer single-strand V9 nanoantibody which can bind and inhibit the human vascular endothelial growth factor.

7 cl, 7 dwg, 7 ex

 

The technical field of the present invention

The present invention relates primarily to the field of molecular medicine and is associated with Monomeric odnoapsidnymi nanoentities. In particular, the present invention relates to a new nanoparticle able to inhibit the activity of growth factor vascular endothelial (isoform A-165) person, and to use this nanoparticle for diagnostic and therapeutic purposes.

The prior art of the present invention

Growth factor vascular endothelial (VEGF) is an important signaling protein involved in vasculogenesis (the de novo formation of the circulatory system of the embryo) and angiogenesis (the growth of blood vessels from pre-existing vessels). From the title of this protein, the activity of VEGF best studied on cells of the vascular endothelium, although it has action and some other cells. For example, VEGF stimulates the migration of monocytes/macrophages, neurons, cancer cells, epithelial cells of the kidney. In vitro experiments have shown that VEGF stimulates mitogenic endothelial cells and migration of cells. VEGF is also a vasodilator and increases capillary permeability, for which the source was named factor vascular permeability.

The VEGF family includes the prototype member of the VEGF-A, placental growth factor (PIGF),VEGF-B, VEGF-C and VEGF-D. Convincing evidence suggests that while the creation and maturation of the vascular wall are highly complex processes that require concerted effects angiopoietins, platelet-derived growth factor B (PDGF-B) and other factors, the effect of VEGF-A is a limiting speed stage of normal and pathological blood vessel growth. Importantly, VEGF-C and VEGF-D regulate angiogenesis lymphatic vessels that emphasizes the unique role of this family of genes in controlling the growth and differentiation of some anatomical components of the vascular system.

VEGF is characterized by significant homology with chains a and PDGF. The gene encoding VEGF-A person, consists of eight exons separated by seven introns. Alternative splicing of exons leads to the formation of four major isoforms - VEGF121, VEGF165, VEGF189 and VEGF206, composed after removal of the signal sequence, respectively, of 121, 165, 189 and 206 amino acids. Alternative splicing regulates the bioavailability of VEGF. By this time gathered a lot of evidence that VEGF165 is the most physiologically competent isoform. Also in the regulation of the bioavailability of VEGF plays an important role in extracellular proteolysis. The plasmin is able to cleave VEGF165 IS VEGF189 and release biologically active product, consisting of the first 110 aminobenzene amino acids.

The main biological effects of VEGF in vitro and in vivo mediated by its interaction with specific receptors. Currently describes two main types of receptor tyrosinekinase role of receptors: VEGFR1 (FLT-1) and VEGFR2 (FLK-1 or KDR). In addition, it is believed that neuropilin can perform the functions of VEGFR3. Currently Central role in ensuring the biological functions of VEGF VEGFR2 plays. Signaling through VEGFR2 starts the processes of proliferation, migration, increases survival of endothelial cells, stimulates angiogenesis and vascular permeability [Ferrara N., K.J. Hillan, Gerber, H.P., Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat. Rev. Drug Discov. 2004; 3:391-400].

Due to the fundamental role of the growth and proliferation of endothelial cells in various human pathologies and the critical role of VEGF in this process it is desirable to develop a method of monitoring the activity of VEGF. This strategy really is practically useful and currently implemented in several different ways, based on blocking the mitogenic signal of VEGF in endothelial cells. These methods [Ellis L.M. & Hicklin D.J. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nature Reviews Cancer. 2008; 8:579-591] include the use of:

antibodies that neutralize the action of VEGF or its receptor through the your linking. In particular, (1) using humanized anti-VEGF monoclonal antibodies [international patent publication WO 98/45331] or Fab'-fragments (drugs bevacizumab (Avastin) and ransomes (Lucentis)), binding with VEGF leads to blocking the ability of VEGF to initiate the transmission of the signal through the corresponding receptor on the surface of endothelial cells, or (2) the use of own antibodies to VEGF, induced by immunization with antigen containing VEGF (conoidea vaccine), the principle of which is similar to the principle of humanized monoclonal antibodies [Rad F.H., Le Buanec h, Paturance S., Larcier P., P. Genne, Ryffel Century, Bensussan, A., Bizzini Century, Gallo R.C., Zagury D., Uzan G. VEGF kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases. Proc. Natl. Acad. Sci. USA. 2007; 104:2837-42];

inhibitors of tyrosine kinases with selectivity of action against the VEGF receptors (e.g., sorafenib and sunitinib). Their action blocks the effect of VEGF by inhibiting the activation of the receptor and subsequent intracellular signal transduction;

soluble forms of VEGF receptors or their hybrids. The action of such agents (e.g., VEGF-trap) based on the binding of VEGF, leading to depletion of free VEGF, capable of interacting with receptors on the cells.

The closest technical solution, which is the basis of the present invention can result in a humanized antibody against VEGF bevacizumab and Lucentis.

However, the basis of the present invention are not classical bivalent antibodies, which is bevacizumab, and small single-stranded nanoparticle, which have several advantages compared to classical antibodies for practical application in the field of diagnostics and therapy of diseases. Because nanoparticle (molecular weight of about 17 kDa) on the order of smaller traditional antibodies, they acquire a number of new positive qualities of practical significance. Polypeptide chain composed of nanoentities forms only one domain not containing the hydrophobic areas. This avoids problems with solubility and proper folding of molecules nanoentities when products prokaryotic cells and leads to significantly lower costs of production compared to traditional methods of obtaining therapeutic monoclonal antibodies in eukaryotic expression systems. Due to the smaller size of the polypeptide chain Manantial more resistant to temperature influences and pH, are stored at room temperature, while traditional multi-domain antibodies require special conditions of storage and transport at 4°C. due To a smaller size nanoparticle have a greater ability to penetrate tissue. Finally, nanoparticle allow the AGCO conduct genetic engineering manipulations for the purpose of subsequent products bespecifically of nanoentities or chimeras, which, in addition to nanoparticle, is another protein with desired properties.

The possibility of obtaining recombinant nanoentities with a given specificity is determined by the existence of the representatives of the family Camelidae functional and has a sufficiently wide range of recognition of the non-canonical antibodies. Non-canonical antibodies consist of a dimer of only one shortened heavy chain of immunoglobulin (without light chains), the specificity of recognition of which is determined by only one variable domain [Hamers-Casterman C., Atarhouch T., Muyldermans, S., Robinson, G., Hamers, S., Bajyana Songa E., Bendahman N., R. Hamers Naturally occurring antibodies devoid of light chains. Nature. 1993; 363:446-448]. Technical implementation of the selection nanoentities, which is a genetically engineered derivative antipersonnel domains of single-chain antibodies camel, based on a highly effective selection process antivirusnaya polypeptide exposed on the particle surface of filamentous phage ("phage display").

Method of phage display is a very effective and widely used technology for screening of large recombinant libraries of peptides and proteins expressed in the composition of the surface protein of filamentous phage [Brissette R & Goldstein N.I. The use of phage display peptide libraries for basic and translational research. Methods Mol. Biol. 2007; 383:203-13; Sidhu S.S. & Koide S. Phage display for engineering and analyzing protein interaction interfaces. Curr. Opin. Strct. Biol. 2007; 17:481-7]. One particularly important application of this technology is the generation of specific recombinant antibodies to various antigens [Hoogenboom H.R. Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 2005; 23:1105-16].

Usually instead of a great many molecules of classical antibodies for display on the surface of phage use a hybrid recombinant single-chain proteins, which represents a random combination of the cloned sequences of the variable regions of the heavy and light chains of immunoglobulins, United short rich in serine and glycine linker sequence. Such a chimeric molecule, if the correct combination of domains, are able to maintain the specificity of the original immunoglobulin, despite entered compared to the native antibody molecule changes. One of the problems with conventional recombinant technology is the need to work with very large libraries of recombinant antibodies, which should be represented all possible combinations of two random variable regions (heavy and light chains of immunoglobulins), United linker sequence. Aside from the issue of representation, there are also obvious and the problem of formation of the correct relative conformation of the two domains, and the problem of the solubility of the individual is variable domains, which often have a tendency to aggregation. These problems can be avoided when using nanoentities, as almost every clone variable domain of single-chain antibodies will in this case be sufficiently antigenocide specificity, corresponding to one of the antibody immunized animal, and can now be selected from a relatively small library of such domains.

Nanoparticle with a given specificity or their derivatives can be used as classical antibodies, in various applications, including, without limitation, detection of antigens (for both research and diagnostic purposes), blocking the activity of a protein antigen-specific delivery by binding to the desired antigen molecules, conjugated with the antibody.

Brief description of drawings

Figure 1. The products of amplification of the cDNA fragments encoding the variable domains of antibodies (arrows)separated by agarose gel containing ethidium bromide (lane P). On the right (lane M contains molecular weight markers indicating their lengths in pairs of nucleotides (BP).

Figure 2. The characteristic pattern of cleavage of plasmid pHEN4 containing the sequence encoding nanoparticle V9, restriction endonuclease HinfI (lane V). To the right (on the cone M) shows the molecular weight markers indicating their lengths in pairs of nucleotides (BP).

Figure 3. Schematic representation of recombinant nanoparticle V9 encoded by plasmid based on modified vector pHEN6. Specify leader sequence periplasmatic localization (peIB), nanoparticle (V9) and (His)6-epitopes.

Figure 4. Cleaning nanoentities V9. Shows the gel electrophoresis in polyacrylamide gel with the addition of LTOs periplasmatic extract of E. coli cells producing nanoparticle V9 with ON - and (His)6-epitopes (1), not contacting Ni-NTA-agarose protein extract (2), proteins dissociate from the Ni-NTA-agarose during the rinse (3) and erwerbende with Ni-NTA-agarose purified nanoparticle V9 in the amount of 1 μg (4). The position of nanoparticle V9 in the gel shown by the arrow. On the right marked 1 µg commercial preparation of lysozyme (5) and protein standards with molecular weights (M). The gel stained with the dye Coomassie Brilliant Blue.

Figure 5. Nanoparticle V9 specifically binds VEGF in enzyme-linked immunosorbent assay. Shows the wells after addition of the chromogenic substrate. 1 - hole without immobilized protein (only blocking solution BSA), 2 - hole with immobilized VEGF, 3 - hole with immobilized immunoglobulins.

6. The proof of the strict relationship between the luminescence intensity and the number of HUVEC cells.

7. Nanoparticle V9 blocks VEGF-dependent proliferation of endothelial cells HUVEC. Cells UVEC subcultured in wells of 96-hole tablet (2000 cells per well) in basal medium for growth of endothelially cells computers-2 (Lonza, USA)containing 2% fetal bovine serum. After attachment of the cells (after 4 hours) was added VEGF to the indicated concentrations (A) or to a concentration of 100 ng/ml (B) and nanoparticle to a concentration of 12.5 μg/ml or Avastin (Ava) to a concentration of 6 µg/ml of cell Proliferation was determined after 96 hours using a set CeliTiter Glo Luminescent Cell Viability Assay (Promega, USA). The data are presented in relative luminescence units as the average of three independent wells±standard deviation.

The disclosure of the present invention

The present invention relates to the recombinant nanoparticle V9, which can specifically bind to VEGF and neutralize its mitogenic activity. The term "VEGF" refers to the 165-amino acid growth factor vascular endothelial And human. The term "nanoparticle" means that the protein, which is the subject of the invention is recombinant variable domain of single-chain antibodies camel.

To obtain a library of single-chain variable domains of antibodies two-humped camel were immunized VEGF or a mixture of proteins containing VEGF.

After immunization of peripheral blood of immunized camel was isolated b-lymphocytes and used them as a source of RNA for cloning of the entire repertoire of variable domains of specific immunoglobulins camel, consisting of a dime is only one heavy chain.

Isolated RNA was used as template in polymerase chain reaction (PCR)combined with reverse transcription, with pairs of primers corresponding to conservative RNA sequences of the heavy chains of immunoglobulins between the signal peptide and the constant plot CH2-domains [Hamers-Casterman et al., 1993; V.K. Nguyen, A. Desmyter, S. Muyldermans Functional heavy-chain antibodies in Camelidae. Adv. Immunol. 2001; 79:261-96; Saerens, D., J. Kinne, Bosnians E., U. Wernery, Muyldermans s, Conrath K. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J. Biol. Chem. 2004; 279:51965-72; Rothbauer, U., K. Zolghadr, Tillib, S., Nowak D., Schermelleh L, A. Gahl, N. Backmann, Conrath K., Muyldermans, S., M.C. Cardoso, H. Leonhardt Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods. 2006; 3:887-9].

The obtained amplification products were separated in agarose gel and isolated from the gel of the PCR products with a size of 600-800 BP, corresponding to the non-canonical antibodies [Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Edition, 1989, Cold Spring Harbour: CSHL Press]. The selected amplification products were used as template in PCR with pairs of primers corresponding to the conservative sequences at the beginning and at the end of this actual variable (indigenousness) domain [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006}. Used in this reaction, the primers also contained an additional sequence corresponding to the areas of recognition, restriction what's endonucleases, accordingly Ncol and Notl.

The obtained amplification products were cloned sites Ncol and Notl in formigny pHEN4 vector [Ghahroudi M.A., Desmyter, A., Wyns L., Hamers R, Muyldermans S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997; 414:521-526], encodes a protein of resistance to ampicillin, for phage display [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006].

Selection of nanoentities, specifically recognizing VEGF, was performed by the method of phage display using immobilized recombinant VEGF (Peprotech), as described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006]. The procedure of selection and subsequent amplification of selected phage particles containing the gene of nanoparticle inside, and expressed nanoparticle in the composition of the surface of phage protein pIII) was repeated, usually three consecutive times. Resulting from this procedure clones nanoentities investigated in terms of their diversity and the required specificity.

Sequences of clones selected nanoentities found in plasmids pHEN4, were grouped according to the similarity of their paintings electrophoretic separated hydrolysis products often sepasi restriction endonucleases. 2-3 representative of each group were used for perchlorovinyl in another expression plasmid vector, which provides connection of the With-end nanoparticle (His)6 epitope.

Thus, the present invention also relates to an expression vector containing the nucleotide sequence encoding nanoparticle V9, and providing his expression with additional(and) epitope(s) on the s-end for detection and selection, and signal peptide at N-end.

Due to the presence of N-end expressed sequence signal peptide (pelB) accumulating recombinant protein (nanoparticle) accumulates in periplasm E.coli that can effectively allocate its method of osmotic shock, do not actually destroying bacterial cells. Recombinant nanoparticle was isolated from periplasmatic extracts using affinity chromatography on a column of Ni-NTA-agarose.

Thus, the present invention also relates to a method of obtaining nanoparticle V9, which includes the following stages:

a) transfection of cells-producer expression vector containing the nucleotide sequence encoding nanoparticle V9, and providing his expression with additional(and) epitope(s) on the s-end for detection and selection, and signal peptide at the N-end;

b) expression of nanoparticle V9 in the cell-producer;

C) isolation of nanoparticle V9 from cell producer.

Dedicated nanoparticle used to analyze their ability in order to nawat preferably VEGF enzyme-linked immunosorbent assay with immobilized recombinant VEGF. Detection contacting VEGF nanoentities was performed using anti-mouse antibodies, secondary antibodies to mouse IgG conjugated with horseradish peroxidase and a chromogenic substrate ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, Sigma).

In another preferred embodiment, nanoparticle V9 is the killer. The term "neutralizing" means that nanoparticle can specifically bind to VEGF and significantly inhibit or cancel the mitogenic activity of VEGF on endothelial cells. Neutralizing nanoparticle of this invention is particularly applicable for prophylactic or therapeutic treatment of unwanted proliferation of endothelial cells and neoangiogenesis by delivery nanoparticle to endothelial cells, the proliferation of which it is desirable to inhibit. Pharmacologically acceptable form, method of delivery and dose nanoparticle V9, administered for preventive or therapeutic purposes, will be determined by a number of factors, including the type of disease and its severity, the tolerance input nanoparticle and the required number of nanoparticle for neutralization of VEGF in the selected route of administration. Confirmation of the existence of this feature is the effectiveness of the drug Avastin, which is a neutralizing VEGF to assicheskie antibody.

The plasmid encoding nanoparticle V9, was isolated from cells of E. coli and nucleotide sequence (SEQ ID NO: 1)encoding nanoparticle was determined by sequencing.

On the basis of nucleotide sequence that encodes nanoparticle V9, determined the amino acid sequence of nanoparticle V9 (SEQ ID NO: 2).

Thus, the present invention also relates to a method of inhibiting proliferation of endothelial cells, providing for the delivery of nanoparticle V9 to endothelial cells, the proliferative activity of which it is desirable to inhibit. Such inhibition may be necessary for prophylactic or therapeutic treatment of diseases in which there is excessive proliferation of the endothelium.

For diagnostic applications, which is another object of the present invention, nanoparticle V9 containing the epitope for detection, can be detected directly using an anti-antibody if the antibody conjugated with horseradish peroxidase or other component that allows the detection, including other enzymes (e.g., phosphatase) and fluorophores, or anti-antibody can be detected using the appropriate secondary antibodies for detection. Nanoparticle V9 can be used in any known method of detection, including direct and nepr is my ELISA, competitive binding, immunofluorescent detection, immunoprecipitation, Western-blotting.

Examples

Example 1

Obtaining a library of single-chain variable domains of antibodies

Immunization

Bactrian camel Camelus bactrianus consistently were immunized 5 times. As the antigen used concentrated conditioned medium of a cell line of Cho, transtorno producing VEGF. For this purpose, the cells were transfusional using reagent Unifectin-56 (Transfection Group, Russia) a plasmid containing cDNA of VEGF under the control of the CMV-promoter. 24 hours after transfection cells were washed in phosphate-saline buffer (PBS) and to obtain conditioned medium containing VEGF, were incubated in medium without serum for 3 days. Conditioned medium was collected, filtered through PES filter with a pore diameter of 0.2 μm (Corning-Costar, USA) and concentrated using a submersible cartridge Immersible CX-10 (Millipore, USA) with a nominal pore size of 10 kDa. The amount of VEGF in the concentrated conditioned medium was determined by staining protein in LTO-polyacrylamide gel dye, Kumasi. For each injection took conditioned medium containing approximately 300 μg of antigen in a final volume of 7.5 ml, brought PBS. The first injection was performed with antigen mixed with complete Freund adjuvant in a 1:1 ratio. Then the antigen mixed with incomplete and what Juventa Freund (1:1) and carried out consistently for another 4 injections, respectively, by 1 month and three times in 2 weeks. Blood samples (150 ml) was performed 5 days after the last injection. To prevent clotting of blood was added to the heparin 35 units/ml and EDTA (2 mm).

The selection of b-lymphocytes

The blood was diluted 2 times the standard saline solution (PBS)containing 1 mm EDTA. 15 ml-step special medium (Histopaque-1077, Sigma) with a density of 1.077 g/ml was layered 35 ml of diluted blood were centrifuged for 20 min at 800 g. Mononuclear cells (lymphocytes and monocytes) was collected from the interphase zone plasma/Histopaque, then washed with a solution of PBS containing 1 mm EDTA.

The allocation of RNA from b-lymphocytes

Total RNA from b-lymphocytes were isolated using TRIzol reagent (Invitrogen). Then poly(A)-containing RNA was isolated by a column of oligo(dT)-cellulose from the total RNA. The concentration of RNA was determined using biophotometer (Eppendorf) and the quality of the selected RNA was checked by electrophoresis in 1.5%agarose gel with formaldehyde.

The reaction of reverse transcription: the synthesis of cDNA on the matrix of poly(A)+RNA isolated from b-lymphocytes

The reverse transcription reaction was performed in 40 μl according to the standard Protocol [Sambrook et al., 1989] using reverse transcriptase inhibitor H - M-MuLV, 1 μg RNA and 1 μg of primer oligo(dT)15 as a seed.

Amplification of fragments of the DNA, encoding the variable domains of antibodies

The products of reverse transcription (1 μl) was used as template in polymerase chain reaction volume of 50 µl, containing two primers CALL001 (5'-gtcctggctgctcttctacaagg-3') and CALL002 (5'-ggtacgtgctgttgaactgttcc-3') in an amount of 20 pmol of the following conditions: 95°C, 90 C, (95°C 30 sec, 59°C for 120 s, 72°C - 90)×30 cycles, 72°C., 300 C. amplification Products were separated in agarose gel containing ethidium bromide (figure 1). Amplification products by size of 600-800 BP, corresponding to the non-canonical antibodies, isolated from the gel using the kit QIAEX II (QIAGEN, USA) and was used as a matrix in the same amplification reaction with primers 5'-ccagccggccatggctgatgtgcagctggtggagtctgg-3' and 5'-ggactagtgcggccgcttgaggagacggtgacctgggt-3', containing an additional sequence corresponding to the recognition sites of restriction endonucleases, respectively Ncol and Notl.

Creating a library of single-chain variable domains of antibodies

The obtained amplification products were cloned sites Ncol and Notl in formigny pHEN4 vector and using as phage-helper phage M13KO7 (New England Biolabs, USA)were phage library with surface expression of single-chain variable domains of the antibodies as described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006].

Example 2

Selection of nanoentities, specifically recognizing VEGF

Selection of NAS of the antibodies was performed by the method of phage display using recombinant VEGF (Peprotech), immobilized at the bottom of the holes 96-well ELISA plate. Used polystyrene immunological tablets with high sorption MICROLON 600 (Greiner Bio-One). To block used 1%BSA (Sigma-Aldrich, USA) and/or 1%nonfat milk (Bio-Rad, USA) in PBS. The procedure of selection and subsequent amplification of selected phage particles containing the gene of nanoparticle inside, and expressed nanoparticle in the composition of the surface of phage protein pill) was repeated, usually three consecutive times. All manipulations were performed as described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006].

Example 3

Identification of nanoparticle V9

Sequences of clones selected nanoentities found in plasmids pHEN4, were grouped according to the similarity of their paintings electrophoretic separated hydrolysis products of restriction enzyme Hinfl. One of the groups of clones were characterized by restriction pattern shown in figure 2. The cDNA sequence of nanoentities were defined for the three members of this group were found identical and coding the same nanoparticle named V9 (SEQ ID NO:1 and 2).

Example 4

Products nanoparticle V9

cDNA of nanoparticle V9 was periglomerular in the expression plasmid vector is a modified vector pHEN6 [Conrath K.E., Lauwereys, M., Galleni, M., Matagne, A., Frère J.M., J. Kinne, L. Wyns, S. Muydermans Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob. Agents Chemother. 2001; 45:2807-12], to join With the end of nanoparticle polyhistidine (His)6 epitope (immediately following the ON-epitope encoded in the vector pHEN6). Due to the presence of N-end expressed sequence signal peptide (pelB) accumulating recombinant protein (nanoparticle) accumulates in periplasm bacteria, which enables him to select the method of osmotic shock, do not actually destroying the bacterial cell (figure 3). Products nanoentities was performed in E. coli (strain BL21). Expression was induced by adding 1 mm indolyl-beta-D-galactopyranoside and the cells were incubated with vigorous stirring for 7 hours at 37°C or overnight at 29°C. Nanoparticle V9 was isolated from periplasmatic extracts using affinity chromatography on Ni-NTA-agarose using the system for cleaning QIAExpressionist (QIAGEN, USA). Results purification of nanoparticle V9 is shown in figure 4.

Example 5

Nanoparticle V9 associated with VEGF

The ability nanoparticle V9 to bind VEGF was examined by the method of enzyme immunoassay with immobilized recombinant VEGF (Peprotech) according to standard Protocol. As control was used wells without immobilized VEGF and immobilized immunoglobulins (especificacion). Detection contacting VEGF nanoentities was performed using anti-mouse antibodies, secondary antibodies to mouse IgG conjugated with horseradish peroxidase and a chromogenic substrate ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, Sigma).

Figure 5 presents the results of the analysis, from which it follows that nanoparticle V9 specifically binds VEGF, but not immunoglobulins or BSA, used as a blocking agent.

Example 6

Nanoparticle V9 inhibits VEGF-induced phosphorylation of the receptor VEGFR2

The action of VEGF is realized through its binding to the primary receptor, VEGFR2, resulting in his autophosphorylation and activation [Meyer M, Clauss M, Lepple-Wienhues, A., Waltenberger J., H.G. Augustin, Ziche M, Lanz C., Büttner, M., Rziha H.J., Dehio C. A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases. EMBO J. 1999; 18:363-74]. Activation of the receptor VEGFR2 is required for VEGF-stimulated proliferation, chemotaxis and survival of endothelial cells in vitro and angiogenesis in vivo [Kärkkäinen M.J. & Petrova T.V. Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis. Oncogene. 2000; 19:5598-605; Rahimi N., Dayanir V., K. Lashkari Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J. Biol. Chem. 2000; 275:16986-92; Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors. Biochem. Soc. Trans. 2003; 31:20-4]. So autophosphorylate is the W VEGFR2 in response to the processing of VEGF can be seen as a measure of Pro-angiogenic actions of VEGF, and blocking VEGF-dependent autophosphorylation nanoentities V9 as proof of his neutralizes VEGF-a activity. VEGFR2 person transtorno expressed in cells NECK by transfection with an appropriate expression vector containing the full-size cDNA VEGFR2 person under the control of the CMV-promoter. 18 hours after transfection cells were washed in PBS and incubated in medium without serum for 24 hours. Then the cells were treated with 30 ng/ml VEGF in the medium without serum, 30 ng per point) or VEGF in the same concentration, preincubation for 60 min with 10 µg of nanoparticle V9. After 5 min the cells were literally and the degree of activation of VEGFR2 was assessed by the number of phosphorylated on tyrosine 951 VEGFR2 by the method of Western blotting with antibodies that specifically recognize phosphorylated on tyrosine 951 VEGFR2 (Cell Signaling, USA). Figure 6 shows that preincubate VEGF with nanoentities V9 leads to a significant suppression of VEGFR2 phosphorylation at tyrosine-951, which indicates the ability of nanoparticle V9 not only communicate, but also to inhibit the action of VEGF.

Example 7

Nanoparticle V9 blocks VEGF-dependent proliferation of endothelial cells

Endothelial cells HUVEC were seeded into wells of 96-hole tablet (2000 cells per well) in basal medium for growth of endothelial cells computers-2 (Lonza). After attachment of cells (che the ez 4 hours) was added VEGF concentrations up to 1, 1.5, 3 or 100 ng/ml and nanoparticle V9 to a concentration of 12.5 μg/ml or, as control, bevacizumab (Avastin) to a concentration of 6 µg/ml Number of cells was determined after 96 hours. To determine the number of HUVEC cells used set of CellTiter Glo® Luminescent Cell Viability Assay production company Promega (Madison, Wl, USA) according to the Protocol of the kit manufacturer (Promega Technical Bulletin Part# TB288). Generated in the reaction of the fluorescent signal is directly proportional to the number of cells per well (Promega Technical Bulletin Part# TB288). In addition, we carried out a study of linearity due to the number of HUVEC cells in well with the generated fluorescent signal for the range in the number of HUVEC cells, overlying the number of cells per well at the time of analysis. As can be seen in Fig.6, in the range from 250 to 5000 cells per well the luminescence signal linearly (r2=0,9935) is associated with the number of cells per well. Visible (figa)that at concentrations of VEGF 1-3 ng/ml nanoparticle V9, as used in the quality control of the drug Avastin, is able to inhibit the proliferation of HUVEC cells. This effect nanoparticle V9 on the proliferation of HUVEC cells is not due to their non-specific toxicity or toxicity of the impurities contained in the drug nanoentities, as in similar conditions with an excess of VEGF (100 ng/ml) in the presence of nanoentities not nablyudavshimisya proliferative activity of cells HUVEC (figb).

1. Nanoparticle V9, characterized by amino acid sequence SEQ ID NO:2 and capable of binding growth factor vascular endothelial person and block its action.

2. The nucleotide sequence encoding nanoparticle V9 according to claim 1.

3. The nucleotide sequence according to claim 2, representing the SEQ ID NO:1.

4. The expression vector containing the nucleotide sequence according to claim 2 and ensuring the expression of nanoparticle V9 with additional(and) epitope(s) on the s-end for detection and selection, and signal peptide at N-end.

5. The method of obtaining nanoparticle V9 according to claim 1, which includes the following stages:
a) transfection of cells-producer vector according to claim 4;
b) expression of nanoparticle V9 in the cell-producer;
C) isolation of nanoparticle V9 from cell producer.

6. Method of inhibiting proliferation of endothelial cells,
providing for the delivery of nanoparticle V9 according to claim 1 to the endothelial cells.

7. The use of nanoparticle V9 according to claim 1 as a diagnostic reagent for the qualitative and quantitative determination of VEGF in the sample.



 

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FIELD: medicine.

SUBSTANCE: immunological detection and quantitative analysis of sequential changes in protein levels of VEGF-165 in obtained patient's samples taken in course of time are conducted, where increasing levels of the protein VEGF-165 in course of time indicate progression of disease or adverse reaction to the therapy, and where decreasing levels of protein VEGF-165 in course of time indicate remission of the disease or a positive reaction to the therapy.

EFFECT: method enables to conduct noninvasive analysis of levels of circulating VEGF-165 which serves as a valuable prognostic indicator of disease outcome.

22 cl, 2 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to obstetrics and can be used in the 3rd trimester of pregnancy for predicting clinical effectiveness in severe gestosis. Substance of the invention consists in that starting from 28 weeks of pregnancy peripheral venous blood of a woman suffering from severe gestosis is analysed for the content of CD16+CD3-cells. If the related values are less than 9%, conventional therapy of severe gestosis is predicted as efficient, while the value 9% or more show the absence of clinical effectiveness in gestosis.

EFFECT: application of the method allows predicting effectiveness of conventional therapy in severe gestosis from 28 weeks of pregnancy with high accuracy, sensitivity and specificity that enable to determine approach to further prenatal care and delivery, to reduce risk of fetal pathology, maternal and perinatal mortality.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immunotechniques. Method of human blood serum testing for OncoMarker MUC1 is offered for diagnostics of breast cancer (BC) by direct solid-phase bivalent immune photometric analysis using pair of homogeneous antibodies M3F1/M3B11. Formed antigen-antibody complexes are detected using Mab-peroxidase conjugate followed by introduction of stain TMB, and standard OncoMarker is human fat lactoglobule antigen MUC1. Method allows for primary diagnostics of BC stages II and III and for prediction of secondary process development (relapses), and for efficient therapy in determined diagnosis. Application of declared method allows to detect 56-63% BC patients BC by blood serum analysis.

EFFECT: possibility of primary diagnostics of BC stages II and III and prediction of secondary process development (relapses).

4 ex, 2 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medical diagnostics. For purpose of acute bacterial enteric infections diagnostics, lymphocytic suspension is tested for acute enteric infection (AEI) agent antigen by indirect immunoperoxidase method. Centrifugated blood cells are applied on glass slide (smear), dried up at room temperature, fix in pure acetone, and then processed in 3% H2O2 for 20 minutes. Blood cells are incubated in blocking normal serum, incubated with poly- or monoclonal antibodies to required antigenes at t 37°C. Then they are processed with reagents of polymeric detection system: Super Enhacer™ Reagent and further with Poly-HRP Reagent. Then they are processed with 3,3-diaminobenzidine-tetrachloride (DAB). Brown granules in lymphocytes and monocytes indicate required antigen, namely infection diagnostics.

EFFECT: method application provides increased accuracy and reduced time of diagnostics of acute bacterial enteric infections.

3 dwg, 1 ex

FIELD: medicine, pediatrics, immunology.

SUBSTANCE: in patients' peripheral blood one should detect expression level of adhesion molecules of CD11β and CD54 lymphocytes due to indirect immunofluorescence technique at applying monoclonal antibodies and at the content of CD11β molecules being above 26.34% and CD54 molecules being above 33.26% one should detect active stage of acute pyelonephritis. The method is simple and of high information value. It enables to evaluate the values of leukocytic adhesion and their transendothelial migration quickly and objectively for 2.5 h after blood sampling, moreover, it is of high information value for proving the activity of microbial-inflammatory process in case of acute pyelonephritis and provides rational complex of therapeutic procedures in due time.

EFFECT: higher accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine, pediatrics, immunology.

SUBSTANCE: in patients' peripheral blood one should detect the expression level of CD95 lymphocytes markers due to indirect immunofluorescence technique and at the content of CD95 markers being above 21.31% one should detect chronic flow of pyelonephritis. The method is simple and of high information value. It enables to evaluate quantitative marker of affected mechanisms of apoptosis at chronic pyelonephritis quickly and objectively for 2.5 h after blood sampling, moreover, it is of high information value for differential diagnostics of chronic pyelonephritis and provides rational complex of therapeutic procedures in due time.

EFFECT: higher efficiency of diagnostics.

1 ex, 1 tbl

FIELD: clinical diagnosis, in particular in vitro determination of skin tuberculin high-grade sensitivity.

SUBSTANCE: skin tuberculin high-grade sensitivity is determined based on alteration of fluorescent intensity of common leucocytal CD45 antigen and isoforms thereof CD45RA and CD45RO in test probe (after incubation of peripheral blood with 2TE tuberculin solution) in contrast to control probe with physiological solution by using monoclonal antibodies labeled with fluorescein isothiocyanate (FITC) and laser flow cytofluorometry.

EFFECT: new method for in vitro determination of skin tuberculin high-grade sensitivity.

1 tbl, 4 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention proposes hybridomas producing monoclonal antibodies showing specificity to human epiregulin. Invention discloses monoclonal antibody recognizing specifically human epiregulin with the sensitivity limit 10 pg/ml. Also, invention describes methods for specific detection of human epiregulin in a sample in vitro and a method for detection of cells expressing human epiregulin in extracellular fluid in vitro by using monoclonal antibody to human epiregulin. Invention provides a simple and highly sensitive method for detection of human epiregulin that can be used in diagnosis of human epiregulin-expressing tumors.

EFFECT: valuable biological and medicinal properties of antibody and hybridoma.

8 cl, 7 dwg, 8 ex

FIELD: medicine, neonatology.

SUBSTANCE: in umbilical cord blood at the moment of a child's birth one should detect the ratio of the parameters for relative content of CD3+HLA-DR+ against CD3+ lymphocytes and at its value being equal to 8.1% or above it one should diagnose perinatal hypoxic CNS lesion at accuracy of 89.28%. Application of the present method enables to diagnose perinatal hypoxic CNS lesion in mature neonatals in gestosis-suffering women at earlier terms.

EFFECT: higher accuracy of diagnostics.

3 ex, 1 tbl

FIELD: immunology, biotechnology.

SUBSTANCE: invention relates to antibodies showing specificity to anomalous processed form of human tau protein that differs by conformation from the normal tau protein and doesn't bind with normal tau protein. Also, invention relates to conformational distinctive tau proteins ("tauones") and diagnostic and therapeutic aspects related to Alzheimer's disease and related taupathies. Proposed antibodies are produced by hybridomas DC-11 or Dc-11/1 deposited in ECACC at numbers 00082215 and 00082216. Also, invention described truncated forms of human tau protein that are truncated by N- and/or C-end and comprise amino acid residues from amino acid 300 to amino acid 400 in the longest isoform of human tau protein (441 amino acids residues). Above mentioned truncated forms of human tau protein can be recognized specifically by antibodies described above. Also, invention describes a method for assay of truncated forms of tau protein in a patient biological sample using a set comprising a proposed antibody and suitable container. Using the proposed invention provides a suitable target for medicinal preparations with early therapeutic effect used in Alzheimer's disease and other taupathies.

EFFECT: valuable medicinal properties of proteins.

11 cl, 15 dwg, 10 ex

FIELD: chemistry.

SUBSTANCE: method of producing diphtheria toxin or its mutant or fragment involves a fermentation step where the Corynebacterium diphtheriae strain is grown in a fermenter while stirring in order to maintain a homogenous culture and with limited aeration such that partial pressure of oxygen (pO2) in the culture falls to a level which is 4% lower on the bigger part of the fermentation step. Diphtheria toxin or its mutant or fragment is isolated from the culture. The invention also relates to a method of preparing a pharmaceutical composition for treating or preventing diphtheria, which includes a step for fermentative production of the toxin and mixing it with a pharmaceutically acceptable carrier after isolation.

EFFECT: use of the invention leads to high output of diphtheria toxin or mutant (for example, CRM 197), ensures high output of diphtheria toxin when the culture medium additionally contains iron or complex initial substances of varying quality.

20 cl, 6 dwg, 8 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of Marburg haemorrhagic fever. A strain of hybrid animal cells Mus musculus L. 3F9 is formed, which is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR. The hybridoma strain produces monoclonal antibodies which are specific to the VP35 protein of the Marburg virus (Popp strain) (hereinafter MCA). MCA 3F9 produced by hybrid animal cells Mus musculus L. 3F9 relate to a subclass of immunoglobulins IgGI, having a heavy 55 kDa and a light 25 kDa chain and having a unique feature of detecting the VP35 protein of the Marburg virus (Popp strain) in a "sandwich" immunoenzymometric system format owing to antigen "capture" properties and simultaneously be an indicator, labeled biotin. The antigen epitope for MCA 3F9 produced by the 3F9 hybridoma is localised between 252 and 278 aminoacid residues.

EFFECT: invention enables to obtain MCA with specificity to VP35 protein of the Marburg virus (Popp strain), suitable for immunodiagnosis of Marburg haemorrhagic fever.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of human cytomegalovirus. The strain of hybrid animal cells Mus musculus L.5F10 is obtained by merging mouse myeloma cells p3-X63/Ag8.653 (NS/1) with mouse spleen cells BALBc, immunised by an affinity purified recombinant protein pp65. The hybridoma strain is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR and is used as a producer of monoclonal antibodies for detecting the pp65 protein of human cytomegalovirus.

EFFECT: invention enables to widening of range of strains of hybrid cells Mus musculus L - producers of MCA for detecting the pp65 protein of human cytomegalovirus and production of domestic diagnostic test-systems for detecting cytomegaly.

2 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: strain of Propionibacterium freudenreichii subsp.shermanii Ac-103 is deposited into the Russian national Collection of microorganisms used in veterinary medicine and animal husbandry numbered VGNKI - 08/02/1957 DEP. This strain is a producer of feed protein.

EFFECT: invention enables to eliminate pollution at production of protein feed, to increase relative protein yield, to reduce energy costs at preparation of protein feed, to simplify medical equipment, to dispose wastes of manufactures using natural raw materials.

1 tbl, 9 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

3 dwg, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in agriculture when making feed. The Lactobacillus plantarum 578/26 strain is deposited in the collection of Russian State Centre for Quality and Standardisation of animal medicines and feed under number VGNKI 08.02.54.-DEP. This strain is a feed protein producer.

EFFECT: invention prevents environmental pollution when making protein feed, increases protein specific output, reduces power consumption during protein feed production, simplifies and speeds up the manufacturing process, simplifies the equipment, enables recycling production wastes used natural material.

2 tbl, 9 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to a process and apparatus for isolating and purifying protein of interest in a stream of a tissue culture liquid obtained from a continuous perfusion fermentation process. The proposed apparatus, in which sterile conditions are maintained, includes a continuous perfusion fermentation system, a continuous particle removal system integrated with the perfusion fermentation system and adapted for continuous reception of tissue culture liquid therefrom and continuous production of clarified tissue culture liquid, and a continuous purification system integrated with the particle removal system and adapted for continuous reception clarified tissue culture liquid therefrom and constant production of the extracted product which contain the protein of interest, where the continuous purification system is an ultrafiltration system. The process involves obtaining heterogeneous tissue culture liquid mixture containing the protein of interest during a continuous perfusion fermentation process, continuous removal of large particle impurities from the liquid mixture to obtain clarified tissue culture liquid containing protein of interest, and purification of the protein from the clarified culture liquid through ultrafiltration. Specific flow rate of the tissue culture liquid mixture during continuous perfusion fermentation, continuous removal of impurities and continuous purification is kept constant.

EFFECT: design of an efficient method of isolating and purifying protein.

9 cl, 17 dwg, 2 tbl

FIELD: biotechnologies.

SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.

EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.

17 cl, 1 dwg, 7 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: plasmid contains a DNA fragment encoding laccase C1 of ligninolytic fungus Trametes hirsuta or a DNA fragment hybridised with SEQ ID N0:1 in tough conditions, under control of promoter functioning in this cell. The resulting cell is a producer of laccase C1. The invention also refers to method of laccase obtaining using to the indicated cell.

EFFECT: obtaining laccase with high catalytic activity.

6 cl, 3 dwg, 1 tbl, 5 ex

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