Nanoantibodies amh1, amh2 binding antigen of mycoplasma homints, method of producing them, method of treating mycoplasma hominis infection
SUBSTANCE: there are presented antibodies specifically binding the lipid-associated antigen of M.hominis with the characterised amino acid and nucleotide sequences, as well as a method of treating a Mycoplasma M.hominis infection involving administering to said mammal a therapeutically effective amount of the nanoantibodies.
EFFECT: invention can find further application in treating the mycoplasma infection.
4 cl, 2 tbl, 6 ex, 5 dwg
The technical field of the present invention
The invention relates to the field of biotechnology and medicine, in particular the production and use of nanoentities to block the infection caused by pathogenic microorganisms, in particular Mycoplasma hominis.
The prior art of the present invention
Mycoplasmas (class Mollicutes) - the smallest pathogenic bacteria, the characteristic feature of which is the lack of a cell wall. The representatives of this family are the cause of various diseases of the respiratory and urogenital tract, and acute and chronic joint diseases in humans and animals [Prozorovsky SV, Rakovsky IV, 1995]. One of the features of mycoplasmas is their ability to persist in vivo for a long period without any clinical manifestations. This ability of mycoplasmas is due to a number of features that allow them to effectively "escape" from the immune system of the host. For mycoplasmas peculiar molecular mimicry and phenotypic plasticity, which leads to the fact that Mycoplasma is not effectively recognized and eliminated by the immune system of the host. This allows the mycoplasmas to persist in the host organism for a long time, which can often be the cause of chronic diseases [Barile M, 199, Razin, S., 1991, 1998]. Another characteristic of Mycoplasma is their ability for long-term persistence in vitro in cultures of eukaryotic cells. Being obligate membrane parasites, they are stored in cell cultures for decades.
At the present time shows that of the 14 species of Mycoplasma, the natural host is human, five species (M.hominis, M.pneumoniae, M.genitalium, M.fermentans and U.urealyticum) associated with various human diseases. The fight against these infections is very difficult due to emerging resistance to antibiotics and the characteristics of the responses of the organism [Prozorovsky, 1995].
Clinical manifestations of Mycoplasma infection are not specific and very diverse. In many cases, Mycoplasma cause latent infection. Under the influence of various factors causing latent infection can become chronic or recurrent acute [Prozorovsky, 1995]. Currently, it is believed that the pathogens causing chronic diseases associated with chronic inflammation, is one of the key factors influencing tumor progression [Coussens L., 2002, DeMarzo, A., 2004, 2007, Gupta, S., 2004, Karin M., 2006, Nelson W., 2003, Platz, E., 2004, K. Sfanos, 2008, Sutcliffe, S., 2008, F. Wagenlehner, 2007].
Mycoplasma hominis causes about half of nongonococcal infections in Addition, in 70% of patients with gonococcal infections detected M.hominis. M.hominis associated with abnormalities of the urogenital tract in men and the upper part of the urogenital tract in women. For example, M.hominis is the main causative agent of nongonococcal urethritis, represtation, vaginitis, endometritis, an inflammation of the pelvic region, cervicitis, infertility, underweight newborns (Cassell et al. (1991) Clin. Perinatol. 18:241-262; Cassell et al. (1984) Adv. Exp. Med. Biol. 224:93-115; and Cassell et al. (1983) Sex. Transm. Dis. 10:294-302).
M.hominis refers to trudnosorbiruemye microorganisms, which makes it bacteriological diagnosis long-term (from 2 to 6 days) and expensive.
Due to the mentioned problems, it is desirable to develop a method for the treatment of mycoplasmal infection that is not associated with antibiotic use, as well as how the specific detection of Mycoplasma different from the classical culture method.
As the nearest analogues technical solutions that form the basis of the present invention, can result:
1) human monoclonal antibodies against bacterial toxins, described in the patent ("Human Monoclonal Antibodies Against Bacterial Toxins - US Patent 4689299"). The patent on obtaining and production of hybrid cell lines secreting monoclonal antibodies against bacterial toxins, and method of using the obtained monoc the national antibodies to protect against bacterial (diphtheria and tetanus) toxins;
2) monoclonal antibodies against Mycoplasma pneumoniae (Monoclonal antibodies against Mycoplasma pneumoniae, hybridomas producing these, methods for the preparation thereof and the use thereof - US Patent 5641638). In the patent described a monoclonal antibody able to specifically bind the antigen P2 Mycoplasma M.pneumoniae and cell hybridoma that produce these monoclonal antibodies.
These technical solutions as the closest to the claimed composition active ingredient of the pharmaceutical composition and method of its use is selected by the authors of the present invention for the prototype.
The disadvantages of the prototype are:
1) no effective method of generation and selection of antibodies;
2) large size, which leads to lower permeability;
3) structural features do not allow to identify "hidden" epitopes of antigens;
4) the limitations of genetic manipulations, adaptation for specific tasks, the ability to create multivalent and multifunctional derivatives.
Thus, in the prior art there is an urgent need to develop effective specific antibodies against Mycoplasma, hominis (M.hominis).
The present invention was the creation of new antibodies able to bind antigens of Mycoplasma M.hominis and block multiplication of this bacteria.
Postavljena problem is solved for MF is t, that creates nanoparticle, specifically linking the lipid-associated antigen M.hominis and having the amino acid sequence of SEQ ID NO:2. Create nanoparticle, specifically linking the lipid-associated antigen M.hominis and having the amino acid sequence of SEQ ID NO:4. Thus nanoparticle having the amino acid sequence of SEQ ID NO:2, suppresses the infection caused by Mycoplasma M.hominis, and also nanoparticle having the amino acid sequence of SEQ ID NO:4, and inhibits infection caused by Mycoplasma M.hominis. A method of treating infection in a mammal caused by Mycoplasma M.hominis is specified in the introduction to the mammal a therapeutically effective amount of nanoparticle having the amino acid sequence of SEQ ID NO:2. A method of treating infection in a mammal caused by Mycoplasma M.hominis is specified in the introduction to the mammal a therapeutically effective amount of nanoparticle having the amino acid sequence of SEQ ID NO:4.
The basis of the present invention are not classical bivalent antibodies, which are considered as a prototype and small nanoparticle, which have several advantages compared to classical monoclonal antibodies for practical use in treatment of diseases. Because nano is nitela (molecular weight of about 12-15 kDa) on the order of smaller traditional antibodies, they acquire a number of new positive qualities of practical importance. There are effective ways to get (and selection) of such antibodies against various (including netcommunity) antigens of Mycoplasma, and thanks to its low immunogenicity of nanoparticle can be used to treat infections caused by pathogens of this group.
Full equivalent of the term "nanoparticle" for the purposes of the present invention is subsumed into General use designation "nanotesla"entered the firm ABLYNX (NANOBODYTM), and "single-domain mini-antibody" and "a single domain nanoparticle".
Recombinant nanoparticle is obtained on the basis of a special non-canonical single-chain antibodies existing in the norm along with the classical antibodies in animals of the family of camelids (and in some species of cartilaginous fishes). These specific antibodies consist of a dimer of only one shortened (excluding the first constant region SN) the heavy chain immunoglobulin and fully functional in the absence of the light chain of the immunoglobulin. For the specific recognition and binding of the antigen when it is necessary and sufficient for only one variable domain (VHH, "nanoparticle", "nanobody" or a single domain nanoparticle) of this antibody. Organization variable domains (VHH) of the non-canonical antibodies largely dobna the that variable domains (VH) classical antibodies (human VH domains of antibodies of the IgG3 subclass are particularly pronounced homology to the VH and VHH camelids). In both cases, V-domains consist of four conservative frame sections (FR, "framework regions")surrounding the three hypervariable segment (define complementarity, CDR, from a "complementarity determining regions"). In both cases, the domains form the typical V-domain immunoglobulin spatial structure of the two beta-layers (sheets), one of four amino acid chains, and the second of the five [E.A. Padlan X-Ray crystallography of antibodies. Adv. Protein Chem. 1996; 49: 57-133. Muyldermans, S., Cambillau, C., L. Wyns Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains./ "Recognition of antigens by single-domain fragments of antibodies: superfluous luxury of paired domains. TIBS 2001; 26: 230-235]. In this structure, all three hypervariable area are clustered on one side of the V-domain (where they are involved in antigen recognition) and are located in the loops connecting the beta-structure. However, there are important differences relating to the operation of VHH in the format of a single domain. So, hypervariable sites CDR1 and CDR3 significantly increased in the case of VHH. Often in hypervariable sites VHH detected cysteine residues, and are present in two areas (mostly in CDR1 and CDR3, at least - in the CDR2 and CDR3). In the study of cristalli the definition of VHH structures have been shown these cysteine residues form a disulfide bond, which leads to an additional stabilization of the structure of loops of a given antigen. The most explicit and reproducible hallmark VHH are four substitutions of hydrophobic amino acid residues on the hydrophilic second frame section (Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly, numbered according to Kabat). This framed area in the case of the VH domain is highly conserved, enriched in hydrophobic amino acid residues and are particularly important for the formation of ties with variable domain VL light chain. VHH domain in this respect is very different: the replacement of hydrophobic amino acids on the hydrophilic make it impossible for the Association VHH and VL. These replacements also explain the high solubility VHH, nanoparticle, when it is obtained in the form of recombinant protein [Tillib SV "Camel nanoparticle" is an effective tool for research, diagnostics and therapy. Molecular biology 2011; 45(1): 77-85].
Compared with the traditional and pure recombinant antibodies camel nanoparticle have a number of advantages, suggesting a great potential for their future use in various research and in the creation of new biotechnological devices, as well as for clinical purposes for the diagnosis and treatment of diseases.
Characteristic especially the authorities of nanoentities, defining a great potential for their use for a variety of practical applications in immunobiotechnology, are the following [see review: Tillib SV "Camel nanoparticle" is an effective tool for research, diagnostics and therapy. Molecular biology 2011; 45(1): 77-85]:
1) a highly efficient method of generating and breeding nanoentities;
2) small size, ~2×4 nm, 13-15 kDa (enhanced permeability of the cell);
3) structural features (the ability to form unusual for classical antibody paratope that allows to communicate with the recesses and the active centers of proteins); can be used to identify "hidden" epitopes or epitopes, which can not be recognized significantly larger conventional antibodies;
4) high expression yield, efficiency developments in large quantities. Usually nanoparticle develop in periplasm bacteria E.coli (1-10 mg from 1 liter of culture). The possibility of their effective practices in yeast, plants, and cells of mammals;
5) simplicity of various genetic manipulations, adaptation for specific tasks, the ability to create multivalent and multifunctional derivatives;
6) low immunogenicity; the opportunity cost to "humanize" the antibody without noticeable is Oteri their specific activity.
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, et al. Naturally occurring antibodies devoid of light chains./ "Existing in nature antibodies without light chains". Nature 1993; 363:446-448]. Technical implementation of the selection nanoentities, which is genetically engineered derivatives of the antigen-recognition domain of single-chain antibodies camel, based on a highly efficient procedure for the selection of antigen-binding polypeptide exposed on the particle surface of filamentous phage ("phage display").
Method of phage display is a very effective and widely used technology for functional screening of large recombinant libraries of DNA sequences encoding peptides and proteins with desired properties and expressed in the composition of the surface protein of filamentous phage [Brissette R & Goldstein NI. The use of phage display peptide libraries for basic and translational research. /"Using peptide phage-display libraries for pound the metal of research and research translation. Methods Mol Biol. 2007; 383:203-13; Sidhu SS & Koide S. Phage display for engineering and analyzing protein interaction interfaces./ "Phage display for design and analysis of interactions of protein domains". Curr Opin Struct Biol. 2007; 17:481-7]. One particularly important application of this technology is the generation of specific recombinant antibodies to various antigens [Hoogenboom HR. Selecting and screening recombinant antibody libraries. / Selection and analysis of libraries of recombinant antibodies". 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 serine/glycine-rich 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 link the nuclear biological chemical (NBC sequence. Aside from the issue of representation here is clear and the problem of formation of the correct relative conformation of the two domains, and the problem of the solubility of the individual variable domains, which often have a tendency to aggregation. The above-mentioned problems can be avoided when using nanoentities, as almost every clone variable domain of single-chain antibodies will in this case have a certain antigen-binding 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, but not limited to, 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. Also nanoparticle can be a source modules (blocks) more complex multimodal drugs. It is possible to unite in one multivalent derived two, three or more monovalent primary nanoentities. These combine into a single structure nanoparticle can bound yatsa with the same epitope of the target antigen, and with its different epitopes or even with different antigens on target. It is also possible combo unite into one design nanoentities and other molecules or drugs with obtaining multifunctional drugs [Conrath KE, Lauwereys M, Wyns L, Muyldermans S. Camel single-domain antibodies as modular building units in bispecific and left-hand drive vehicles antibody constructs. / "Camel single-domain antibodies as modularly building units in bispecific and bivalent structures of antibodies". J Biol Chem. 2001 Mar 9; 276 (10): 7346-50; Zhang J, Tanha J, Hirama T, Khieu NH, To R, Tong-Sevinc H, Stone E, Brisson JR, MacKenzie CR. Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. / Pentaerythrit single-domain antibodies from phage libraries: a new strategy for rapid receipt reagents antibodies with high avidity". J Mol Biol. 2004 Jan 2; 335 (1): 49-56; Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, Revets H. Efficient cancer therapy with a nanobody-based conjugate. / Effective cancer therapy conjugates on the basis of Manotel". Cancer Res. 2004 Apr 15; 64 (8): 2853-7; Baral TN, Magez S, Stijlemans B, Conrath K, Vanhollebeke B, Pays E, Muyldermans S, De Baetselier P. Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor. / "Experimental therapy of African trypanosome using human trypanosomiasis factor, conjugating with nanotesla". Nat. Med. 2006 May; 12 (5): 580-4; Coppieters K, Dreier T, Silence K, Haard HD, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Wiele CV, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factoralpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. / Rich anti-TNFalpha VHH proteins isolated from camelids, demonstrate a high affinity to the inflamed joints in a murine model of collagen-induced arthritis". Arthritis Rheum. 2006 Jun; 54 (6): 1856-66]; multimerization with the introduction of additional amino acid sequences of the interacting protein domains, such as lacinova the zippers [Harbury W., Zhang T., Kim P.S., et al. A switch between two-, three - and four-stranded coiled coils in GCN4 leucine zipper mutants. / "D between two-, three - and four-chain coiled structures in mutants GCN-"latinboy lightning". Science, 1993, 262:1401-1407; Shirashi T., Suzuyama k., Okamoto, H. et al. Increased cytotoxicity of soluble Fas ligand by fusing isoleucine zipper motif. / "Enhanced cytotoxicity of soluble Fas-ligand due to its connection with theme of "isoleucinol lightning". Biochem. Biophys. Res. Communic. 2004, 322: 197-202; Chenchik, A., Seth William page, A., Komarov, A., Natarajan V. Reagents and methods for producing secreted bioactive peptides. / "Reagents and methods for the production of bioactive secreted peptides". 2010. US Patent Application 20100305002] or sequences of small proteins that form stable complexes [Deyev SM, Waibel R, Lebedenko EN, Schubiger AP, Plückthun A. Design of multivalent complexes using the barnase*barstar module. / "The design of multivalent complexes using the module barnase-barstar". Nat Biotechnol. 2003, 21(12):1486-92].
It was also shown [Vincke, C., R. Loris, Saerens d, et al. // J. Biol. Chem. 2009. V.284. No. 5. P.3273-3284]that it is possible to "humanize" these camel nanoparticle without C matney loss of their specific activity, after a small number of point substitutions of amino acids. This opens up the potential widespread use of nanoentities as a means of passive immunization for prevention of various infectious diseases [J. Wesolowski, Alzogaray V., Reyelt J. et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. / "Single domain antibodies: a promising eksperimentalnye and therapeutic tools in the field of infection and immunity". Med. Environ. Immunol. 2009; 198, 157-174].
The disclosure of the present invention
The method of producing nanoentities linking antigens of Mycoplasma M.hominis, carried out on the basis of the selection of the method of phage display, genetically engineered modifications encoding these antibody sequences and use as the producer of the active substance (antibody) of E. coli bacteria. Nanoparticle (molecular weight of about 12-15 kDa) on the order of smaller traditional antibodies and are fully functional antigen-binding units, with a number of new positive qualities of practical significance. Nanoparticle is a single-domain protein that is highly soluble and has a high stability in a wide range of temperatures and pH). This avoids problems with solubility and proper folding of antibody molecules by product procure the political cells and leads to significantly lower costs of production compared to traditional methods of obtaining therapeutic monoclonal antibodies in eukaryotic expression systems. Considerably easier as well as for storing and transporting antibodies compared with significantly less stable traditional antibodies. Due to a smaller size nanoparticle have a greater ability to penetrate tissue. Finally, nanoparticle make it easy to carry out 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.
Although mainly for illustrative purposes, the authors present invention focuses on the production of antibodies using E. coli, the average expert in the art it will be obvious that under the scope of the present invention fall and other types of systems implementing the present invention not expressly stated herein. For example, as a eukaryotic producer can be used yeast culture or any other system, is evident in this technical field.
The method of producing nanoentities described in examples 1 and 2. The choice of the ways of implementation to obtain nanoentities with the claimed properties of prokaryotic expression system, in accordance with one of preferred embodiments of the present invention due to the following factor is mi:
1) high expression yield, efficiency developments in large quantities due to the expression of nanoentities in periplasm bacteria E.coli (1-10 mg from 1 liter of culture);
2) simplicity of various genetic manipulations, adaptation for specific tasks, the ability to create multivalent and multifunctional derivatives;
3) high economic profitability. The authors of the present invention proceed from the fact that, as is well known to the average expert in the art, the primary source of the received sequence nanoentities can then be adapted or "rich" in a different way for future practical use.
So, nanoparticle can be a source modules (blocks) more complex multimodal drugs. It is possible to unite in one multivalent derived two, three or more monovalent primary nanoentities. These combine into a single structure nanoparticle can communicate both with the same epitope of the target antigen and its different epitopes or even with different antigens on target. It is also possible combo unite into one design nanoentities and other molecules or drugs with obtaining multifunctional drugs; multimerization by introducing additional is sustained fashion amino acid sequence, interacting protein domains, such as lacinova the zippers, or sequences of small proteins that form stable complexes. For modulating properties of drug nanoparticle, for example, to increase the lifetime or improved method of purification, the resulting compounds may be introduced an additional amino acid sequence. Average expert in the art it will be obvious that such modifications and other options antibodies underlying the present invention fall under the scope of the present invention, as are the structural and functional variants nanoentities. Thus, the authors of the present invention is meant by the term "nanoparticle" as the primary source derived, "minimum" amino acid sequence nanoentities and their modifications resulting from the above-mentioned adaptations or "formatting" and their variants. The term "variant antibodies" for the purposes of the present invention means a polypeptide that contains the changes in amino acid sequence, i.e. deletions, insertions, additions or substitutions of amino acids, provided that it retains the desired activity of the protein, for example at least 10% of the activity of the parent nanoparticle. A number of changes in version belosevic from the position or the type of amino acid residue in the three-dimensional structure of the protein. The amount of change may be, for example, from 1 to 30, more preferably from 1 to 15, and most preferably, from 1 to 5 changes in the sequence of the original nanoparticle. These changes can occur in regions of the polypeptide that are not critical to its function. This is possible due to the fact that some amino acids have a high homology with each other, and therefore the tertiary structure or activity of the protein is not violated by such a change. Therefore, as a variant protein can be a protein that is characterized by a homology of at least 70%, preferably at least 80%, more preferably at least 90%and most preferably at least 95% relative to the amino acid sequence of the original nanoparticle while maintaining the activity of the polypeptide. Homology between amino acid sequences can be established using well-known methods, for example using sequence alignment computer program BLAST 2.0, which calculates three parameters: the account, the identity and similarity.
Substitution, deletion, insertion, addition or substitution of one or several amino acid residues will represent a conservative mutation or conservative mutations, provided that the activity of the protein when it is protected. An example of a conservative mutasa(s) is a conservative substitution(s). "Conservative amino acid substitution" is a substitution in which the amino acid residue is substituted with amino acid residue having a similar side chain. In this field of technology is defined family of amino acids with similar side chains. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). As hypervariable regions of nanoentities determine their specific interaction with the antigen, it is homologous replacement of amino acids in these areas can lead to several different sequences nanoentities, which have identical or similar properties. Thus, the average expert in the art it will be obvious that the volume under the m of the present invention are subject not only the application sequence nanoentities, but those that can be obtained by substitutions of amino acids in the hypervariable sites (indicated in the list of sequences as CDR), but very similar in properties, amino acids (conservative substitutions).
DNA fragments that encode essentially the same functional polypeptide can be obtained, for example, by modifying the nucleotide sequence of the DNA fragment encoding the original nanoparticle, for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a specific site will be deleterows, substituted, inserted or added. The DNA fragments, modified as described above, can be obtained using traditional processing methods with the aim of obtaining mutations.
DNA fragments that encode essentially the same functional polypeptide original nanoparticle, can be obtained by ekspressirovali DNA fragments with mutations described above, and determine the activity of the expressed product.
Substitution, deletion, insertion or addition of nucleotides described above, also include mutations that occur in nature and, for example, due to variability.
The polypeptides of nanoentities according to the present invention can be encoded by a large variety of molecules of nucleic Ki the lot, what 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. Any of these degenerate molecules encoding nucleic acids may be part of cassettes expressing antibodies declared in accordance with the present invention and falling under the scope of the present invention.
Obtain functionally active nanoentities that recognize antigens M.hominis shown in examples 3, 4, 5.
The effectiveness of treatment of mycoplasmal infections of selected pharmaceutically active amount of antibodies demonstrated in example 6.
Brief description of drawings
Figure 1 shows the nucleotide and amino acid sequences of the two selected nanoentities, aMh1 and aMh2 received in parallel in the same way and has the desired properties: the ability to specifically bind Mycoplasma hominis. Nucleotide sequence of cDNA encoding two selected nanoparticle were identified (aMh1 - SEQ ID NO: 1; aMh2 - SEQ ID NO: 3), and from them were derived the corresponding amino acid sequence selected nanoentities (aMh1 - SEQ ID NO: 2; for aMh2 - SEQ ID NO: 4). In azannyh sequences are underlined, respectively (from left to right, from N - to C-end) hypervariable sites CDR1, CDR2 and CDR3 antivirusnaya sequences selected nanoentities.
Figure 2 presents electrophoregram polyacrylamide gel with antigenic material proteins of Mycoplasma hominis (M.hominis) strain H-34, used for immunization of a camel.
Figure 3 and 4 shows the results of ELISA analysis of recognition selected by nanoparticulate aMh1, aMh2 immobilized in the wells of immunological die lipid-associated membrane proteins of Mycoplasma. As a control used cells with immobilized protein ovalbumin chickens. It is seen that the antibodies specifically bind with the antigens of Mycoplasma hominis M.hominis, and, with lower affinity (figure 4), Mycoplasma oral M.orale and Mycoplasma arginini M.arginini and do not interact with the lipid-associated membrane proteins of other species of Mycoplasma.
Numbers from 1 to 8 marked columns corresponding to different species of Mycoplasma and negative control of the experiment:
1 - Mycoplasma hominis (Mycoplasma, hominis),
2 - Mycoplasma orale (Mycoplasma oral),
3 - Mycoplasma arginini (Mycoplasma arginini),
4 - Mycoplasma arthritidis (Mycoplasma arthritidis),
5 - Mycoplasma pneumoniae (Mycoplasma, pneumonia),
6 - Mycoplasma fermentans (Mycoplasma fermentans),
7 - Ureaplasma urealiticum (Ureaplasma urealyticum),
8 - negative control, digits and 2 are marked accordingly nanoparticle aMh1, aMh2.
Figure 5 illustrates the results of enzyme immunoassay recognition selected by nanoparticulate aMh1, aMh2 M.hominis in paraformaldehyde fixed infected cell cultures. It is seen that the antibodies specifically associated with M.hominis in the infected culture and do not interact with uninfected control cells. Figures 1 and 2 are marked accordingly nanoparticle aMh1, aMh2, figure 3 corresponds to the negative control, the numbers from 1 to 3 columns marked, sootvetstvetstvovat infected cells to uninfected cells and pinkunicorn with antibodies to the cells.
The embodiments of the present invention
Obtaining a library of single-chain variable domains of antibodies
Bactrian camel Camelus bactrianus consistently were immunized 5 times by subcutaneous administration of antigenic material mixed with an equal volume of complete (first injection) or incomplete (if other injections) 's adjuvant adjuvant. As the antigen used the drug lipid-associated proteins of Mycoplasma hominis (M.hominis), strain H-34 (figure 2)allocated to standard fractionation of the proteins in TX-114 (Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution., J Biol Chem. 1981 Feb 25; 256(4): 1604-7). The second immunization was performed 3 weeks after the first, then at intervals of two weeks Provo is or even three immunization. Blood samples (150 ml) was performed 5 days after the last injection. To prevent clotting of blood was added 50 ml of a standard salt solution (PBS)containing heparin (100 units/ml) and EDTA (3 mm).
The blood was diluted 2 times the standard saline solution (PBS)containing 1 mm EDTA. 35 ml of diluted blood was layered on the step special medium (Histopaque-1077, Sigma) with a density of 1.077 g/ml volume of 15 ml and perform the centrifugation 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.
Total RNA from b-lymphocytes were isolated using TRIzol reagent (Invitrogen). Then, on a column of oligo(dT)-cellulose from the total RNA was purified poly(A)-containing RNA. The concentration of RNA was determined using biophotometer (Eppendorf) and checked the quality of the selected RNA by electrophoresis in 1.5%agarose gel with formaldehyde.
The reverse transcription reaction was performed according to standard Protocol [Sambrook et al., 1989] using reverse transcriptase H-M-MuLV and primer oligo(dT)15as seed.
The products of reverse transcription were used as matrix in the two-stage polymerase chain reaction and the resulting amplification products were cloned sites Ncol(Pstl) and Notl in amigny vector, as described previously [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006]. Proceedure selection was also similar to that in the mentioned works. It was based on the method of phage display, in which the phage-assistant used the phage M13KO7 (New England Biolabs, USA).
Selection of nanoentities, specifically recognizing M.hominis
Selection of nanoentities was performed by the method of phage display using the drug lipid-associated proteins M.hominis, strain H-34, immobilized on the bottom of the holes 96-well ELISA plate. Used polystyrene immunological die 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 pIII) was repeated, usually three consecutive times. All manipulations were performed as described in the publications Tillib S.V., Ivanova TI, Vasiliev L.A. 2010. Fingerprinty analysis selection "Manantial" method of phage display using two variants of phage-helpers. Acta Naturae 2010; 2 (3): 100-108; Hamers-Casterman C, Atarhouch, T., Muyldermans, S. et al. Nature 1993; 363: 446-448.; V.K. Nguyen, A. Desmyter, Muyldermans S. Adv. Immunol. 2001; 79: 261-296; Saerens, D., Kinne j, Bosmans E., U. Wernery, uyldermans S., Conrath K. J Biol Chem. 2004; 279: 51965-51972; Rothbauer, U., K. Zolghadr, Tillib, S., et al. Nature Methods 2006; 3: 887-889].
Sequences of clones selected nanoentities were grouped according to the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of the amplified sequences nanoentities three parallel customease restriction endonucleases (Hinfl, Mspl, Rsal). The cDNA sequence of nanoentities (SEQ ID NO: 1 and 3) were identified (figure 1). In these sequences are underlined, respectively (left to right) hypervariable sites CDR1, CDR2 and CDR3 antigen-binding sequences selected nanoentities.
The cDNA sequence selected nanoparticle, periglomerular in the expression plasmid vector is a modified vector pHEN6 [Conrath KE, Lauwereys M, Gallenj M, Matagne A, Frère JM, Kinne J, Wyns L, Muyldermans S. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. / The "beta lactamase inhibitors derived from single-domain fragments of antibodies induced in Camelids". Antimicrob Agents Chemother. 2001; 45:2807-12], which allows joining With the end of nanoparticle (His)6 epitope (immediately following the ON-epitope encoded in the vector pHEN6). Due to the presence of N-end expressed sequence signal peptide (peIB) accumulating recombinant protein (nanoparticle) accumulates in lane the plasma bacteria, that allows him to select the method of osmotic shock, do not actually destroying bacterial cells. 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 was isolated from periplasmatic extracts using affinity chromatography on Ni-NTA-agarose using the system for cleaning QIAExpressionist (QIAGEN, USA).
Detection of specific antigens using nanoentities, specifically recognizing M.hominis.
Demonstration of binding nanoentities with lipid-associated proteins M.hominis
The ability of nanoentities to bind lipid-associated proteins M.hominis was tested using the method enzyme immunoassay with immobilized lipid-associated proteins M.hominis, according to the standard Protocol. As control was used wells with immobilized protein a chicken ovalbumin or bovine serum albumin (non-specific proteins). Detection of bound peroxidase aMh1 and aMh2 was performed using anti-mouse antibodies, secondary antibodies to mouse IgG conjugated with horseradish peroxidase and a chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine, Sigma).
Figure 3 presents the results of analysis, from which it follows that nanoparticle specifically bound to the immobilized lipid-associated proteins M.hominis, but not associated with ovalbumin was used as a blocking agent.
To demonstrate the binding specificity of nanoentities with lipid-associated proteins M.hominis, M.pneumoniae, M.orale and M.arginini
The ability of nanoentities to bind lipid-associated proteins .hominis was tested using the method enzyme immunoassay with immobilized lipid-associated proteins M.hominis, M.arginini, M.arthritidis, M.orale, M.fermentans, M.pneumoniae, U.urealiticum according to the standard Protocol. As control was used wells with immobilized protein a chicken ovalbumin or bovine serum albumin (non-specific proteins). Detection of bound peroxidase aMh2, aMh1 was performed using anti-mouse antibodies, secondary antibodies to mouse IgG conjugated with horseradish peroxidase and a chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine, Sigma).
Figure 3 and 4 presents the results of the analysis, from which it follows that nanoparticle specifically bound to the immobilized lipid-associated proteins of Mycoplasma hominis (M.hominis) and, with lower affinity, Mycoplasma pneumoniae (M.pneumoniae), Mycoplasma oral (M.orale) and Mycoplasma arginini (M.arginini), but not associated with lipid-associated proteins other the species of Mycoplasma.
Demonstration of binding nanoentities eukaryotic cells infected with Mycoplasma, hominis (M.hominis), in vitro
Eukaryotic cell culture MCF7 was infected M.hominis by adding culture M.hominis into the culture medium and incubation of MCF7 cells in this environment. Then cells were sown on cultural tablet and recorded 0,8% paraformaldehyde according to the standard Protocol. The ability of nanoentities aMh3, aMh1 contact M.hominis, persistent on the surface of eukaryotic cells, was tested using the method of immunoassay according to the standard Protocol. As control was used wells with fixed uninfected cells. Detection of bound peroxidase aMh1, aMh2 was performed using anti-mouse antibodies, secondary antibodies to mouse IgG conjugated with horseradish peroxidase and a chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine, Sigma).
Figure 5 presents the results of the analysis, from which it follows that nanoparticle specifically associated with M.hominis, persistent on the surface of eukaryotic cells, but not in contact with an uninfected cells.
Thus, stated in accordance with the present invention the pharmaceutical composition has proven its applicability for detection M.hominis in vitro.
To demonstrate the effectiveness of terap whitesage actions nanoentities for the treatment of mycoplasmal infections mouse strain BALB/C mice (18-20 g) with synchronized ovulation cycle were infected vnutrivaginalno M.hominis line MY17386, at a dose of 1*10^7 on the mouse, as described previously (Taylor-Robinson D, Furr PM. Further observations on the murine model of Mycoplasma hominis infection. / "Further observations in the mouse model of infection with Mycoplasma hominis. J Environ Med. 2010 Aug; 59 (Pt 8):970-5. Epub 2010 May 6). One week after infection, mice received daily endovaginal douching volume of 100 μl FBI with nanoparticulate aMh1, 2 in the amount of 70 μg/mouse/day (minimum active amount of antibodies capable of blocking mycoplasmal infection) within 5 days, the dose of nanoentities was selected in accordance with literature data, according to which for therapeutic purposes antibodies used in doses of from 30 to 100 μg per mouse (Xiao X, Zhu Z et al. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.PLoS One. 2010 Oct 13; 5(10):e13047; Chen Z, Moayeri M, Purcell Monoclonal Antibody Therapies against Anthrax. RToxins (Basel). 2011 Aug; 3(8): 1004-19. Epub 2011 Aug 15). Control groups of mice syringing carried out 100 ál of the FBI. Later, 2 days after the last douching organs of the reproductive system of mice were homogenized using a homogenizer with the principle of the bead mill, the homogenates used to determine the titer of mycoplasmas by culture method of sowing.
In table 1 and table 2 presents the results of cultural sowing M.hominis from homogenates of the genital organs of mice. From the presented results it follows that nanoparticle aMh1 and aMh2 have a reliable inhibitory effect on m is coplestone endovaginal infection, that leads to lower titers of mycoplasmas in homogenates of organs of the reproductive system. Thus, the demonstrated efficacy of treatment of Mycoplasma infection using nanoentities aMh1, 2. Table 1 presents the demonstration of the effectiveness of nanoparticle aMh1 for the treatment of mycoplasmal infections caused M.hominis.
|Douching FBC PFU/ml M.hominis||A douche nanoparticulate aMh1, PFU/ml M.hominis|
Table 2 presents the demonstration of the effectiveness of nanoparticle aMh2 for the treatment of mycoplasmal infections caused M.hominis.
|Douching FBC PFU/ml M.hominis||A douche nanoparticulate aMh2, PFU/ml M.hominis|
Thus, in these examples revealed: the creation of new antibodies able to bind antigens of Mycoplasma M.hominis; the retrieval method; a method of treating urogenital mycoplasmosis caused by a mammal by Mycoplasma M.hominis, from which it is evident that the task is achieved.
1. Nanoparticle, specifically linking the lipid-associated antigen M.hominis and having the amino acid sequence of SEQ ID NO:2.
2. Nanoparticle, specifically linking the lipid-associated antigen M.hominis and having the amino acid sequence of SEQ ID NO:4.
3. Nanoparticle according to claim 1, characterized in that it suppresses the infection caused by Mycoplasma M.hominis.
4. Nanoparticle according to claim 2, characterized in that it suppresses the infection caused by Mycoplasma M.hominis.
5. A method of treating infection in a mammal caused by Mycoplasma M.hominis, including the introduction of a given mammal a therapeutically effective amount of nanoparticle according to claim 1.
6. A method of treating infection in a mammal caused by mycoplas the Oh M.hominis, includes introduction to the specified mammal a therapeutically effective amount of nanoparticle according to claim 2.
SUBSTANCE: what is presented is a diagnostic technique for preeclampsia in a pregnant woman with chronic arterial hypertension on her 24-40 weeks of pregnancy. Using ELISA, blood serum is examined for the content of IgG autoantibodies against duplex DNA, thrombocyte membrane antigens TrM-001-15 and TrM-015-12, renal tissue antigens KiM-05-300 and KiS-07-120 and liver mitochondrial antigen NMMR. Further, each value of the examined antibodies is described for the autoantibodies ratio in the test serum to the control serum. If the value is 0.55 or less of at least three types of the autoantibodies, one of which refers to the antibodies in kidney tissue, and the other - to the duplex DNA, preeclampsia is diagnosed in the pregnant woman.
EFFECT: invention provides the effective diagnostic technique for preeclampsia in pregnant women with chronic arterial hypertension on their 24-40 weeks, and the improvement of the perinatal outcomes.
SUBSTANCE: lachrymal fluid and blood serum are examined by enzyme immunoassay using specific test systems. Higher levels of metalloproteinase-9 (MMP-9) exceeding 52.5 ng/ml in lachrymal fluid and 274.49 ng/ml in blood serum; higher levels of a complex of metalloproteinase-9 and its tissue inhibitor (MMP-9/TIMP-1) exceeding 0.19 ng/ml in lachrymal fluid and 4.93 ng/ml in blood serum, and higher levels of secretory immunoglobulin A (slgA) exceeding 47.38 mg/l in lachrymal fluid and 2.1 g/l in blood serum are the criteria to diagnose primary open-angle glaucoma.
EFFECT: use of the declared method can effectively diagnosing primary open-angle glaucoma.
6 dwg, 2 tbl, 1 ex
SUBSTANCE: detection system for detecting target molecules includes a sensor chip (1), having on its detecting surface (33) an immobilised target molecule or a capturing molecule for target molecules and a soluble reagent layer (5), having a labelled molecule for binding with the target. The group of inventions also relates to a sensor chip (1) and a method of detecting target molecules in a sample using said sensor chip.
EFFECT: high sensitivity and accuracy of analysis while cutting duration of analysis.
19 cl, 8 dwg, 2 ex
SUBSTANCE: patient's blood serum taken on the first three days following the myocardial infraction is examined for the level of a tissue inhibitor of type 1 metalloproteinase (TIMP-1) and the concentration of vascular cell adhesion molecules (sVCAM-1) to calculate a repair coefficient (RC) by formula: RC=TIMP 1 /sVCAM1, and if the CR values exceeds 0.94, the developing left ventricular postinfarction dilatation is predicted.
EFFECT: early prediction of the developing left ventricular postinfarction dilatation, including of the symptom-free forms.
3 ex, 1 tbl
SUBSTANCE: invention describes a method for measuring the immunomodulatory properties of cosmetic products, which involves the change determination of saliva dysenteric microbe antibody titer in saliva passive hemagglutination test (PHT); and the change determination of the antibody titer is specified in the saliva samples before the face, hands or body treatment with a cosmetic product and after that; the individuals being tested are divided into two groups; the first group of individuals with low antibody titer 1:128 or less are determined with the increased saliva antibodies after the face and body treatment with the cosmetic product, i.e. the immunomodulatory properties, and vice versa, the second group of individuals having antibody titers 1:256 or higher shows either no reaction, or the normalised content of the saliva humoral immunity factors.
EFFECT: method is simple, accurate, cost-saving and safe.
3 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: presented invention refers to medical microbiology, particularly to molecular-genetic typing of the strains Helicobacter pylori. What is presented is a method for differentiation of the strains H.pylori by multilocal VNTR-typing with the PCR involving oligonucleotide primers on VNTR-comprising loci of H.pylori - HpA, HpD, HpE and HpF; the strains H.pylori are differentiated by a number of repetitions in the amplified fragments in each VNTR-comprising locus of H.pylori - HpA, HpD, HpE and HpF that enables genetic typing of the studied strains.
EFFECT: what is presented is the method for differentiation of the strains Hpylori by multilocal VNTR-typing.
1 tbl, 3 ex
SUBSTANCE: what is involved is the immunogenetic analysis of venous blood of the patients with chronic myeloid leukemia treated with a first-generation tyrosine kinase inhibitor, Gleevec. Polymerase chain reaction PCR-SSP is used to identify the genes of the class II HLA system, locus B. If observing any specificities HLA-DRB1*15(02) or HLA-DRB1*16(02), the favourable outcome of chronic myeloid leukemia with an optimal response developing over the control period (6, 12 18 months of therapy) is predicted. In observing the specificities DRB1*11(05) or DRB1* 14(06), the unfavourable outcome of chronic myeloid leukemia with a failure to achieve an optimal response is predicted.
EFFECT: higher accuracy of prediction of the outcome of chronic myeloid leukemia with underlying Gleevec therapy.
9 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. What is presented is a monoclonal antibody or a fragment thereof, which causes NK-cell induced lysis of human target cells carrying HLA-E on their surface, optionally conjugated with a detectable marker. The antibody or the fragment thereof are specific to NKG2A, have heavy and light chains, each of which contains 3 CDR and does not bind specifically: human NKG2C or NXG2E and Fc receptor. There are described: a pharmaceutical composition, a method for recovering the NK-mediated lysis of target cells, a conjugate, a set and a method for detection based on the use of the antibodies. What is disclosed is a composition for treating autoimmune and inflammatory diseases.
EFFECT: use of the invention can find application to stimulate the NK-cells leading to the lysis of dendritic cells, which contribute to the pathology of autoimmune and inflammatory diseases, that can find application in medicine.
36 cl, 4 dwg, 5 tbl, 12 ex
SUBSTANCE: for the purpose of the prediction of the delayed bone consolidation accompanying extrafocal osteosynthesis, preoperative blood CD3+ lymphocytes and CD 19+ lymphocytes are counted, and then the CD3+ lymphocytes/CD 19+ lymphocytes index is calculated. If the relation is more than 6.6 units, the postoperative delayed bone consolidation is predicted, and the value being 6.6 units and less enables predicting the normal clinical course of osteogenesis.
EFFECT: use of this method allows for the preoperative prediction of the bone consolidation pattern.
SUBSTANCE: for the purpose of early prediction of bronchopulmonary dysplasia, the 3-7-day newborn's blood serum is examined for interleukine-6 and interleukine-1 receptor antagonist. If interleukine-6 appears to increase more than 24 pg/ml, while the interleukine-1 receptor antagonist is more than 560 pg/ml, developing bronchopulmonary dysplasia is predicted.
EFFECT: early prediction of bronchopulmonary dysplasia, and respectively, the adequate therapeutic correction in the newborns with very low or extremely low body weight.
1 tbl, 6 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discloses a method for preparing an anthrax diagnostic serum by hyperimmunisation of ox producers with an antigen of the strain Bacillus anthracis M-71. Hyperimmunisation is conducted in increasing doses: first subcutaneous introduction of the antigen 100-120 mln microbial cells together with saponin 2.5-3 mcg; 12-14 days later, the antigen is introduced intracutaneously in a dose of 2.5-3.0 bln microbial cells together with saponin 2.5-3 mcg; 6-7 days later, every 3-4 days, the antigen is introduced intravenously 12-14 times in increasing doses 10.0 to 210 bln. microbial cells. It is followed by blood sampling, keeping at temperature 37-38°C for 2-3 hours, placing in a fridge at 2-8°C for 3-5 days, separating serum. The prepared serum is preserved in 5-7% carbolic acid in isotonic solution in ratio (9-10):1 respectively. The ready serum is sterile and has an AR titre min. 1:1000, a CFT titre min. 1:20. A diagnostic set for anthrax diagnosis comprises the antigen of the strain Bacillus anthracis M-71, the anthrax diagnostic serum and healthy bovine's native serum.
EFFECT: invention provides higher specific activity of the serum and diagnostic effectiveness for an anthrax agent.
5 cl, 1 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discloses a number of polynucleotides and polypeptides β-hemolytic streptococci, in particular polypeptides and polynucleotides of Streptococcus pyogenes, and based on them immunogenic compositions, used for prevention or reduction of symptoms of colonisation or infection, caused by β-hemolytic streptococci. Immunogenic composition (versions) contains mixture of two or more polypeptides, encoded by sequence of nucleic acid (NA), which has, at least, 90% identity of sequence of NA, selected from group, consisting of peptidase C5a (SCP), open reading frame (OPC)554, OPC 1218, OPC 1358 and OPC 2459. One of versions of immunogenic composition contains polypeptide SCP, polypeptide peptidylpropylisomerase and, at least, one other polypeptide. Also disclosed are methods of protecting susceptible mammal against colonisation or infection, caused by β-hemolytic streptococcus, by immunisation of immunogenic composition by invention.
EFFECT: invention provides immunogenic compositions and methods for prevention or reduction of symptoms of infections, caused by β-hemolytic streptococci of group A, B, C and G, as well as ensures immunity to wide spectrum of bacteria BHS.
41 cl, 16 dwg, 2 tbl, 3 ex
SUBSTANCE: invention discloses a purified and/recombinant antigen polypeptide possessing toxin activity, recovered from Clostridium perfringens with specified amino acid sequence. The invention discloses the recovered or recombinant polynucleotide coding such polypeptide, an expression vector and a host cell expressing the polypeptide. The invention discloses a method for preparing the polypeptide, an antibody specifically bound with the polypeptide, immunogenic compositions and vaccines containing the given polypeptide or a polynucleotide thereby providing a specifically immune response to the polypeptide. There are disclosed a method for inducing the immune response, a method of determining the fact whether an individual has been exposed to a pathogen (versions), a method of screening, an agonist or an antagonist modulating activity of the polypeptide, a method of animal vaccination, e.g. hens for inducing active immunity, as well as passive immunity in hen off-springs which becomes less sensitive to clostridial diseases. What is disclosed is a transgenic plant containing the exogenous polynucleotide coding the polypeptide under the invention, applicable for animal feeding.
EFFECT: polypeptide is used as an ingredient of a forage or a beverage for preventing a disease caused by bacteria expressing the polypeptide under the invention.
39 cl, 8 dwg, 6 tbl, 11 ex
SUBSTANCE: hybrid cultured cell strain of the animals Mus museums 13F8 is produced by immunisation of Balb/c mice. The mice are immunised by four introductions of the recombinant antigen preparation F1 100 mcg/mouse. The third post-immunisation day is followed by splenocyte hybridisation of the immunised mice (1x108 cells) and mice myeloma cells P3-X63 Ag/8-653 (1×107cells). Polyethylene glycol (Sigma, the USA) is used as a fusion agent. The hybridisation is followed by hybridoma selection, screening, cloning and cryopreservation. Hybridoma is deposited in the State Collection of Pathogens and Cell Cultures of GKPM-Obolensk, No. N-18.
EFFECT: hybrid cultured cell strain producing monoclonal antibodies is applicable for constructing the based plague agent test systems.
7 dwg, 4 tbl, 8 ex
SUBSTANCE: polypeptide (versions) immunogenic with respect to meningococcal infections contains: an amino acid sequence at least 90 % identical to a sequence presented in the description (SEQ ID NO: 32), or said amino acid sequence, or a fragment of 80 sequenced amino acids of said sequence. What is described is an antibody which contacts with the polypeptide under the invention and which may be used as a drug. What is described is nucleic acid of the preset structure which codes the polypeptide or its versions and which may be used for treating or preventing a disease and/or an infection caused by Neisseria meningitides. The invention provides additional polypeptides applicable in advanced vaccines for preventing and/or treating meningococcal meningitis. The peptides can also find application in diagnosing of the disease and as targets of antibiotics.
EFFECT: higher clinical effectiveness for meningococcal meningitis.
19 cl, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: drug preparation for treating infectious diseases accompanied by neurotoxic disorders including neuroinfectious and neurodegenerative diseases is presented in the form of a pharmaceutical composition and contains an activated potentiated form of human gamma interferon (IFN-γ) antibodies and an activated potentiated form of brain-specific S-100 antibodies. The pharmaceutical composition is presented in a solid dosage form - in the form of granules. As pharmaceutically acceptable additives, it contains lactose, microcrystalline cellulose and magnesium stearate, and each of the components of very-low-dose affinity purified antibodies are used in the form of a mixture of various, preferentially homoeopathic dilutions 1/100. A method of treating and preventing infectious diseases accompanied by neurotoxic disorders involves the introduction into a body of the activated potentiated form of IFN-γ antibodies, and an enhancing agent in the form of the activated potentiated form of very-low-dose affinity purified brain-specific S-100 antibodies.
EFFECT: use of the invention enables enhanced immune response at height of the disease, ensures effective rehabilitation and prevents recurrences of the disease.
14 cl, 4 dwg, 1 tbl, 2 ex
SUBSTANCE: invention describes novel polynucleotide and amino acid sequences of Brachyspira hyodysenteriae, which can be used to diagnose diseases in animals, caused by B. hyodysenteriae, to treat or prevent diseases associated with infection with B. hyodysenteriae. The invention describes a cell containing a plasmid containing a polynucleotide, for treating and preventing a disease associated with infection of an animal with B. Hyodysenteriae. The invention describes immunogenic and vaccine compositions for generating immune response in an animal, which contain a polypeptide, a polynucleotide, a cell or a plasmid for treating or preventing infection of animals by B. hyodysenteriae, as well as sets of instruments for diagnosis which contain a monoclonal antibody, capable of biding the disclosed polypeptide or a polypeptide or polynucleotide. The invention enables to successfully diagnose diseases caused by B. hyodysenteriae, prevent or treat animals infected with B. hyodysenteriae. The sequences described herein can be used for diagnosis and therapeutic and/or preventive treatment of animals from diseases caused by other types of Brachyspira, including B. intermedia, B. suantatina, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii and B. pilosicoli.
EFFECT: high efficiency of using the composition.
39 cl, 4 tbl
SUBSTANCE: strain of hybridioma is obtained by immunization of mice of line BALB/c. Mice are immunised by conventional method by double with thirty day exposition subcutaneous introduction of inactivated spores of B. Anthracis strain "СТИ-1". On the third day after last buster-injection carried out is hybridisation of splenocytes of immune mice (1×108 cells) with cells of mouse myeloma P3-X63 Ag/8-653 (1×107 cells), using for fusion polyethylene glycol (Sigma, USA). After hybridisation carried out are selection, screening, cloning and cryopreservation of hybridoma. Obtained hybridoma is deposited in collection of microorganisms of FSSE SRC AMB under number H10. Invention relates to biotechnology and can be used for obtaining monoclonal antibodies (MCA) to spores Bacillus anthracis. Strain productivity is 20-50 mcg/ml of monoclonal antibodies (MCAla in ascitic liquid- 5-10 mg/ml. Hybridoma produces MCA, related to IgGl subclass of mouse immunoglobulins and spores specific to Bac.anthracis.
EFFECT: increase of strain productivity.
7 dwg, 2 tbl, 9 ex
SUBSTANCE: invention discloses peptides for detecting Mycobacterium tuberculosis, which consist of less than 18 amino acids and are capable of binding with the antibody against GRDIKVQFQSGGNNSPAV peptide (all sequences are given in a list of sequences). One of the peptides contains an NSPAX sequence, where X denotes methionine. More preferably, the peptides contain an NNSPAV sequence or have the type SGGNNSPAX, where X denotes methionine, leucine, alanine or valine. The invention describes a nucleotide sequence which codes the disclosed peptides, as well as an antibody or fragment thereof, capable of binding with said peptide. The invention discloses methods of detecting M.tuberculosis, involving ELISA substitutive analysis of a sample using a peptide capable of binding with the antibody against GRDIKVQFQSGGNNSPAV peptide or using the antidody.
EFFECT: use of the invention simplifies tests to determine anti-tuberculosis antibodies in a population owing to the artificially modified peptide sequences of T-cell epitope Ag85B M tuberculosis.
27 cl, 5 dwg
SUBSTANCE: there are described polypeptides eliciting an immune response on H.influenzae. There is offered an antibody selectively binding specified polypeptides. The invention also concerns an immunogenic composition containing the described polypeptides.
EFFECT: invention can be used in medicine for treating or preventing a disease or an infection caused by Hinfluenzae, such as otitis media.
9 cl, 3 tbl
SUBSTANCE: invention relates to a pyridine thio-derivative of general formula (I) or pharmaceutically acceptable salts thereof: , where R1 and R2, respectively, denote a hydrogen atom; R3 and R4, respectively, denote a hydrogen atom or a C1-8alkyl group; X denotes -O- or -S-; Y denotes a C1-12alkyl group, which can be substituted with a substitute selected from a group consisting of a hydroxyl group, C1-8alkoxy group or -(R5-O)n-R6 (where R5 denotes a C1-5alkylene group, R6 denotes a C1-8-alkyl group which can be substituted with a halogen atom, and n denotes an integer from 1 to 2) and Z denotes a hydrogen atom. The invention also relates to pharmaceutical compositions based on said compounds, having antibacterial activity towards Helicobacter pyroli.
EFFECT: obtaining novel compounds and a pharmaceutical composition based on said compounds, which can be used in medicine to prevent or treat diseases in which Helicobacter pylori participates, specifically gastritis, gastric ulcers, duodenal ulcers, gastric MALT lymphoma, hyperplastic gastric polyps or gastrointestinal malignant diseases.
10 cl, 3 tbl, 14 ex